Cell Death in Biology and Diseases: Series Editors

Cell Death in Biology and Diseases
Series Editors
Xiao-Ming Yin
Zheng Dong
For further volumes:

http://www.springer.com/series/8908
Daniel E. Johnson
Editor
Cell Death Signaling

in Cancer Biology
and Treatment
Editor
Daniel E. Johnson
Division of Hematology/Oncology
Department of Medicine
University of Pittsburgh
Pittsburgh
PA, USA
ISBN 978-1-4614-5846-3 ISBN 978-1-4614-5847-0 (eBook)

DOI 10.1007/978-1-4614-5847-0
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Library of Congress Control Number: 2012952022
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This book is dedicated to my loving wife
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Series Preface
Cell death, or conversely cell survival, is a major biological phenomenon. Just

as with cell proliferation and cell differentiation, cell death is a choice that a cell
has to make, sometimes voluntarily, other times accidentally. As such, cell death
serves a purpose in the biology of a multicellular organism. The machinery of
cell death and that of cell protection are evolutionarily conserved and their ele-
ments can even be found in single-celled organism. The disruption of cell death
mechanisms can often cause developmental abnormalities. Factors that can trigger
cell death are diverse and the cell death process is intricately connected with other
biological processes. Cell death directly contributes to the pathogenesis of many
diseases, including cancer, neurodegenerative diseases, and tissue injury in organ
failure.
The study of cell death and cell survival has become a multidisciplinary sub-
ject, which requires expertise from all fields of the modern biology. Exploring
the role of cell death in disease development and the modulation of cell death
for the prevention and treatment of devastating disease demands constant updat-
ing of our knowledge through the broadest interactions among all investigators,
basic and clinical. The rapid expansion of our knowledge in this field has gone
beyond what could be summarized in a single book. Thus, this timely series Cell
Death in Biology and Diseases summarizes new developments in different areas
of cell death research in an elaborate and systemic way. Each volume of this series
addresses a particular topic of cell death that either has a broad impact on the field
or that has an in-depth development in a unique direction. As a whole, this series
provides a current and encyclopedic view of cell death.
We would like to sincerely thank the editors of each volume in the series and
the authors of each chapter in these volumes for their strong commitment and
great effort towards making this mission possible. We are also grateful to our
team of professional Springer editors. They have worked with us diligently and
vii
viii Series Preface
creatively from the initiation and continue this on the development and produc-
tion of each volume of the series. Finally, we hope the readers will enjoy the read-
ing, find the content helpful to their work, and consider this series an invaluable
resource.
Xiao-Ming Yin MD, Ph.D.

Department of Pathology and Laboratory Medicine
Indiana University School of Medicine
Indianapolis, IN 46202, USA
Zheng Dong Ph.D.

Department of Cellular Biology and Anatomy
Georgia Health Science University and Charlie Norwood VA Medical Center
Augusta, GA 30912, USA
Book Preface
Over the past few decades, it has become widely appreciated that genetically
programmed, active cell death processes are critically important for immune-
mediated removal of infected or transformed cells, as well as self-elimination of
damaged cells. This includes removal via cellular suicide of cells that have been
damaged by conventional anti-cancer therapies such as chemotherapy and radia-
tion. Concurrently, it has been determined that defects in cell death pathways can
promote tumor development and progression, including the development of resist-
ance to chemotherapy, radiation, immunotherapies, and biologic agents. These
resistance mechanisms have devastating consequences for the successful treatment
of cancer patients. A key goal in the development of new therapeutic strategies
and agents for the treatment of cancer is to achieve selective and efficient killing
of tumor cells. Assisting this goal are numerous ongoing studies and discoveries
that have elucidated the molecular mechanisms of cell death pathways, including
the extrinsic and intrinsic apoptosis pathways, and the proteins that act to regulate
these pathways. Understanding of these cell death pathways is now making it pos-
sible to identify the specific cell death defects that occur in cancer cells, and is
leading to the development of novel strategies and agents to overcome or correct
these defects in cancer patients.
This volume summarizes current understanding of the molecular mechanisms
that govern cell death in normal and malignant cells, and introduces cutting-edge
opportunities for achieving selective killing of cancer cells by targeting these
mechanisms in patient’s tumors. A particular emphasis is placed on data emerging
from the translation of basic research findings to clinical trials. The book begins
by describing many of the common cell death defects that have been identified
in primary patient specimens, including aberrant overexpression of anti-apoptotic
proteins and oncoproteins, and mutation or loss of expression of pro-apoptotic
proteins and tumor suppressors. The unique bioenergetics of cancer cells and
unique characteristics of the tumor microenvironment are then discussed, along
with opportunities for novel therapeutic interventions that are afforded by these
distinctive features. The role of autophagy in regulating cancer cell death and
current progress in targeting autophagy to improve responsiveness to conven-
tional anti-cancer therapies is also presented. Additionally, a rapidly expanding
field implicates microRNAs in the regulation of cell death proteins and pathways.
ix
x Book Preface
Recent descriptions of aberrant microRNA expression in cancer cells and the

potential for targeting microRNAs are summarized. Further chapters describe
preclinical and clinical approaches currently being used to target DNA repair
pathways and protein chaperones as a means to provoke tumor cell death. New
discoveries regarding the complex interplay between dying cancer cells and the
immune system are also discussed, with an eye towards optimizing these interac-
tions for therapeutic advantage. Finally, recent progress in targeting specific com-
ponents of cell death signaling pathways is reviewed. These chapters highlight
exciting advancements in the targeting of sphingolipid signaling, Bcl-2 family
members, IAPs, death receptor signaling, the proteasome, and survival signaling
mediated by the PI3K/AKT and RAS/RAF/MEK/ERK pathways.
Cell Death Signaling in Cancer Biology and Treatment will be particularly
beneficial to biochemists, molecular biologists, and systems biologists inter-
ested in basic mechanisms of cell death signaling, the nature of cell death defects
in cancer, and the impact of basic biological processes on cell death regulation.
Scientists interested in the translation of findings from basic cell death research
to clinical trials will appreciate the emphasis each chapter places on these impor-
tant advances. Moreover, industry and academic scientists interested in anti-can-
cer drug development will learn of unique opportunities and approaches being
taken to develop and evaluate highly selective agents with potent killing activities
against tumors cells resistant to radiation or conventional chemotherapy drugs.
The work presented in this book is built on the foundation of many excellent
studies and discoveries from a vast number of scientists. I wish to acknowledge
those whose work is not presented, or whose area is not a specific focus of this
book. In addition, I would like to thank the inspirational guidance of the late
Stanley Korsmeyer, who introduced me to the fascinating field of cell death. I am
particularly thankful for the authors who are respected leaders in their areas of
research. Special thanks also go to my close colleagues in this field, with whom
I have had many helpful discussions, including Pam Hershberger, John Lazo,
Changyou Li, Hannah Rabinowich, Shivendra Singh, Xiao-Ming Yin, Jian Yu, Yan
Zang, and Lin Zhang. This book represents the first in a new book series entitled
Cell Death in Biology and Diseases (Springer) with Series co-editors Xiao-Ming
Yin and Zheng Dong. I am indebted to the co-editors for helping to develop the
content of this book, as well as to Aleta Kalkstein and Renata Hutter, editors from
Springer. Lastly, I wish to thank my children, Rachel, Josiah, and Matthew, for
their patience, inspiration, and laughter.
Daniel E. Johnson Ph.D.

Departments of Medicine and Pharmacology
and Chemical Biology
University of Pittsburgh and the University
of Pittsburgh Cancer Institute
Pittsburgh
PA 15213 USA
Contents
1 Defective Apoptosis Signaling in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . 1

Daniel E. Johnson
2 The Warburg Effect and Beyond: Metabolic Dependencies

for Cancer Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
David M Hockenbery, Mark Tom, Cori Abikoff
and Daciana Margineantu
3 Emerging Opportunities for Targeting the Tumor–Stroma

Interactions for Increasing the Efficacy of Chemotherapy. . . . . . . . . . 53
Rajesh R. Nair, Anthony W. Gebhard and Lori A. Hazlehurst
4 The Role of Autophagy in Drug Resistance and Potential

for Therapeutic Targeting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Reshma Rangwala and Ravi Amaravadi
5 MicroRNAs in Cell Death and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . 117

Jong Kook Park and Thomas D. Schmittgen
6 Targeting DNA Repair Pathways for Cancer Therapy. . . . . . . . . . . . . 137

Conchita Vens and Robert W. Sobol
7 Molecular Chaperones and How Addiction Matters

in Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Monica L. Guzman, Maeve A. Lowery, Tony Taldone, John Koren III,
Erica DaGama Gomes and Gabriela Chiosis
8 Sphingolipid Metabolism and Signaling as a Target

for Cancer Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Vinodh Rajagopalan and Yusuf A. Hannun
xi
xii Contents
9 Leading Small Molecule Inhibitors of Anti-Apoptotic

Bcl-2 Family Members. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Victor Y. Yazbeck and Daniel E. Johnson
10 SMAC IAP Addiction in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

Matthew F. Brown, Kan He and Jian Yu
11 Harnessing Death Receptor Signaling for Cancer Treatment. . . . . . . . 281

Simone Fulda
12 Proteasome Inhibition as a Novel Strategy for Cancer Treatment. . . . 303

Min Shen and Q. Ping Dou
13 New Agents and Approaches for Targeting the

RAS/RAF/MEK/ERK and PI3K/AKT/mTOR
Cell Survival Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
James A. McCubrey, Linda S. Steelman, William H. Chappell,
Stephen L. Abrams, Richard A. Franklin, Giuseppe Montalto,
Melchiorre Cervello, Ferdinando Nicoletti, Graziella Malaponte,
Clorinda Massarino, Massimo Libra, Jörg Bäsecke, Agostino Tafuri,
Michele Milella, Francesca Chiarini, Camilla Evangelisti,
Lucio Cocco and Alberto M. Martelli
14 Activation of Immune-Mediated Tumor Cell Death

by Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Melanie J. McCoy, Anna K. Nowak and Richard A. Lake
About the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Contributors
Cori Abikoff Division of Clinical Research, Fred Hutchinson Cancer Research

Center, Seattle, WA, USA
Stephen L. Abrams Department of Microbiology and Immunology, Brody School
of Medicine at East Carolina University, Greenville, NC, USA
Ravi Amaravadi Department of Medicine, Perelman School of Medicine,
Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA
Jörg Bäsecke Department of Medicine, University of Göttingen, Göttingen,
Germany
Matthew F. Brown Department of Pathology and Molecular and Cellular Pathol-
ogy Graduate Training Program, University of Pittsburgh School of Medicine, Uni-
versity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
Melchiorre Cervello Istituto di Biomedicina e Immunologia Molecolare, “Alberto
Monroy”, Consiglio Nazionale delle Ricerche, Palermo, Italy
William H. Chappell Department of Microbiology and Immunology, Brody
School of Medicine at East Carolina University, Greenville, NC, USA
Francesca Chiarini Institute of Molecular Genetics, National Research Council-
Rizzoli Orthopedic Institute, Bologna, Italy
Gabriela Chiosis Breast Cancer Service, Department of Medicine, Memorial
Sloan-Kettering Cancer Center, New York, NY, USA; Department of Molecular
Pharmacology and Chemistry, Sloan-Kettering Institute, New York, NY, USA
Lucio Cocco Cell Signalling Laboratory, Department of Human Anatomy, Univer-
sity of Bologna, Bologna, Italy
Q. Ping Dou Department of Pharmacology, Karmanos Cancer Institute, Wayne
State University School of Medicine, Detroit, MI, USA
Camilla Evangelisti Institute of Molecular Genetics, National Research
Council-Rizzoli Orthopedic Institute, Bologna, Italy
xiii
xiv Contributors
Richard A. Franklin Department of Microbiology and Immunology, Brody

Simone Fulda Institute for Experimental Cancer Research in Pediatrics, Goethe-
University, Frankfurt, Germany
Anthony W. Gebhard Molecular Pharmacology and Physiology Program and
Molecular Oncology Program, H Lee Moffitt Cancer Center, Tampa, FL, USA
Erica DaGama Gomes Breast Cancer Service, Department of Medicine, Memo-
rial Sloan-Kettering Cancer Center, New York, NY, USA
Monica L. Guzman Division of Hematology and Oncology, Weill Cornell Medical
College, New York, NY, USA
Yusuf. A. Hannun Stony Brook University, Stony Brook, NY, USA
Lori A. Hazlehurst Molecular Pharmacology and Physiology Program and Mo-
lecular Oncology Program, H Lee Moffitt Cancer Center, Tampa, FL, USA; Cancer
Biology Program, University of South Florida, Tampa, FL, USA
Kan He Department of Pathology, University of Pittsburgh School of Medicine,
University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
David M. Hockenbery Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA
Daniel E. Johnson Departments of Medicine and Pharmacology and Chemical
Biology, University of Pittsburgh, University of Pittsburgh Cancer Institute, Pitts-
burgh, PA, USA
John Koren III Breast Cancer Service, Department of Medicine, Memorial Sloan-
Kettering Cancer Center, New York, NY, USA
Richard A. Lake School of Medicine and Pharmacology, National Centre for As-
bestos Related Diseases, The University of Western Australia, Crawley, WA, Aus-
tralia; St John of God Health Care, Subiaco, WA, Australia
Massimo Libra Department of Biomedical Sciences, University of Catania,
Catania, Italy
Maeve A. Lowery Gastrointestinal Service, Department of Medicine, Memorial
Sloan-Kettering Cancer Center, New York, NY, USA
Graziella Malaponte Department of Biomedical Sciences, University of Catania,
Catania, Italy
Daciana Margineantu Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA
Alberto M. Martelli Institute of Molecular Genetics, National Research Council-
Rizzoli Orthopedic Institute, Bologna, Italy; Cell Signalling Laboratory, Depart-
ment of Human Anatomy, University of Bologna, Bologna, Italy
Contributors xv
Clorinda Massarino Department of Biomedical Sciences, University of Catania,

Catania, Italy
Michele Milella Regina Elena National Cancer Institute, Rome, Italy
Melanie J. McCoy School of Medicine and Pharmacology, National Centre for
Asbestos Related Diseases, The University of Western Australia, Crawley, WA,
Australia ; St John of God Health Care, Subiaco, WA, Australia
James A. McCubrey Department of Microbiology and Immunology, Brody
Giuseppe Montalto Department of Internal Medicine and Specialties, University
of Palermo, Palermo, Italy
Rajesh R. Nair Molecular Oncology Program, H Lee Moffitt Cancer Center, Tam-
pa, FL, USA
Ferdinando Nicoletti Department of Biomedical Sciences, University of Catania,
Catania, Italy
Anna K. Nowak School of Medicine and Pharmacology, The University
of Western Australia, Crawley, WA, Australia ; St John of God Health Care,
Subiaco, WA, Australia; Department of Medical Oncology, National Centre
for Asbestos Related Diseases, Sir Charles Gairdner Hospital, Nedlands, WA,
Australia
Jong Kook Park Division of Pharmaceutics, College of Pharmacy, Ohio State
University, Columbus, OH, USA
Vinodh Rajagopalan Department of Biochemistry and Molecular biology,
University of South Carolina, Charleston, SC, USA
Reshma Rangwala Department of Medicine, Perelman School of Medicine,
Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA
Thomas D. Schmittgen Division of Pharmaceutics, College of Pharmacy, Ohio
State University, Columbus, OH, USA
Min Shen Department of Pharmacology, Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI, USA
Robert W. Sobol Department of Pharmacology and Chemical Biology, Hillman
Cancer Center, University of Pittsburgh Cancer Institute, University of Pittsburgh
School of Medicine, Pittsburgh, PA, USA; Department of Human Genetics, Univer-
sity of Pittsburgh School of Public Health, Pittsburgh, PA, USA
Linda S. Steelman Department of Microbiology and Immunology, Brody School
of Medicine at East Carolina University, Greenville, NC, USA
Agostino Tafuri Department of Cellular Biotechnology and Hematology, Sapi-
enza, University of Rome, Rome, Italy
xvi Contributors
Tony Taldone Breast Cancer Service, Department of Medicine, Memorial Sloan-

Kettering Cancer Center, New York, NY, USA
Mark Tom Division of Clinical Research, Fred Hutchinson Cancer Research Cent-
er, Seattle, WA, USA
Conchita Vens Division of Biological Stress Response, The Netherlands Cancer
Institute, Amsterdam, NL, The Netherlands
Victor Y. Yazbeck Department of Medicine, University of Pittsburgh and the Uni-
versity of Pittsburgh Cancer Institute, Pittsburgh, USA
Jian Yu Department of Pathology and Molecular and Cellular Pathology Gradu-
ate Training Program, University of Pittsburgh School of Medicine, University of
Pittsburgh Cancer Institute, Pittsburgh, PA, USA
Chapter 1
Defective Apoptosis Signaling in Cancer
Daniel E. Johnson
Abstract Apoptosis is critically important during development, facilitating the

sculpting and molding of tissues, and in the adult, acting to maintain homeostasis
of cell numbers. Apoptosis also plays a key role in immune-mediated elimination
of infected or transformed target cells. In addition, apoptosis drives cellular suicide
following damage to DNA or other cell components, including damage resulting
from treatment with chemotherapy or radiation. In view of the fundamental impor-
tance of apoptotic cell death, it is, perhaps, not surprising that defects in apoptosis
signaling are involved in a number of human diseases, including the development
and progression of human malignancies. Efforts to promote therapeutic elimina-
tion of cancer cells via induction of apoptosis will benefit from more complete
understanding of normal apoptosis signaling and the defects in apoptosis which
frequently occur in human tumors. This chapter will describe the elucidation of
the intrinsic and extrinsic apoptosis signaling pathways and focus on the defects in
these pathways that have commonly been observed in cancers.
1.1 Characterization of a Human Disease and Biochemical

Studies Unravel Apoptosis Signaling Pathways
The elimination of normal or neoplastic cells via induction of a cell death program
has been recognized since the 1960s, with the term “apoptosis” first being used
to describe this process in 1972 [1]. Early studies defined a number of ordered
morphologic changes in cells undergoing apoptosis, including cell shrinkage,
chromatin condensation, membrane blebbing, and eventual breakup of the cell
into membrane encapsulated apoptotic bodies [1, 2]. In vivo, these apoptotic bod-
ies were observed to be engulfed and degraded by macrophages or neighboring
cells [1, 3, 4]. The early characterization of apoptotic cells also identified a few
D. E. Johnson (*)
Departments of Medicine and Pharmacology and Chemical Biology, University of Pittsburgh
and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
e-mail: [email protected]
D. E. Johnson (ed.), Cell Death Signaling in Cancer Biology and Treatment, 1

Cell Death in Biology and Diseases, DOI: 10.1007/978-1-4614-5847-0_1,
X.-M. Yin and Z. Dong (Series eds.), Cell Death in Biology and Diseases
2 D. E. Johnson
biochemical changes, including externalization of plasma membrane phospholip-

ids, activation of cellular DNAses, and degradation of genomic DNA to oligonu-
cleosomal-length fragments visualized as “apoptotic DNA ladders” on agarose
gels [5]. More detailed elucidation of the molecular mechanisms and pathways
of apoptosis has involved a remarkable convergence of investigations, including
examination of an apoptosis-deficient human cancer, genetic studies in C. elegans,
and biochemical studies in multiple organisms.
Follicular lymphomas are a form of B-cell malignancy and represent one of the
most common cancers of the human immune system. Initially, follicular lympho-
mas exhibit a relatively indolent phenotype. However, later during the course of
disease they become highly aggressive, which is likely due to the acquisition of
secondary genetic alterations. Cytogenetic analyses discovered that greater than
85 % of follicular lymphomas, as well as approximately 20 % of diffuse B-cell
lymphomas, harbor a specific chromosomal translocation, the t(14;18) transloca-
tion [6, 7]. Molecular cloning of the t(14;18) breakpoint revealed juxtaposition of
the IgH chain locus with sequences encoding a gene that was designated b cl-2,
resulting in overexpression of wild-type Bcl-2 protein [8–11]. It was initially
assumed that Bcl-2 would act as a typical oncoprotein, promoting rapid prolifera-
tion of cells. However, enforced overexpression of Bcl-2 in transfected cell lines
did not markedly impact cell cycle status or proliferation. Instead Bcl-2 overex-
pression acted to markedly inhibit cellular apoptosis, including apoptosis induced
by withdrawal of essential cytokines or neurotrophic factors, or treatment with
apoptotic stimuli such as chemotherapy or radiation [12–22]. Overexpression of
Bcl-2 in the B cells of transgenic mice resulted in extended B-cell survival and
an expanded B-cell compartment [23]. Initially, these mice exhibited an indolent
phenotype, followed by development of more aggressive malignant lymphomas
[24]. Collectively, these studies identified Bcl-2 as the first oncoprotein that acts
by inhibiting cellular apoptosis.
In unrelated studies, Horovitz and colleagues were performing mutational
screens to identify genes that regulate apoptosis in a population of neuronal cells
that consistently undergo apoptosis during C. elegans development. These stud-
ies identified several genes that were important for negatively regulating apop-
tosis and several that were required for apoptosis to occur. Among the genes that
inhibited apoptosis was ced-9 [25]. Sequence analyses determined that the protein
encoded by ced-9 bore close homology to human Bcl-2. Moreover, the human
bcl-2 gene effectively replaced ced-9 function in mutant C. elegans worms [26].
Among the genes that were required for efficient apoptosis were ced-3 and ced-4
[27]. Sequencing revealed that the protein encoded by ced-3 was closely related to
a previously identified human protease responsible for processing of IL-1β [28–30].
Those who had previously been working with this protease, termed ICE (for
interleukin-1β converting enzyme), had determined that it was a cysteine protease
with a specificity for cleaving after aspartate residues and had developed a num-
ber of peptide inhibitors of ICE [29, 30]. It was quickly demonstrated that ICE
promoted apoptotic cell death when overexpressed in cell culture [31]. In a ddition,
peptide inhibition of cellular ICE was found to potently inhibit cell death resulting
1 Defective Apoptosis Signaling in Cancer 3
Fig. 1.1 Activation of the Apoptosis Stimulus

caspase protease cascade
during apoptosis
Activation of “initiator”
caspases
Activation of
“executioner”
caspases
Cleavage of caspase substrate proteins

And destruction of the cell
from a variety of apoptotic stimuli, underscoring the importance of ICE and ICE-
like proteases in apoptosis execution [32–37]. Subsequently, more than a dozen
proteases related to ICE have been cloned and have come to be known as caspases
(reviewed in [38]). The term caspases refers to the fact that these proteases are
“c-asp-ases”. The caspases have conveniently been assigned names of caspase-1
through caspase-14 [38]. Normally, caspases exist as inactive zymogens in cells.
However, in response to an apoptotic stimulus, a subset of caspases, called the initi-
ator caspases (e.g., caspase-8 and caspase-9), undergo processing to active enzyme
forms (Fig. 1.1). The activated initiator caspases then cleave and activate execu-
tioner caspases, of which the most common are caspase-3 and caspase-7 [39, 40].
Activated executioner caspases cleave specific cellular substrate proteins promoting
the eventual destruction of the cell.
Following the identification of caspases as executioners of apoptosis, it was
soon determined that overexpression of Bcl-2 could act to prevent caspase pro-
tease activation in the cell. But how was Bcl-2 preventing caspase activation,
and what are the mechanisms for caspase activation in the absence of Bcl-2?
To address these questions, Wang and colleagues performed cellular fractiona-
tion studies to identify proteins that could promote activation of a procaspase in
cell-free extracts [41]. Remarkably, cytochrome c, a mitochondrial protein, was
found to be capable of promoting caspase activation in conjunction with a sec-
ond protein that was given the name Apaf-1 (apoptotic protease activating factor
1) [41, 42]. Interestingly, Apaf-1 was found to bear close homology with Ced-4
protein from C. elegans [43]. Further studies revealed that during the course of
apoptosis caused by chemotherapy, radiation, or cytokine withdrawal, cytochrome
c is released into the cytosol, and this release is prevented in cells overexpressing
Bcl-2 [41, 42, 44, 45].
A further foundational development in the understanding of apoptosis signal-
ing has come with the identification of the tumor necrosis factor (TNF) family of
death ligands. This family includes proteins such as TNF, Fas ligand, and TRAIL
(TNF-related apoptosis-inducing ligand), which bind to cognate plasma membrane
4 D. E. Johnson
Death ligand/Death receptor

(extrinsic apoptosis pathway)
procasp-8/-10 caspase-8/-10
(intrinsic apoptosis pathway)
Chemotherapy/Radiation
“DISC” procasp-3 caspase-3
Damage
Cleavage of caspase
substrate proteins
cytochrome c
release DNA fragmentation
Cell destruction
caspase-9
Apaf-1
procasp-9
“Apoptosome”
Fig. 1.2 The extrinsic and intrinsic apoptosis pathways
receptors, promoting caspase activation and cell death (see Chap. 11). The identifi-
cation of this receptor-mediated pathway of apoptosis, in conjuction with the early
discoveries regarding Bcl-2, caspases, Apaf-1, and cytochrome c, has provided the
foundation for detailed delineation of the extrinsic and intrinsic pathways of apop-
tosis described in the section below.
1.2 The Intrinsic and Extrinsic Apoptosis Pathways
Based on the seminal discoveries described above, two pathways of apoptosis

execution have subsequently been delineated, the extrinsic (or death receptor-
mediated) pathway and the intrinsic (or mitochondrial-mediated) pathway
(Fig. 1.2). The extrinsic apoptosis pathway is activated following binding of
a death ligand to its cognate cell surface receptor. The simplest example of this
pathway is illustrated by Fas-mediated signaling (Figs. 1.2 and 1.3). Expression
of Fas ligand is primarily restricted to activated immune cells, as well as immune-
privileged sites such as testis and eye. Fas ligand is predominantly expressed as a
membrane-spanning protein. The binding of Fas ligand to its receptor, Fas, results
in trimerization of the receptor protein, bringing together the three cytoplasmic
regions (Fig. 1.3; reviewed in Chap. 11). Within the cytoplasmic region of Fas, and
other death receptors, is a domain called the death domain (DD). Trimerization
of the Fas DDs results in recruitment of the DD in the adaptor protein FADD
(Fas-associated death domain protein), beginning a process of forming a larger
complex referred to as the death-inducing signaling complex (DISC; Fig. 1.2).
Fig. 1.3 DISC formation
and caspase-8 activation
Fas ligand (death ligand)
Fas (death receptor)
FADD
Procaspase-8
FADD also contains a domain called a death-effector domain (DED) which then
recruits the DED found in the prodomain of the zymogen form of the initiator cas-
pase, caspase-8 (or the initiator caspase-10). Recruitment of procaspase-8 to the
DISC results in a slight conformational change in the zymogen protein, resulting
in modest activation of the enzyme activity and proximity-induced proteolytic pro-
cessing of procaspase-8 proteins present in the DISC (Fig. 1.3) [46–48]. This pro-
cessing removes the inhibitory prodomain and produces large and small caspase-8
subunits. A heterotetrameric complex consisting of two large subunits and two
small subunits comprises the fully activated caspase enzyme (Fig. 1.3) [38]. Once
the initiator caspases, caspase-8 or caspase-10, are activated, they then cleave
and activate executioner caspases, including the primary executioner, caspase-3
(Fig. 1.2) [38]. The activated executioners cleave specific substrate proteins,
resulting in activation of cellular DNAses and proteolytic destruction of the cell.
The intrinsic apoptosis pathway is activated by a variety of cellular insults,
including withdrawal of essential cytokines or neurotrophic factors, or treat-
ment with chemotherapy or radiation. In the case of agents causing cellular
damage (e.g., chemotherapy drugs), the damage is detected by the cell, and a
signal (described in Sect. 1.4.3) is relayed to the mitochondria, causing release
of cytochrome c into the cytosol (Fig. 1.2) [41]. Once released, the cytosolic
cytochrome c associates with the cytoplasmic adapter protein Apaf-1 and the
zymogen form of the initiator caspase, caspase-9, forming a complex referred
to as the apoptosome [42]. Formation of the apoptosome results in a slight con-
formational change in procaspase-9, causing sufficient activation to promote
autoprocessing to active caspase-9 [48]. Active caspase-9 cleaves and activates
downstream executioner caspases, and at this point, the intrinsic and extrinsic
apoptosis pathways converge. In some instances, earlier cross-talk between the
intrinsic and extrinsic pathways can occur. Active caspase-8 from the extrinsic
pathway is known to cleave Bid protein, and the Bid cleavage product (tBid) can
6 D. E. Johnson
Damage
Cleavage of caspase
substrate proteins
cytochrome c
release X DNA fragmentation
Cell destruction
caspase-9
Apaf-1
procasp-9
“Apoptosome”
Fig. 1.4 Bcl-2 inhibition of the intrinsic apoptosis pathway
migrate to the mitochondria and stimulate cytochrome c release and activation of

the intrinsic pathway [49, 50].
The Bcl-2 oncoprotein acts to negatively regulate the intrinsic apoptosis
pathway, in part due to its prominent localization as an integral membrane pro-
tein in the outer mitochondrial membrane (Fig. 1.4). In preventing the release of
cytochrome c from mitochondria (discussed in further detail in Sect. 1.4.1), Bcl-2
prevents formation of the apoptosome and activation of caspase-9 [44, 45].
In subsequent sections of this chapter, we will focus on genetic and epigenetic
alterations that directly impact the expression or activity of specific components
of the extrinsic and intrinsic apoptosis pathways. We will focus, in particular,
on alterations that have been observed or confirmed in primary human tumor
specimens.
1.3 Defects in Caspase Signaling
In view of the critical importance of caspases to both the extrinsic and intrinsic
apoptosis pathways, one might predict that the genes encoding these proteases
would be targets for genetic alterations impacting the function or expression of
caspases in human tumors. Indeed, a considerable body of evidence indicates that
this is likely the case, particularly for the genes encoding the initiator caspases,
caspase-8 and caspase-10. A smaller number of publications also point to muta-
tions or reduced expression of the initiator, caspase-9, and the executioners, cas-
pase-3 and caspase-7.
1.3.1 Mutation or Dysregulated Expression of Caspase

Proteases
Mandruzzato et al. [51] reported in 1997 a mutant form of caspase-8 expressed

in a patient with head and neck carcinoma. In this mutant, the stop codon was
altered, and an additional 88 amino acids were added to the C-terminus of the
protein. Functional testing demonstrated reduced ability of the mutant protein to
trigger apoptosis. Subsequently, Soung et al. [52] identified a specific frameshift
mutation in the caspase-8 gene (1224_1226delTG) which resulted in premature
termination within small subunit domain of the procaspase-8 protein. In hepatocel-
lular carcinoma (HCC), 9/69 (13 %) patients were found to harbor this inactivating
mutation. Mutation of caspase-8 has also been observed in 5/98 (5.1 %) colorectal
carcinoma specimens and 13/162 (8.0 %) gastric carcinomas [53, 54].
A large number of publications have reported reduced expression or hypermeth-
ylation of the caspase-8 gene in human tumors. In neuroblastoma, several groups
have observed loss of caspase-8 expression or gene hypermethylation in a majority
of patients [55–60]. However, the relationship in neuroblastoma between reduced
caspase-8 expression or gene methylation and either MYCN amplification or poor
prognosis remains controversial, as some reports support a strong correlation while
others indicate a lack of correlation [55, 58, 59]. A majority of medulloblastoma
tumors also exhibit hypermethylation of the caspase-8 gene [61, 62], and loss of
caspase-8 expression has been shown to correlate with poor prognosis in childhood
medulloblastoma [63]. In pituitary adenomas, 19/35 (54 %) patients exhibited
hypermethylation of the caspase-8 gene [64]. Hyerpmethylation of the caspase-8
gene, albeit in a minority of patients, has also been reported in bladder cancer [65],
HCC [66, 67], and small-cell lung cancer (SCLC) [68]. In pediatric malignancies,
hypermethylation of the caspase-8 gene has been observed in a majority of neuro-
blastomas, medulloblastomas, retinoblastomas, and rhabdomyosarcomas [61].
Mutations in the caspase-10 gene have been detected in autoimmune diseases
as well as tumors. In 1999, Wang et al. identified inactivating caspase-10 point
mutations in autoimmune lymphoproliferative syndrome (ALPS) type II [69]. A
frameshift mutation resulting in premature caspase-10 termination has been iden-
tified by Tadaki et al. [70] in a patient with systemic juvenile idiopathic arthritis.
In solid tumors, caspase-10 mutations have been observed in 3/99 (3.0 %) gastric
cancers [71] and 2/47 (4.3 %) colon cancers [72]. Mutations in caspase-10 have
also been detected in hematopoietic malignancies, including non-Hodgkin’s lym-
phomas (17/117; 14.5 %) [73], T-acute lymphoblastic leukemia (1/13; 7.7 %) [74],
and multiple myeloma (1/22; 4.5 %) [74].
Considerably less information has been reported on the mutational status of
caspase-9, caspase-3, and caspase-7 in human tumors, perhaps reflecting a reduced
prevalence of mutations of these caspases in cancer. An extensive analysis of the
human caspase-9 gene by Soung et al. [75] in 180 gastric cancers, 104 colorectal
cancers, and 69 lung adenocarcinomas detected a total of only three mutations,
but none of these mutations resulted in an amino acid change. A similar analysis
8 D. E. Johnson
Fig. 1.5 XIAP inhibition of
caspases in healthy cells
XIAP
procasp-3 caspase-3
X
Cleavage of caspase
X substrate proteins
DNA fragmentation
Cell destruction
caspase-9
XIAP
detected somatic mutations of the caspase-3 gene in 4/98 (4.1 %) colon carcino-
mas, 4/181 (2.2 %) non-small-cell lung cancers (NSCLCs), 2/129 (1.6 %) non-
Hodgkin’s lymphomas, 1/28 (3.6 %) multiple myelomas, 1/80 (1.3 %) HCCs, and
2/165 (1.2 %) gastric carcinomas [76]. Inactivating mutations of caspase-7 have
been reported in 1/33 (3.0 %) head and neck carcinomas, 1/50 (2.0 %) esophageal
carcinomas, and 2/98 (2.0 %) colon carcinomas [77].
1.3.2 Aberrant Expression of IAPs
The intracellular activities of the initiator, caspase-9, and the executioners, caspase-3
and caspase-7, are negatively regulated by certain members of the inhibitor of
apoptosis (IAP) protein family (reviewed in [78, 79] and Chap. 10). The IAP fam-
ily is comprised of eight members (XIAP, cIAP1, cIAP2, NAIP, survivin, MLIAP,
BRUCE, and ILP2), with each characterized by the presence of 1 or 3 baculovi-
rus IAP repeat (BIR) domains (see Chap. 10). Four members of this family (XIAP,
cIAP1, cIAP2, MLIAP2, and ILP2) also contain carboxy-terminal RING domains,
which possess E3 ubiquitin ligase activity. Among the IAPs, XIAP has potent and
direct inhibitory activity against caspase-9, caspase-3, and caspase-7, while cIAP1
and cIAP2 can act to indirectly inhibit the activities of these caspases [80].
XIAP directly binds active caspase-3 and caspase-7 with high affinity via two
interaction sites. The BIR2 domain of XIAP binds to an IBM (IAP-binding motif)
that becomes exposed in the processed/active proteases [81, 82]. In addition, a
linker region between the BIR1 and BIR2 domains binds to the active site of cas-
pase-3 and caspase-7 and prevents the access of caspase substrate proteins [81–84].
In the case of caspase-9, the BIR3 domain of XIAP binds to the monomeric form
of caspase-9, preventing dimerization and full activation of the enzyme [85, 86].
As shown in Fig. 1.5, XIAP-mediated inhibition of any caspase-9, caspase-3,
or caspase-7 activities present in healthy growing cells prevents undesirable
procasp-8/-10 caspase-8/-10 XIAP

Damage
Cleavage of caspase
substrate proteins
cytochrome c &
SMAC release DNA fragmentation
Cell destruction
caspase-9
Apaf-1
procasp-9
XIAP
“Apoptosome”
Fig. 1.6 SMAC overcomes XIAP to promote the intrinsic apoptosis pathway
activation of the caspase protease cascade and death of the cells. However, dur-
ing activation of the intrinsic apoptosis pathway, the mitochondrial protein SMAC
(second mitochondria-derived activator of caspases), is released from the mito-
chondria in conjunction with cytochrome c. The amino-terminal domain of the
processed SMAC protein contains a tetrapeptide sequence (AVPI) that binds
tightly to XIAP and displaces XIAP from bound caspases, leading to the activa-
tion of caspase-9, as well as caspase-3 and caspase-7 (see Fig. 1.6; reviewed in
Chap. 10). While this is the scenario in cells that efficiently undergo apoptosis via
the intrinsic apoptosis pathway, it is now evident that many tumors overexpress
XIAP (discussed below), thwarting the ability of limiting levels of SMAC to over-
come XIAP inhibition of the caspase cascade (see Fig. 1.7).
cIAP1 and cIAP2 act to indirectly inhibit caspase-9, caspase-3, and caspase-7
via two distinct mechanisms. First, the cIAP1 and cIAP2 proteins are capable of
binding to SMAC and can thereby sequester SMAC protein and prevent displace-
ment of XIAP from bound caspases [80]. Second, acting via their RING domains,
cIAP1, cIAP2, as well as XIAP, can promote ubiquitination of caspase-3 and cas-
pase-7 [87–90]. The ubiquitination of these executioner caspases results in inhibi-
tion of their activities and their eventual degradation via the proteasome.
Aberrant overexpression of XIAP, as well as other IAPs, has been observed in
multiple solid tumor and hematopoietic malignancies. In several cases, overex-
pression of XIAP has been shown to correlate with treatment resistance or poor
prognosis. XIAP is overexpressed in HCC, where its expression has been linked
to metastasis, recurrence, and poor prognosis [91–93]. In advanced head and neck
cancer, XIAP correlates with resistance to cisplatin-based regimens and poor
prognosis [94]. Similarly, XIAP overexpression correlates with chemoresistance
10 D. E. Johnson

XIAP XIAP
XIAP XIAP
s XIAP ssXIAP
ssXIAP sXIAP
s XIAP
IAPs
X
Damage
Cleavage of caspase
X substrate proteins
cytochrome c &
SMAC release DNA fragmentation
Cell destruction
caspase-9
Apaf-1
procasp-9 IAPs
IAPs
IAPs IAPs
XIAP
“Apoptosome” IAPs
XIAP
Fig. 1.7 XIAP overexpression in cancer overwhelms SMAC to prevent caspase activation
in pancreatic cancer [95]. XIAP also serves as a negative prognostic indica-

tor of survival in both colorectal cancer [96] and renal cell carcinoma [97–99].
Overexpression of XIAP has also been detected in NSCLC [100–102], but the sig-
nificance of this expression remains unclear. In one study, XIAP expression in rad-
ically resected NSCLC was found to be inversely correlated with proliferation and
directly correlated with favorable prognosis [100], while another study reported
a lack of correlation between XIAP and response to chemotherapy in advanced
NSCLC patients [101]. Additional studies have identified XIAP overexpression in
testicular germ cell tumors [103] and papillary thyroid carcinomas [104].
Among hematopoietic malignancies, overexpression of XIAP is associated with
poor clinical outcome in diffuse large B-cell lymphomas (DLBCL) [105]. XIAP
overexpression has also been observed in myelodysplastic syndromes transform-
ing to acute leukemia [106]. High levels of XIAP in adult acute myeloid leukemia
(AML) correlate with poor response to chemotherapy, unfavorable cytogenetics,
and poor prognosis [107–109]. In childhood de novo AML, XIAP overexpression
also correlates with chemoresistance and poor prognosis [110, 111]. Poor response
to glucocorticoid therapy is observed in childhood T-cell acute lymphoblastic leu-
kemias exhibiting XIAP overexpression [112].
Dysregulated expression of cIAP1, cIAP2, and survivin has been demon-
strated in a large variety of tumors and frequently correlates with poor prognosis
(reviewed in [78, 79, 113]). Interestingly, cIAP2 is overexpressed in MALT lym-
phomas harboring the t(11;18) translocation, which fuses the 3 BIR domains of
cIAP2 with sequences encoding the carboxyl terminus of MALT1 protein [78].
Additionally, amplification of 11q21-q23, which encodes both cIAP1 and cIAP2,
is frequently detected in multiple forms of cancer [78, 79].
1.4 Defects Affecting the Intrinsic Apoptosis Pathway
Activation of the intrinsic apoptosis pathway is fundamentally important for cell

death induced by chemotherapy or radiation. Hence, defects in components of this
pathway can have a profound impact on the sensitivities of tumor cells to these
therapeutic agents. By extension, resistance to chemo- and radiotherapy has a huge
impact on prognosis and survival for cancer patients.
The intrinsic apoptosis pathway is tightly regulated by members of the Bcl-2
protein family [114–117]. Members of this family share homology via the presence
of conserved domains called Bcl-2 homology, or BH domains. While some Bcl-2
family members contain up to 4 BH domains (BH1-4), others contain only a single
BH domain, typically a single BH3 domain. On a functional basis, members of this
family can be divided into 2 major categories, anti-apoptotic proteins and proapop-
totic proteins. The anti-apoptotic Bcl-2 family members include Bcl-2 [10, 118],
Bcl-XL [119], Mcl-1 [120], A1/Bfl-1 [121, 122], and Bcl-w [123]. These proteins
contain multiple BH domains, as well as carboxyl-terminal membrane-anchoring
domains, and act to inhibit cytochrome c release from the mitochondria. The proap-
optotic Bcl-2 family members can be subdivided into two groups, those containing
multiple BH domains (e.g., Bax and Bak), referred to as “multi-domain” proteins,
and those containing only a single BH3 domain (e.g., PUMA, Bik, Noxa, Bid, Bad,
etc.), referred to as “BH3 domain-only” proteins (reviewed in [116, 117]). Genetic
studies have revealed that cells lacking Bax and Bak are highly resistant to stimuli
that activate the intrinsic apoptosis pathway [124, 125], underscoring the impor-
tance of Bax and Bak for chemotherapy- and radiation-induced apoptosis.
1.4.1 Overexpression of Anti-Apoptotic Bcl-2 Family

Members
Overexpression of anti-apoptotic Bcl-2 has been reported in a wide variety of hemat-

opoietic and solid tumor malignancies, a subject that has been reviewed extensively
[116, 117, 126–128]. Among hematopoietic tumors, Bcl-2 overexpression has been
observed in follicular B-cell lymphoma [7, 129], DLBCL [130], AML [131–135],
acute lymphoblastic leukemia (ALL) [127, 136], chronic lymphocytic leukemia
(CLL) [137, 138], anaplastic large cell lymphoma (ALCL) [139], and multiple mye-
loma [140]. Solid tumor malignancies exhibiting overexpression of Bcl-2 include
melanoma [141–143], glioblastoma [144], SCLC [145], and cancers of the bladder
[146–149], colon [150], and prostate [151–153]. Overexpression of anti-apoptotic
Bcl-XL has also been detected in a wide range of cancers, including AML [154, 155],
multiple myeloma [156], melanoma [157, 158], squamous cell carcinoma of the head
and neck (SCCHN) [159, 160], and cancers of the liver [161], breast [162], bladder
[163], colon [164], and pancreas [165, 166]. Importantly, in several of these malig-
nancies, overexpression of Bcl-2 and/or Bcl-XL has been closely correlated with
resistance to chemotherapy or radiation, and poor overall survival [116, 126].
12 D. E. Johnson
Less information is available regarding potential overexpression of anti-apop-

totic Mcl-1, A1/Bfl-1, and Bcl-w in human cancers. However, overexpression of
Mcl-1 has been reported AML [167], ALL [167], multiple myeloma [168, 169],
CLL [170], and ovarian cancer [171]. Moreover, Mcl-1 is rapidly degraded via the
proteasome. Treatment of tumors with proteasome inhibitors results in elevated
Mcl-1 expression, which can act to attenuate the efficacies of these therapeutic
agents [172]. Overexpression of A1/Bfl-1 has been reported in B-cell lymphomas
and gastric carcinomas [122, 173, 174], while Bcl-w overexpression occurs in
colorectal carcinomas [175].
Several different mechanisms have been described which account for Bcl-2
overexpression in cancer. In hematopoietic malignancies harboring the t(14;18)
chromosomal translocation, overexpression results from juxtaposition of the intact
Bcl-2 coding sequence next to cis transcriptional elements from the IgH chain
locus [8–11]. Amplification of chromosomal segments encompassing the bcl-2
gene has been reported to play a role in Bcl-2 overexpression in DLBCL and SCLC
[176, 177]. Gene amplification has also been observed for the genes encoding Bcl-
XL and Mcl-1 [117, 178]. In B-CLL, hypomethylation of the bcl-2 gene promoter
represents an additional mechanism that may contribute to Bcl-2 overexpression
[137].
Recent evidence indicates that expression of Bcl-2 family members can be con-
trolled via expression of microRNAs (see Chap. 5). The microRNAs miR-15a and
miR-16-1 negatively regulate expression of Bcl-2 [179]. In CLL, expression of
miR-15a and miR-16-1 is frequently lost due to deletion of the miR-15a and miR-
16-1 genetic locus encoding these microRNAs [180, 181]. Loss of miR-15a and
miR-16-1 is thought to play a key role in the overexpression of Bcl-2 in CLL, and
potentially other malignancies where this genetic locus is deleted [182, 183].
1.4.2 Mutation or Reduced Expression of Proapoptotic Bcl-2

Family Members
Early efforts to uncover the molecular mechanism whereby Bcl-2 acts to inhibit
apoptosis were stymied by the failure to identify any enzymatic activity associ-
ated with the Bcl-2 protein. The first clue regarding the mechanism of Bcl-2 action
came with the discovery that Bcl-2 binds tightly to the closely related proapop-
totic protein Bax [184]. Subsequent studies have revealed a complex network of
physical interactions between anti-apoptotic Bcl-2 family members and proapop-
totic members of the Bcl-2 protein family [114, 115, 126, 185]. Additional stud-
ies determined that heterodimerization between pro- and anti-apoptotic Bcl-2
family members served to neutralize the apoptosis-inducing activity of the proa-
poptotic protein, and led to the proposal of the “rheostat model” by Korsmeyer
and colleagues [186]. The rheostat model proposed that the relative levels of pro-
versus anti-apoptotic Bcl-2 family members expressed in a cell would determine
whether that cell is prone to undergo apoptosis (i.e., chemotherapy sensitive), or
prone to be apoptosis resistant. The second major clue regarding the mechanism
of Bcl-2 action came with the discovery discussed previously that Bcl-2 (and Bcl-
XL/Mcl-1) acts to prevent cytochrome c release from the mitochondria [44, 45].
The third major clue came from biophysical and biochemical studies of proapop-
totic Bax and Bak. These studies revealed that Bax and Bak can homooligomer-
ize and form small pores in artificial lipid membranes, as well as mitochondrial
membranes in intact cells [187–191]. The pores formed by Bax and Bak were
found to be sufficient in size to allow the release of cytochrome c from mitochon-
dria. The binding of anti-apoptotic Bcl-2 proteins to Bax or Bak prevents their
homooligomerization, explaining how these anti-apoptotic proteins can prevent
cytochrome c release and activation of the intrinsic apoptosis pathway. A large
number of additional studies have shown that certain “BH3 domain-only” proap-
optotic proteins, such as PUMA, tBid, and Bim, can bind directly to Bax or Bak
and induce pore formation. These “BH3 domain-only” proteins are commonly
referred to as “activators” [117, 192–195]. The other “BH3 domain-only” proap-
optotic proteins are called “derepressors” (or “sensitizers”) and exert their effects
by binding to the anti-apoptotic Bcl-2 family members (e.g., Bcl-2, Bcl-XL, and
Mcl-1) [117, 192–195]. In binding to anti-apoptotic proteins, the “derepressors,”
as well as “activators,” cause release of sequestered Bax and Bak, freeing these
proteins up to form pores. In addition, “derepressor” binding can cause the release
of bound “activators,” freeing them up to directly bind and activate Bax and Bak
[196–198].
Just as anti-apoptotic Bcl-2 family members are found to be frequently over-
expressed in human cancers, it is reasonable to predict that the proapoptotic Bcl-2
proteins might be underexpressed or functionally inactivated in tumors. Indeed,
this appears to be the case [117, 199]. Frameshift and inactivating mutations in
the bax gene have been detected in colorectal and hematopoietic malignancies
[200–202]. Mutation of noxa gene has been reported in DLBCL [203], while
bak gene mutations are present in gastric and colon tumors [204]. In mantle cell
lymphomas (MCL), homozygous deletion of bim gene has been observed [205].
Loss of heterozygosity in bik gene occurs in renal cell carcinoma [206].
Promoter methylation also appears to play an important role in reducing the
expression of proapoptotic proteins in cancer. Hypermethylation of bim gene pro-
moter has been reported in Burkitt’s lymphoma (BL) and DLBCL [203, 207];
hypermethylation of puma gene occurs in BL [208]; and hypermethylation of noxa
gene is found in DLBCL [203]. Hypermethylation of bik gene is present in renal
cell carcinomas [206], while colorectal tumors exhibit hypermethylation of hrk
gene [209].
Emerging evidence indicates that expression of proapoptotic proteins is likely
regulated by the expression of specific microRNAs (see also Chap. 5). Expression
of Bim has been found to be downregulated in several types of cancer via
expression of microRNAs encoded by the miR-17-92 and miR-106b-25 clusters
[210–213]. It appears highly likely that regulation of the expression of pro- and
anti-apoptotic proteins by microRNAs will remain an active area of research in the
coming years.
14 D. E. Johnson
Bak
Bak
cytochrome c
release
Bax
Bax
Fig. 1.8 p53 induces PUMA and NOXA to activate the intrinsic apoptosis pathway
1.4.3 Mutation/Deletion of p53
The tumor suppressor protein p53 is often cited as the most commonly mutated
protein in human cancers [214]. Mutations in the p53 gene lead to loss of p53
function as a transcription factor, with frequent conversion to a dominant-negative
inhibitor, or loss of expression of the p53 protein [214]. The prevalence of p53
mutation or loss of expression in cancer is likely due to the critical roles that p53
plays in the regulation of cell cycle arrest, DNA repair, apoptosis, and senescence.
p53 actively promotes apoptosis induction via the intrinsic apoptosis pathway,
particularly in response to DNA damage. In damaged cells, activation of ATM and
ATR kinases leads to phosphorylation of p53 and MDM2, disrupting p53/MDM2
interactions and abrogating MDM2-mediated degradation of p53 via the proteas-
ome [215–221]. This leads to upregulation of the p53 protein, with the majority of
the protein being expressed in the nucleus, but some also appearing in the cytosol
and at the mitochondria.
Upregulated p53 protein in damaged cells activates the intrinsic apoptosis path-
way via both transcription-dependent and transcription-independent mechanisms.
The transcription-dependent mechanism requires functional DNA binding and
transcription factor function of the p53 protein. The genes encoding Bax and Apaf-
1 have been shown to be p53 target genes, and their transcription is moderately
induced in p53-expressing cells [222, 223]. However, although Bax and Apaf-1
are central factors in the intrinsic pathway, p53 induction of these proteins may
play only a modest role in the proapoptotic activity of p53. By contrast, p53 induc-
tion of select proapoptotic “BH3 domain-only” proteins appears to play a major
role in the transcription-dependent mechanism of apoptosis induction by p53.
p53 markedly induces the expression of the “BH3 domain-only” proteins PUMA
[224, 225], NOXA [226], and BID [227]. As illustrated in Fig. 1.8, p53-mediated
induction of PUMA and NOXA results in migration of these proteins to the mito-
chondria, where PUMA can directly activate Bax or Bak, or indirectly activate
these proteins via inhibitory interaction with anti-apoptotic Bcl-2 family members,
including Bcl-2 and Bcl-XL [197, 198, 228–230]. Induced NOXA demonstrates
specificity for binding to anti-apoptotic Mcl-1 and functionally inactivates Mcl-1
by promoting the release of bound proapoptotic proteins [193, 196–198, 229–231].
Transcription-independent activation of the intrinsic apoptosis pathway by p53
occurs via several different mechanisms. Cytosolic p53 directly activates proapop-
totic Bax, leading to mitochondrial membrane permeabilization and cytochrome
c release [232, 233]. p53 present at the mitochondria physically associates with
proapoptotic Bak, disrupting Bak sequestration by Mcl-1 and resulting in Bak acti-
vation [234]. In addition, mitochondrial-localized p53 can bind to Bcl-2 or Bcl-
XL, promoting the release of sequestered proapoptotic proteins [233, 235].
Collectively, the combination of transcription-dependent mechanisms mediated
by nuclear p53 and transcription-independent mechanisms mediated by cytosolic/
mitochondrial p53 confers a potent impact of p53 upregulation on cellular apop-
tosis. Moreover, it is readily apparent why loss of p53 can provide an important
survival advantage for tumors and may enhance resistance to chemotherapy and
radiation. However, it is important to note that activation of the intrinsic apoptosis
pathway is not entirely dependent on p53, as activation of this pathway can also
be observed in some cell types lacking p53. The mechanisms of p53-independ-
ent activation of the intrinsic apoptosis pathway are less well understood, and this
remains an area of active investigation.
1.5 Defects Affecting the Extrinsic Apoptosis Pathway
Similar to what is seen with the intrinsic apoptosis pathway, multiple defects in
the extrinsic (or death receptor-mediated) pathway have been observed in human
tumors. In healthy individuals, the extrinsic apoptosis pathway plays a central
role in immune-mediated elimination of infected or transformed cells. Therefore,
defects in the extrinsic pathway most likely contribute to tumorigenesis primar-
ily by negatively impacting the efficiency of immune surveillance. However, it
is also important to note that in certain cell types, cross-talk occurs between the
extrinsic and intrinsic pathways, meaning defects in one pathway may affect
killing initiated by the other pathway. For example, in cells referred to as type I
cells, stimulation with a death ligand leads to efficient killing via a linear pathway
involving caspase-8 activation, followed by caspase-3 activation. By contrast, in
cells referred to as type II cells, efficient killing by a death ligand also requires
caspase-8-mediated cleavage of Bid, followed by tBid induction of cytochrome c
release and activation of the intrinsic pathway [49, 50, 236]. Thus, in type II cells,
defects in the intrinsic pathway can impair killing initiated by the extrinsic path-
way. In addition, other reports have indicated that defects in the extrinsic pathway
may reduce the sensitivity of leukemic cells to chemotherapy [237–240].
16 D. E. Johnson
As described previously (Sect. 1.3.1), the initiator caspases for the extrinsic

pathway, caspase-8 and caspase-10, are frequently mutated or underexpressed in
human cancers. In the sections below, we will focus on dysregulated expression or
mutation of the death receptors for Fas ligand and TRAIL, the DISC adaptor pro-
tein FADD, and the negative regulator c-FLIP.
1.5.1 Loss or Mutation of Death Receptors
Similar to what is seen for caspase-8 and caspase-10, the expression and function
of Fas death receptor is commonly altered in autoimmune disease and malignant
tumors (reviewed in [241]; see also Chap. 11). In the lpr strain of mice, sequences
from the 3′ LTR of a transposable element are inserted into the second exon of
the fas gene, resulting in expression of defective Fas protein [242–245]. The lpr
mice are characterized by ineffective deletion of self-reactive T cells, lymphopro-
liferation, and development of autoimmune disease [242]. In humans, somatic Fas
mutations are also commonly detected in patients with autoimmune lymphoprolif-
erative syndrome (ALPS) [241, 246, 247]. Loss of heterozygosity in fas gene has
also been reported in human ALPS [248, 249]. Mutations in Fas, typically lead-
ing to an inactive protein, have been detected in both hematologic and solid tumor
malignancies. In hematologic malignancies, Fas mutations have been reported in
cutaneous T cell [250, 251], MALT-type [252, 253], nasal NK/T cell [254], non-
Hodgkin’s [255], DLBC [256] and thyroid lymphomas [257], as well as multiple
myeloma [258]. Solid tumors harboring Fas mutations include bladder carcinoma
[259], malignant melanoma [260], NSCLC [261, 262], squamous cell carcinoma
[263], and cancers of the stomach [264] and testis [265]. Methylation of the fas
gene may be an important mechanism for downregulation of Fas expression in
human tumors, with hypermethylation having been reported in Sezary syndrome
[266] and cancers of the bladder [267], colon [268], and prostate [269].
Apoptosis induced by the death ligand TRAIL is mediated by the death
receptors TRAIL-R1 (also called DR4) and TRAIL-R2 (also called DR5)
(reviewed in [270–272]; see also Chap. 11). As might be expected, reduced
expression and mutation of TRAIL-R1 and TRAIL-R2 has been reported in a
variety of cancers. Mutations in TRAIL-R1 have been detected in non-Hodg-
kin’s lymphoma [273], metastatic breast cancer [274], gastric carcinoma [275],
head and neck cancer [275], NSCLC [275], and osteosarcoma [276]. Mutations
in TRAIL-R2 occur in non-Hodgkin’s lymphoma [273], metastatic breast can-
cer [274], gastric carcinoma [277], and NSCLC [278]. Loss of heterozygosity
in the TRAIL-R2 has been reported in colorectal carcinoma [279], gastric car-
cinoma [277], and NSCLC [278]. Hypermethylation of the TRAIL-R1 gene has
been observed in gastric carcinoma [280], glioma [281], SCLC [282], and ovar-
ian cancer [283].
1.5.2 Regulation of Death Receptor Signaling by Decoy

Receptors, Aberrant Subcellular Trafficking, and p53
Although mutation, loss of heterozygosity, and gene hypermethylation appear

to play important roles in the inactivation of death receptor signaling in
tumors, other mechanisms that contribute to inhibition of the extrinsic apop-
tosis pathway also exist. In particular, decoy receptors for both Fas ligand
and TRAIL have been extensively described (reviewed in [270–272]; see also
Chap. 11). It remains somewhat unclear, however, what role the decoy recep-
tors for TRAIL play in tumor progression. Additionally, work in cell line mod-
els indicates that aberrant subcellular trafficking of TRAIL-R1 and TRAIL-R2
may contribute to resistance to TRAIL. In breast cancer cells, reduced surface
expression of TRAIL-R1 and TRAIL-R2 was found to correlate with consti-
tutive endocytosis of these receptors [284]. In colon cancer cells selected for
resistance to TRAIL, deficient transport of TRAIL-R1 and TRAIL-R2 to the
cell surface was observed [285].
Further evidence indicates that the p53 status of tumor cells may play a role
in sensitivity to TRAIL. The genes encoding TRAIL-R1 and TRAIL-R2 have
been shown to be p53 target genes [286–291]. In both cases, p53 induces gene
expression via binding to intronic sequences [289, 291]. The ability of p53 to
induce TRAIL-R1 and TRAIL-R2 underscores the significance of p53 loss in
human tumors. However, the situation may not be as simple as a direct correla-
tion between wild-type p53 expression and sensitivity to TRAIL. Upregulation of
TRAIL receptors can also occur via p53-independent pathways [287]. Moreover,
the genes encoding decoy receptors for TRAIL also appear to be p53 target genes
[292, 293].
1.5.3 FADD Mutation
The FADD adaptor protein plays an important role in caspase-8 activation fol-
lowing stimulation of the receptors for Fas ligand, TNF, and TRAIL. During Fas-
and TRAIL receptor-mediated activation of caspase-8, FADD is the first protein
recruited to the DISC. During TNF receptor-mediated activation of caspase-8,
FADD recruitment to the DISC follows the recruitment of RIP (receptor-inter-
acting protein), TRADD (TNF receptor-associated death domain protein), and
TRAF-1/-2 (TNF receptor-associated factor). Mutation of FADD protein has been
occasionally observed. Bolze et al. identified a missense mutation in FADD in an
ALPS patient that leads to reduced FADD protein levels and impaired apopto-
sis induction following Fas stimulation [294]. In addition, FADD mutations have
been reported in 4/80 NSCLC patients [262] and 1/98 colon cancer patients [295].
18 D. E. Johnson
1.5.4 Dysregulated c-FLIP Expression
Just as anti-apoptotic Bcl-2 family members and IAPs act as endogenous negative
regulators of cytochrome c release and caspase activities, respectively, the c-FLIP
(cellular FLICE-inhibitory protein) proteins are endogenous inhibitors of death
receptor-mediated caspase-8/caspase-10 activation. Three major c-FLIP pro-
tein isoforms have been reported, c-FLIPL, c-FLIPs, and c-FLIPr (reviewed in
[116, 296, 297]). Similar to procaspase-8 and procaspase-10, all 3 c-FLIP iso-
forms contain two amino-terminal DED domains. The c-FLIPs and c-FLIPr con-
tain only a short carboxyl-terminal sequence following the DED domains. By
contrast, the DED domains of c-FLIPL are followed by sequences closely homol-
ogous to the caspase-8 enzyme, although c-FLIPL is catalytically inactive. A
large number of publications have shown that high levels of c-FLIPL, c-FLIPs, or
c-FLIPr effectively compete with procaspase-8 and procaspase-10 for binding to
FADD or TRADD proteins present in the forming DISC of activated death recep-
tors [296, 297]. In so doing, high levels of the c-FLIP proteins effectively prevent
caspase-8/caspase-10 activation and execution of the extrinsic apoptosis pathway.
Conversely, when expressed at only low levels, c-FLIPL has been shown to heter-
odimerize with procaspase-8, promoting caspase-8 activation [298, 299].
Overexpression of c-FLIP proteins, particularly c-FLIPL, has been detected
in a variety of human tumors, rendering the cancer cells more resistant to apop-
tosis induction by death ligands. Specifically, c-FLIP is overexpressed in breast,
colon, endometrial, liver, ovarian, and prostate cancers, as well as melanomas,
glioblastomas, and NSCLCs (reviewed in [116, 296, 297]). Importantly, high lev-
els of c-FLIPL expression have been determined to correlate with tumor progres-
sion or poor prognosis in colon, endometrial, liver, ovarian, and prostate cancers,
as well as BL [116, 296, 297, 300–302]. The prevalence of c-FLIP overexpres-
sion in human tumors, and the impact of these proteins on the extrinsic apop-
tosis pathway, has made the development of c-FLIP inhibitors an area of active
investigation.
1.6 Conclusions
A remarkable convergence of genetic and biochemical studies in humans, mice,

and C. elegans has led to our current understanding of the mechanisms whereby
cells kill themselves in response to apoptotic stimuli, as well as the mechanisms
responsible for regulation of cell death pathways. Elucidation of the components
of the extrinsic and intrinsic apoptosis pathways has provided the foundation for
investigating cell death defects occurring in human tumors. Multiple components
have been identified which are important for promoting or facilitating apopto-
sis via these pathways (e.g., caspases, p53, cytochrome c, SMAC, proapoptotic
Bcl-2 family members, Apaf-1, death ligands and receptors, FADD, etc.), and
multiple components have been identified that act to negatively regulate apoptosis
(e.g., anti-apoptotic Bcl-2 family members, IAPs, c-FLIP, etc.). Detailed analyses
have determined that several of the proapoptotic proteins involved in the extrin-
sic and intrinsic pathways commonly exhibit reduced expression in a wide vari-
ety of human tumors via loss of gene heterozygosity or promoter methylation.
Additionally, the proapoptotic proteins also exhibit frequent functional inactivation
via mutation. By contrast, many malignancies overexpress the anti-apoptotic pro-
teins that negatively regulate the extrinsic and intrinsic pathways. These findings
are stimulating a flurry of drug discovery efforts (described in subsequent Chapters)
aimed at restoring the expression or activities of proapoptotic proteins, or reduc-
ing the expression or inhibiting the activities of anti-apoptotic proteins in patient
tumors. Moreover, by identifying the location of defects within the apoptosis path-
ways, treatment options can focus on either correcting the defect, or activating the
pathway downstream from the molecular block. Ultimately, the rapid determination
of the apoptosis defects present in a patient’s tumor should allow the development
of effective personalized treatments to restore sensitivity to cell death–inducing
therapies such as chemotherapy, radiation, and therapeutic death ligands.
Acknowledgments This work was supported by National Institutes of Health grants R01
CA137260 and P50 CA097190. A vast number of researchers have made important contributions
to the work described in this review. We apologize to those authors whose work we have not cited.
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Chapter 2
The Warburg Effect and Beyond:
Metabolic Dependencies for Cancer Cells
David M. Hockenbery, Mark Tom, Cori Abikoff

and Daciana Margineantu
Abstract Current definitions of cancer are best realized as a list of traits or hall-
marks, such as tissue invasion, metastasis, cell-autonomous growth, and resist-
ance to apoptosis. A recent update included deregulated cellular energetics as an
emerging hallmark. However, debate about tumor cell metabolism occupied center
stage in the pre-oncogene era. Over the last 15 years, direct links of oncogenes and
tumor suppressor genes to cell metabolism have brought cancer metabolism to the
forefront once again. Current tools provide much greater opportunities for prob-
ing metabolic differences between normal and cancer cells, in some cases reveal-
ing flux through unexpected metabolic pathways. Metabolic networks may also be
truncated, presenting opportunities for selective growth inhibition or death by tar-
geting non-redundant pathways in cancer cells. These “metabolic dependencies”
are not likely to be associated with classically defined oncogenes or computation-
ally derived drivers and thus may require novel strategies for discovery.
2.1 Pre-Molecular Biology Research
Two giants of biochemistry, Otto Warburg and Herbert Crabtree, developed novel
insights into cancer metabolism in the 1920s. Warburg improved on previous man-
ometric techniques to study respiratory quotients in a variety of cell types and tis-
sue slices. Based on his demonstration of a sixfold increase of oxygen uptake in
sea urchin eggs following fertilization, he entertained the notion that tumor growth
could be explained by increased bioenergetic metabolism. In opposition to his
original predictions, the respiratory rate in Flexner rat carcinomas was similar to
normal rat tissues. However, the rate of glycolysis was increased up to 30-fold in
the carcinoma compared with rat liver.
Comparing glycolysis in air and nitrogen, Warburg observed that both tissues
had higher rates of lactate generation in the absence of oxygen. In normoxic
D. M. Hockenbery (*) · M. Tom · C. Abikoff · D. Margineantu

Division of Clinical Research Seattle, Fred Hutchinson Cancer Research Center, WA, USA

36 D. M. Hockenbery et al.
samples, glycolytic rates were at or below the limit of detection in most normal
tissues. In contrast, glycolysis in normoxic cancer tissues still accounted for the
majority of glucose turnover. The ratio of aerobic glycolysis to respiration was
substantially elevated in cancers, expressed in various ways as mole percent glu-
cose metabolized to lactate (~90 %), ATP generated by glycolysis (35–50 %), or
mass of lactate produced (10 % of tissue weight per hour) [1].
Warburg extended these results to human cancer tissues and also demonstrated
that although many adult tissues did not produce lactate even under hypoxic condi-
tions, normal growing tissue (e.g., embryonic samples) had intermediate rates of aer-
obic glycolysis. According to the Pasteur effect, oxygen should inhibit fermentation
(lactate production). Warburg interpreted his findings as demonstrating that respira-
tion was insufficient to suppress glycolysis, either due to limitations in oxygen sup-
ply or due to mitochondrial activity. Since his aerobic experiments were conducted in
thin tissue slices in which oxygen diffusion was not rate limiting, the problem seemed
to Warburg to be intrinsic to mitochondria. Furthermore, if respiration was insuffi-
cient for the cellular energy demand, selection of cells with high glycolytic rates for
survival was possible. Several normal tissues, including retina, kidney medulla, car-
tilage, bone marrow, skin, fibroblasts, intestinal mucosa, placenta, and proliferating
thymocytes, have been demonstrated to have high rates of aerobic glycolysis. In fact,
Warburg argued against using aerobic glycolysis as a specific test for cancer cells [2].
The central observation that cancer cells have high rates of aerobic glycolysis,
now known as the Warburg effect , has been repeated numerous times and is the
basis for the use of 2-deoxy-2-(18F) fluoro-D-glucose positron emission tomogra-
phy (PET) scans for tumor staging. However, mitochondrial dysfunction as a basis
for the Warburg effect is still debatable. The Warburg effect is inhibited by treat-
ment with mitochondrial uncouplers, indicating a well functioning Krebs cycle
and electron transport chain. Anaerobic glycolysis occurs at higher rates in cancer
cells than aerobic glycolysis, while the ratio of the decrease in lactic acid produc-
tion to oxygen consumption in normoxia is similar in cancer and normal tissues.
Thus, if energy demand remains constant, the efficiency of oxidative phosphoryla-
tion at suppressing glycolysis is similar in tumors and normal tissues.
However, it has been suggested that cancer cells may have reduced mitochon-
drial reserve in response to glycolytic inhibition [3]. Isolated mitochondria from
cancer cell lines with pronounced Warburg effects have been shown to exhibit
specific defects in substrate utilization, respiratory control ratios, and mitochon-
drial content [4, 5]. Reduced expression of the β subunit of the mitochondrial
F1FO ATPase has been linked to breast, lung, and colon adenocarcinomas with
poor prognosis [6–8]. Upregulated expression of the ATPase inhibitor IF1 has also
been reported in cancers [9, 10]. Aside from intrinsic differences in mitochondrial
function, several regulatory mechanisms restricting mitochondrial oxidative phos-
phorylation in cancer cells have been identified.
The Crabtree effect refers to the ability of glucose to inhibit respiration
in cancer cells, as opposed to the stimulation of respiration noted in normal tis-
sues [11]. This dynamic regulation provides another perspective on the glycolytic
shift in cancer cells identified by Warburg. Typically observed in cells in which
2 The Warburg Effect and Beyond: Metabolic Dependencies for Cancer Cells 37
glucose metabolism by glycolysis equals or exceeds oxidation in the TCA cycle,

the Crabtree effect has been proposed to represent competition between glycolysis
and oxidative phosphorylation for limiting substrates such as ADP and phosphate,
and the regulation of respiration by the energy charge [ATP]/[ADP][Pi] [12, 13].
Although ATP is also a negative feedback regulator of glycolysis at phosphofruc-
tokinase-1 (PFK-1), the allosteric activator fructose-2,6-bisphosphate (F-2,6-BP) is
overproduced in many cancer cells and lessens the effect of ATP [14]. Thus, high
rates of ATP synthesis can suppress oxidative phosphorylation to a greater extent
than glycolysis under these circumstances. Like aerobic glycolysis, the Crabtree
effect is also observed in normal cell types, such as proliferating thymocytes [15].
2.2 Glycolytic Regulation in Cancer
Current hypotheses rationalize the association of aerobic glycolysis and cancer

by the high demand for glycolytic intermediates in rapidly growing cells. Glucose
is utilized for the production of nucleosides (the pentose phosphate pathway pro-
duces ribose; 3-phosphoglycerate is precursor for glycine; serine and glycine are
utilized in one carbon folate metabolism), amino acids (pyruvate is transaminated
to produce alanine, 3-phosphoglycerate yields serine, glycine, and cysteine), and
reducing power (NADPH from pentose phosphate pathway). Paradoxically, sev-
eral steps in the glycolytic pathway are regulated in cancer to increase the levels of
glycolytic intermediates by slowing downstream enzyme turnover. However, some
cancer cells can have similar growth rates on slowly metabolized carbohydrates,
such as fructose and galactose, in which case most metabolism is diverted to the
pentose phosphate pathway, with minimal lactate production [16]. This suggests
that glycolysis in the presence of glucose operates in large excess to the require-
ments for anabolic reactions.
The final enzyme in glycolysis, lactate dehydrogenase, produces lactic acid,
which is transported out of the cell rather than serving as a synthetic intermedi-
ate. Two ideas to explain the high rates of lactic acid production have been pro-
posed. The first involves an “overflow” phenomenon, in which the rate of pyruvate
generation exceeds the capacity of the mitochondrial TCA cycle metabolism [17].
Alternatively, LDH may fulfill the requirement for regeneration of NAD+ for gly-
colysis to proceed, in the setting of low activity of malate–aspartate and glycerol-
3-phosphate mitochondrial shuttles for reducing equivalents [18].
In vivo, poor perfusion due to the disordered tumor microvasculature may
evoke a hypoxic response associated with expression of the hypoxia-inducible
transcription factors (HIFs), upregulation of glycolysis, and diversion of substrate
fuels away from mitochondrial metabolism. However, unless there is some mecha-
nism for “fixing” this response, it seems unlikely to account for aerobic glycolysis
measured under normoxic conditions.
The alternative explanation that increased glycolysis is matched to an increased
ATP demand is less favored in the face of evidence that tumor cells exhibit
adaptations that diminish ATP generation. Pyruvate kinase catalyzes the transfer of
phosphate from phosphoenolpyruvate (PEP) to ADP, generating pyruvate and ATP.
Originally described by Sato in 1978 [19], the pyruvate kinase splicing isoform
M2 (PK-M2) is highly expressed in cancers, embryonic tissues, and several adult
tissues, including adipocytes, retina, and lung [20]. PK-M2 is active as a tetramer,
but mostly occurs as inactive dimer in cancer cells. Tetramer formation is induced
allosterically by the proximal glycolytic intermediate, fructose-1,6-bisphosphate
(F-1,6-BP), but opposed by tyrosine phosphorylation of PK-M2 or interactions
with phosphotyrosine-bearing proteins at the F-1,6-BP-binding site [21–24].
Reduced activity of PK-M2 increases the diversion of upstream glycolytic inter-
mediates into anabolic pathways, including the pentose phosphate pathway [25],
as well as slowing ATP generation. Interestingly, substitution of the PK-M1 splic-
ing isoform increases glucose oxidation and reduces lactate production by an
unexplained mechanism [23].
Reduction in the PK-M2 activity in cancer cells raises questions as to the
source of pyruvate utilized for lactate generation in aerobic glycolysis. Analogous
to bacterial pathways, the substrate for PK-M2, PEP can be used as a phosphate
donor in protein kinase reactions [26, 27]. One of the protein substrates for PEP-
dependent phosphorylation is the glycolytic enzyme phosphoglycerate mutase
(PGAM1), which is phosphorylated on the catalytic histidine (H11) involved in
transferring phosphate to the C-2 position of 3-phosphoglycerate. This reaction
produces pyruvate as a product in the absence of ATP generation. Thus, cancer
cells can switch to an alternative glycolytic pathway that does not produce net ATP.
Finally, the concept that the predominant function of glycolysis is ATP produc-
tion can be questioned by a study of glucose deprivation in IL-3-dependent cells
[28]. Glucose is necessary for cell growth and proliferation in many cell types.
In this example, glucose was shown to be required for N-linked glycosylation of
the IL-3 receptor, required for its cell surface localization and in turn, uptake of
an alternative substrate fuel, glutamine. Glucose could be replaced by the specific
product of the hexosamine biosynthetic pathway, N-acetylglucosamine, enabling
glutamine uptake and cell growth.
Three major pathways are involved in deregulating glycolysis in cancer, the
transcription factors c-Myc and HIF-1, and the serine/threonine kinase, Akt . The
c-Myc oncoprotein is overexpressed in more than 50 % of human cancers and
binds to 15 % of all gene promoters in the genome. Among the pathways con-
trolled by c-Myc is glycolysis, with 9/10 glycolytic enzymes and glucose trans-
porters type 1, 2, and 4 identified as Myc targets. Myc-dependent cell cycle entry
and transformation are associated with increased lactic acid production [29, 30].
Stable isotope labeling studies indicate that Myc directs glucose carbons to mul-
tiple anabolic pathways, including nucleotide, amino acid, and lipid biosynthesis
[31, 32]. N-Myc appears to share similar targets in glycolysis [33].
The hypoxia-inducible transcription factor, HIF-1α, is degraded by the ubiq-
uitin–proteasome system following oxygen-dependent proline hydroxylation.
HIF-1α is overexpressed in 13/19 common tumor types [34]. HIF-1 targets include
glycolytic enzymes, glucose transporters and the pyruvate dehydrogenase kinase
(PDK1) that inactivates pyruvate dehydrogenase, required for pyruvate oxidation

in the TCA cycle. HIF-1 can also be stabilized in normoxia by mutations in succi-
nate dehydrogenase and fumarate hydratase that lead to product inhibition of pro-
line hydroxylases [35], increased concentrations of pyruvate and lactate [36], and
mTOR -dependent translation [37].
Although HIF-1 and c-Myc may have antagonistic functions [38], studies in a
Burkitt’s lymphoma model demonstrated a cooperative effect on glycolysis [39].
The Akt serine/threonine kinases are activated downstream of phospho-
inositide 3-kinase (PI3K ) and direct both glucose uptake and metabolism.
Glucose transporters Glut 1 and 4 recycle between insulin-responsive storage
compartments and the plasma membrane via Akt phosphorylation of the Rab
GTPase activity TBC1D proteins [40, 41]. Akt also activates 6-phosphofructo-
2-kinase (PFK-2), the enzyme responsible for synthesis of fructose-2,6-bisphos-
phate, by phosphorylation [42]. Finally, Akt promotes association of hexokinases
I and II with mitochondria [43], increasing glucose phosphorylation, by prevent-
ing GSK3β phosphorylation of the voltage-dependent anion channel (VDAC)
[44]. Overexpression of c-Myc , HIF-1α, and constitutively active Akt stimulates
aerobic glycolysis [29, 45–47].
2.3 Therapeutic Strategies Based on Glycolysis Inhibition
Inhibition of glycolysis is often lethal in tumor cell lines in vitro, prompting both
early and renewed interest in this strategy for cancer treatment. The glucose analog,
2-deoxy-D-glucose (2-DG), is phosphorylated by hexokinase, but in the absence
of further glycolytic transformations, 2-DG-6-P accumulates in cells and inhibits
hexokinase. 2-DG was first administered to human cancer patients [48] with a vari-
ety of cancers and has since been studied in early stage clinical trials in patients
with glioblastomas [49] and advanced solid malignancies [50]. Novel LDH-A inhib-
itors have been reported [51, 52]. A peptide inhibitor of PK-M2 is in clinical tri-
als for melanoma and renal cell carcinoma (www.thallion.com). Several classes of
inhibitors of the PI3K–Akt signaling pathway are in clinical trials, including PI3K
inhibitors, Akt inhibitors, and dual PI3K–mTOR inhibitors [53]. A common theme
in cell death following glucose deprivation or glycolytic inhibition is an increase in
mitochondrial generation of reactive oxygen species, with decreased generation of
NADPH reducing equivalents in the pentose phosphate pathway [54, 55].
2.4 Alternative Mitochondrial Fuels
Mitochondrial substrate fuels are not restricted to pyruvate-derived acetyl-CoA.

The role of glutamine as a major fuel for cancer cell mitochondria was recognized
more than 50 years ago [16]. Glutamine is initially metabolized to glutamate by
Fig. 2.1 Truncated and reversed TCA cycles (red) with glutaminolysis, involving malic enzyme
producing pyruvate and NADPH, and reductive carboxylation of α-ketoglutarate to citrate
phosphate-dependent glutaminase, which can be converted to α-ketoglutarate

using either glutamate dehydrogenase or transaminase enzymes. Glutamine
entry into the TCA cycle can replenish intermediates (anaplerosis) or serve as a
source of acetyl-CoA from pyruvate generated by malic enzyme from malate
(Fig. 2.1). The latter reaction is one of the major sources of NADPH generation
in cancer cells, used for fatty acid and cholesterol biosynthesis, ribonucleotide
reduction, and reduction of GSSG to GSH, in addition to the pentose phosphate
pathway. Glutamine can also be utilized for lipid biosynthesis, with conver-
sion of mitochondrial citrate to cytosolic acetyl-CoA by ATP citrate lyase. Two
paths for citrate production from glutamate are available, a truncated TCA cycle
(α-ketoglutarate to citrate) or reductive carboxylation to isocitrate. ATP citrate
lyase activity is activated by Akt -dependent phosphorylation. The energy yield
from glutamine metabolism is 6-12 ATP/glutamine, depending on the inclusion of
NADP+ -dependent glutamate and malate dehydrogenases in the reaction path-
way. Glutamine metabolism accounts for 65 % or higher of ATP production in
many cancer cells. Chemical inhibition of glutaminase slows growth or kills out-
right cells dependent on glutamine, especially hypoxic cells or cells that lack other
pathways to generate α-ketoglutarate [56, 57].
Several nonessential amino acids can be derived from glutamate (proline, argi-
nine, aspartate, and asparagine). Glutamine contributes its γ-nitrogen to nucleotide
and hexosamine synthesis and its α-nitrogen to alanine and aspartate in transami-
nation reactions. Glutamate is utilized in glutathione synthesis, and glutamine
exchange with leucine and other essential amino acids is essential for mTOR
activity [58]. However, a significant portion of glutamine nitrogen and carbon is
transported extracellularly, as glutamate, alanine, and lactate, indicating that, like
glycolysis, glutaminolysis is an inefficient process in cancer cells [59].
c-Myc has emerged as a major driver of glutamine metabolism. Glutamine
deprivation is sufficient to kill Myc-overexpressing cells [60, 61]. Surprisingly,
glutamine-deprivation-induced cell death involves depletion of TCA cycle
intermediates, but not ATP deficiency, glutathione depletion, or DNA dam-
age, and can be partially rescued with antioxidants [60, 62]. The anaplerotic
requirement for glutamine is highlighted by the glutamine-independent growth
of tumor cells expressing pyruvate carboxylase, an anaplerotic enzyme associ-
ated with glucose metabolism [63]. Glutaminase translation is suppressed by
two microRNAs, miR-23a and miR-23b, that are in turn targets for Myc tran-
scriptional repression [62]. Myc also increases the expression of glutamine
transporters [61].
Although there are differences in the regulation of glucose and glutamine
metabolism, one mechanism for dual control is the basic helix-loop-helix leu-
cine zipper transcription factor, MondoA (also known as MLXIP), related to Myc
[64]. In glucose-containing growth media, MondoA functions as a transcrip-
tional activator for TXNIP, which suppresses glucose uptake and aerobic glyco-
lysis. However, the addition of glutamine converts MondoA to a transcriptional
repressor, increasing glucose uptake. The ability of a cell-permeable analog of
α-ketoglutarate to similarly affect MondoA transcriptional activity suggests
that the levels of TCA cycle intermediates may be monitored to adjust glucose
uptake and metabolism. Recent data also indicate that Nrf2, a transcription factor
responding to oxidative stress, directs both glucose and glutamine utilization in
anabolic pathways, such as the pentose phosphate pathway and nucleotide syn-
thesis [65].
Tumor cells engage in de novo fatty acid synthesis, in contrast to importation of
circulating lipids by most normal cells in the body [66, 67]. Several recent reports
have indicated that cancer cells may also exhibit high rates of peroxisomal and
mitochondrial β-oxidation [68, 69]. Prostate cancers with low 2-deoxy-2-fluoro-
D-glucose (FDG) avidity on PET scans may engage in fatty acid β-oxidation as a
principal bioenergetic pathway [69]. Respiration in glioblastoma cells is inhibited
30–40 % by treatment with the carnitine palmitoyltransferase-1 (CPT1) inhibitor,
etomoxir, with a 50 % decrease in ATP levels [70]. Fatty acid β-oxidation has been
associated with uncoupling protein-2 expression and chemoresistance [71, 72].
The brain CPT1C isoform is upregulated in non-small-cell lung cancers and con-
fers resistance to hypoxia, glucose deprivation, and the mTOR inhibitor, rapamy-
cin [73]. Adipocytes promote the growth of ovarian cancer cells by mobilizing free
fatty acids for β-oxidation by tumor cells [74]. Inhibition of fatty acid β-oxidation
(etomoxir, ranolazine) or fatty acid biosynthesis (cerulenin, C75, orlistat) is selec-
tively cytotoxic to some cancer cells [70, 75].
2.5 Screening for Additional Metabolic Dependencies
The Recon 1 genome-scale metabolic reconstruction includes 1,496 open reading

frames and 3,311 metabolic and transport reactions [76]. Enzymes are optimal tar-
gets for drug development, with active sites and allosteric binding pockets well
suited for drug interactions. The extent of metabolic differences between cancer
and normal cells is currently not known, although it is evident that the Warburg
effect is only one aspect of “cancer metabolism”. In addition to glutaminolysis
and lipid metabolism, cancer cells exhibit auxotrophy for specific amino acids and
increased NAD+ turnover [77–80].
Several approaches have been employed recently to identify metabolic path-
ways that are required for growth or survival in cancer cells, but not normal cell
types. Analogous to other strategies for identifying “driver” genes in cancer, analy-
sis of gene expression may reveal candidate targets for functional genomic stud-
ies. Possemato et al. generated a high-priority set of 133 metabolic and transporter
genes based on high expression in cancers versus normal tissues, high expres-
sion in aggressive breast cancers, and expression in stem cells [81]. Tumorigenic
breast cancer cells were transduced with pools of shRNAs targeting the 133 genes
and grown as orthotopic tumors in mice. 16 genes were identified with signifi-
cant depletion of targeting shRNAs during 28 days of in vivo growth. One of the
genes, phosphoglycerate dehydrogenase (PHGDH), is amplified in breast can-
cers and melanomas, and PHGDH expression is associated with poor prognosis
in ER-negative breast cancers. PHGDH catalyzes the oxidation of the glycolytic
intermediate, 3-phosphoglycerate, to phospho-hydroxypyruvate, which can subse-
quently be converted to serine. Serine is used for the synthesis of phosphatidyl-
serine, sphingosine, cysteine, and glycine, as well as protein synthesis. Additional
stable isotope analysis demonstrated increased serine biosynthetic flux in can-
cer cells overexpressing PHGDH, accounting for 8–9 % of total glycolytic flux.
RNAi-mediated suppression of PHGDH in these cells induced non-apoptotic cell
death and reduced tumor growth in vivo. Finally, metabolomic studies demon-
strated large decreases in α-ketoglutarate concentrations in these cells, indicating
that phosphoserine aminotransferase provides a major route for conversion of glu-
tamate to α-ketoglutarate.
Unlike transcription factors or signal transduction pathways, metabolic enzyme
activity is not manifested by accumulation of downstream gene products or post-
translationally modified substrates, but rather by increased substrate to product
flux. Furthermore, enzyme regulation often includes allosteric effects. Locasale
et al. used [U-13C] glucose stable isotope metabolic flux measurements to identify
high levels of glycine enrichment in cancer, but not immortalized, cell lines [82].
As in the previous study, PHGDH amplification was observed to correlate with
high flux in the serine biosynthetic pathway.
Finally, in silico network models of cancer metabolism based on flux bal-
ance analysis have been used to predict metabolic pathways required for growth
in specific genetic contexts [83]. Germline mutations of the TCA cycle enzyme,
fumarate hydratase (Fh1), cause hereditary leiomyomatosis and renal cell can-
cer (HLRCC). The accumulation of fumarate inhibits prolyl hydroxylases, lead-
ing to an increase in HIF expression under normoxic conditions. Incorporation
of cellular growth as consumption of biosynthetic precursors in the in silico
model allowed testing of specific gene knockouts for effects on growth. Frezza
et al. predicted 24 reactions to be synthetically lethal with Fh1 deletion, 18 of
which involved heme metabolism. Heme biosynthesis utilizes TCA-derived suc-
cinate, providing an alternative route to fumarate synthesis. Subsequent analysis
of Fh1−/− cells demonstrated increased excretion of bilirubin, the degradation
product of heme. Only three enzymes involved in heme degradation were overex-
pressed in Fh1−/− cells, highlighting the power of the computational approach to
identify significant pathways in the absence of expression criteria. Several strate-
gies to inhibit heme pathway flux reduced growth of Fh−/− cells.
Toward an unbiased screening strategy, we have generated a chemical library
with inhibitors for enzymes of intermediary metabolism, as validated in the litera-
ture by direct enzymatic assay. Inhibitors were chosen from the BRENDA enzyme
database (www.brenda-enzymes.org) and review of published literature, purchased
from Sigma-Aldrich or other chemical supply houses, or donated by academic
labs. Inhibitors of 585 enzymes have been identified. Each inhibitor is plated
at 100X EC50 concentration in DMSO. Transcriptome analysis in mouse and
human databases indicates ~750 enzymes of intermediary metabolism encoded
in the genome with unique EC numbers [84]. Such a library has several applica-
tions relevant to cancer research. Screening cancer cell lines versus normal cel-
lular counterparts for loss of viability or cell growth can reveal critical metabolic
dependencies associated with the transformed phenotype. Altered expressions
of metabolic enzymes, in particular, as revealed by Gene Set Enrichment analy-
ses, are frequently observed in RNA microarray profiles. As yet, there is no rapid
method to determine the essentiality of a metabolic pathway for cancer cell viabil-
ity. Metabolomic studies of cancer cells are increasingly being reported. A chemi-
cal inhibitor library can rapidly ascertain which pathways connecting to a given
metabolite are casually linked to cell growth or viability.
We have tested 217 causally compounds out of a total of 585 in our enzyme
inhibitor compound library in the MCF10A cell line expressing Myc-ERTAM. As
shown in Fig. 2.2, inhibitors are available for a high proportion of enzymes in
major metabolic pathways. MCF10A-MycERTAM cells were treated with tamoxifen
to activate Myc for 24 h and compared with comparably treated vehicle controls.
Cell metabolism was evaluated using a Seahorse analyzer, which demonstrated a
shift toward glycolytic metabolism in tamoxifen-treated, but not control, cells.
Cells were tested with each compound in triplicate at a single dose, based on
published IC80 values for enzymatic inhibition. Cell viability was assessed after
24 h by Alamar Blue assay. Log2-transformed results were analyzed according to
SSMD scores (strictly standardized mean difference; [85]) with respect to nega-
tive vehicle controls. The results are graphed in Fig. 2.3 (high SSMD values cor-
respond to selective killing of tamoxifen-treated cells). A secondary assay for cell
number (Hoechst 33342 fluorescence of adherent cells) confirmed these results.
Fig. 2.2 Comparison of enzymes in major primary metabolic pathways and availability of

specific inhibitors
Fig. 2.3 Plot of SSMD scores for 217 compounds tested against MCF10A-MycER cells +/− tamox-
ifen, in relation to negative (vehicle) controls
The top hits include aldehyde dehydrogenase, malic enzyme, aromatic L-amino
acid decarboxylase, and threonine tRNA ligase. Aldehyde dehydrogenases are
markers for cancer stem cells. Disulfiram, the aldehyde dehydrogenase inhibitor
in the screen, is reported to have anticancer activity [86], although the relevant tar-
get has not been validated. Aromatic L-amino acid decarboxylase (also known as
L-dopa decarboxylase) is expressed in several epithelial cancers in addition to neu-
roendocrine tumors and has co-activator activity for the androgen receptor [87, 88].
Borrelidin, the threonine tRNA ligase inhibitor, induces apoptosis in acute lympho-
blastic leukemia cells associated with the activation of the GCN2 stress kinase and
proapoptotic CHOP transcription factor [89].
2.6 Summary
It is not surprising, based on the intimate role of catabolic and anabolic metab-
olism in furnishing biochemical energy and building blocks for macromolecu-
lar synthesis and repair, that metabolic pathways are vital to cancer cell growth
and survival in different tumor microenvironments. What we have learned more
recently is that metabolic pathways can also direct chromatin structure and gene
expression, differentiation, and stemness [90–95], typified by the discovery of the
oncometabolite, D-2-hydroxyglutarate (2HG). Specific gain-of-function mutations
in the isocitrate dehydrogenase-1 and dehydrogenase-2 enzymes occur in glio-
blastomas and acute myeloid leukemia and alter enzymatic function to produce
2HG from α-ketoglutarate (α-KG) [96]. 2HG is an inhibitor of α-KG-dependent
Jumonji-C domain histone demethylases [97, 98] and is associated with increased
histone methylation and altered gene expression [99, 100]. Comprehensive map-
ping of cancer cell metabolism is at an early stage, but current knowledge suggests
that the Warburg effect may be the “tip of the iceberg”. The ultimate value of
cancer therapies directed against metabolic dependencies is still to be determined,
as the links between metabolism and cell death, and extent of metabolic flexibil-
ity are largely unknown. Further exploration of metabolic networks in cancer will
require novel strategies, but seems likely to yield novel targets for cancer therapy.
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Chapter 3
Emerging Opportunities for Targeting
the Tumor–Stroma Interactions
for Increasing the Efficacy
of Chemotherapy
Rajesh R. Nair, Anthony W. Gebhard and Lori A. Hazlehurst
Abstract It has become evident that tumor cells utilize survival signals that ema-
nate from the tumor microenvironment to aid in survival and disease progression.
Experimental evidence indicates that these same pathways contribute to de novo
drug resistance. Identification of the mechanisms underlying the recruitment of
accessory cells and survival signals provided by normal cells has provided a novel
area for drug discovery for increasing the efficacy of cancer therapy.
3.1 Introduction
Cancer is characterized as a disease that is initially driven by the expression of

oncogenes and attenuation of tumor suppression genes expressed in the malig-
nant tumor cell. From these observations came the term oncogene addiction which
implies that cancer cells require the activity of specific oncogenes for survival and
growth. Furthermore, this premise infers that if we can specifically target onco-
genes, then we could more effectively treat cancer. Early chemotherapeutic agents
typically target rapidly dividing cells, a phenotype typically driven by oncogene
expression. However, DNA-damaging agents are associated with toxicity and the
emergence of resistance can limit the overall success of these agents. Resistance
to DNA-damaging agents includes overexpression of drug transporters, increased
DNA repair, increased metabolism, and failure to eliminate dormant cancer cells,
as well as the cancer stem cell population [1]. It was envisioned that targeting spe-
cific oncogenes may reduce toxicity and increase efficacy over standard therapy
R. R. Nair · A. W. Gebhard · L. A. Hazlehurst

Molecular Oncology Program, H Lee Moffitt Cancer Center, Tampa, FL, USA
A. W. Gebhard · L. A. Hazlehurst
Molecular Pharmacology and Physiology Program, Tampa, FL, USA
L. A. Hazlehurst (*)
Cancer Biology Program, University of South Florida, Tampa, FL, USA

54 R. R. Nair et al.
due to increased specificity for the cancer cell as well as reduce the emergence of
multi-drug resistant phenotypes associated with standard therapy.
In support of this potential promise of targeted agents are data generated in
chronic myeloid leukemia (CML) which is driven by the BCR-ABL oncogene
[2, 3]. A myriad of experimental data support the findings that the expression of
the fusion oncogene BCR-ABL is sufficient for transformation and disease pro-
gression using transplant and transgene in vivo model systems [4, 5]. The identi-
fication of BCR-ABL as driving transformation of CML provided an ideal target
for drug discovery, and first- and second-generation BCR-ABL inhibitors have
provided proof-of-principle that this approach may indeed lead to clinical success
[6]. However, it became clear that although BCR-ABL inhibitors are very effec-
tive when treating patients in the early chronic stage of the disease, BCR-ABL
inhibitors were not curative and similar to DNA-damaging agents did not bypass
the emergence of drug resistance. Because CML is initially driven by one driver
oncogene, it represents an ideal disease to delineate de novo drug resistance and
the emergence of acquired resistance. Acquired resistance to BCR-ABL inhibitors
includes point mutations in what is commonly referred to as the gatekeeper region
of the molecule [7]. These mutations result in reduced affinity of drug binding
and thereby render the cells insensitive to the compound. The identification of the
mutations, along with structural information, led to the development of second-
generation compounds predicted to be active in the presence of specific gatekeeper
mutations, and indeed, these molecules do show promising activity in the subset of
patients harboring the T315I gatekeeper mutation [8]. Other mechanisms reported
to contribute toward resistance to BCR-ABL inhibitors include increased expres-
sion of transporters, quiescence, and progression of the disease to blast phase
which typically is the result of expression of additional driver mutations and sub-
sequent resistance to BCR-ABL inhibitors.
Of particular interest for this review is the observation that even in patients
exquisitely sensitive to BCR-ABL inhibitors, minimal residual disease can be
detected at the molecular level. Subsequent to the identification of BCR-ABL came
the identification of activating EGFR mutations in lung cancer and head and neck
cancer and expression of BRAF in melanoma. Again, the identification of these
driver oncogenes led to the rapid translation of specific kinase inhibitors. However,
within the landscape of the complexity associated with lung cancer, colon can-
cer, and melanoma, these inhibitors, albeit effective, demonstrated shorter clinical
response time before the emergence of resistance was a clinical concern [9–12].
Thus, it became clear that targeted therapies which potently inhibit the activity of
a driver oncogene do not circumvent the emergence of drug resistance. The emer-
gence of drug resistance can be caused by mutations and overamplification of tar-
gets which are mechanisms that contribute to acquired resistance. However, more
recently, attention has shifted toward the role of the tumors inherent ability for
co-opting and recruiting normal cells into the niche to provide an adaptive envi-
ronment that favors the survival of tumor cells [13], and hence, as the niche is con-
ditioned by the tumor, the dependency of cancer cells on the activity of a specific
oncogene is diminished. This review will focus on mechanisms associated with
3 Emerging Opportunities for Targeting 55
components of the tumor cell microenvironment which contribute the maintenance

of minimal residual disease and resistance to cancer chemotherapy.
The tumor microenvironment is a complex network consisting of multiple cell
types including cancer-associated fibroblasts, immune accessory cells, and endothe-
lial cells that support growth and invasion through production of cytokines, growth
factors extracellular matrixes, and the formation of angiogenic blood vessels. Some
of these same pathways which tumor cells initially co-opt to support growth and
survival can also contribute to resistance to standard therapy and targeted therapy
[14]. Components of the tumor microenvironment known to contribute to drug
resistance will be discussed in the first half of this chapter. These interactions pro-
vide novel targets for drug discovery for increasing the efficacy of cancer therapies,
and these strategies will be discussed in the second half of this review.
3.2 Cellular Components of the Tumor Microenvironment

Aiding Drug Resistance
3.2.1 Mesenchymal Stroma Cells

and Cancer-Associated Fibroblasts
Mesenchymal stem cells (MSCs) are characterized by their ability to differentiate

into osteoblasts, chondrocytes, adipocytes, muscle cells, and reticular fibroblasts.
MSCs are considered a rare population contained within the bone marrow estimated
at 0.01–0.001 % of all mononuclear cells in the bone marrow [15]. Co-culturing
of hematopoietic cells with either MSCs or bone marrow stroma cells has been
shown to induce a multi-drug resistant phenotype in a variety of hematopoietic
tumors including myeloma, acute myeloid leukemia, chronic myeloid leukemia, and
chronic lymphoblastic leukemia (CLL) [16–20]. The mechanism of resistance var-
ies depending on the drug investigated and the tumor type. For example, in CML,
resistance toward BCR-ABL inhibitors is associated with the activation of the JAK/
STAT3 pathway [16, 20]. In contrast, resistance associated with topoisomerase II
inhibitors correlates with a reduction in the formation of cleavable complex and
DNA double-strand breaks [21, 22]. Multiple cytokines and growth factors can initi-
ate the JAK/STAT3 pathway, and correspondingly, MSCs are known to secrete many
of these same factors including IL-6 and VEGF (See Table 3.1 for a complete list).
STAT3 can be activated by cytokines and growth factors [23–26]. Furthermore, the
activation of STAT3 can be amplified in the context of β1 integrin–mediated adhe-
sion [27]. Thus, the activation of STAT3 can be adaptively activated by multiple
components of the tumor microenvironment, which is typically rich in the levels of
inflammatory cytokines and extracellular matrixes. The activation of STAT3 induces
increased expression of anti-apoptotic Bcl-2 family members such as Bcl-2, Bcl-
XL, and Mcl-1. Additionally, STAT3 increases the expression of inhibitors of apop-
totic machinery like survivin and c-IAP2 [28–32]. STAT3 is proximally activated via
the tyrosine kinase receptor–associated Janus kinase (JAK) [33–35], resulting in the
Table 3.1 Factors secreted by cultured stromal cells that activate STAT3

Effect Molecule References
Survival and angiogenic VEGF [197–199]
Adhesion, survival, and angiogenic IL-6 [27, 198, 200]
Immunomodulation and differentiation LIF [201–203]
Adhesion and survival SDF-1 [204–207]
Chemoattractant and survival IL-8 [208, 209]
Chemoattractant MCP-1 [210–212]
Survival SCF [200, 205, 213, 214]
Survival G-CSF [215–217]
Survival GM-CSF [216, 218, 219]
Immunomodulation and survival HGF [220–222]
Survival and angiogenic FGF-1,2 [223–226]
phosphorylation at the Tyr705 residue in the C-terminal domain [33–35]. The acti-
vated phosphorylated STAT3 then undergoes homodimerization by reciprocal inter-
action between the SH2 domain of one monomer and the phosphorylated Tyr705
residue of its dimerizing partner. STAT3 dimer can translocate to the nucleus and bind
specific DNA sequences and regulate the transcription of the responsive gene [36, 37].
Targeting of STAT3 could potentially occur at multiple points of regulation
including inhibition of the activating receptor kinase or cytokine receptor, via inhi-
bition of JAK kinases or by disrupting the dimerization or subsequent DNA bind-
ing. Specifically, disrupting the dimerization and DNA binding has proven to be a
difficult task using small molecule design. Experimental evidence indicates that
in some tumors, IL-6 plays a dominant role in conferring resistance. For example,
in neuroblastoma, Ara et al. found that blocking IL-6 signaling using a blocking
IL-6Rα antibody reversed resistance to the topoisomerase II inhibitor, etoposide
when neuroblastoma cells were cultured in the presence of MSCs [38]. In multiple
myeloma, IL-6 has been shown to be critical for cellular survival via activation
of the JAK/STAT3 pathway and subsequent increased expression of the anti-
apoptotic family member Bcl-XL [39]. More recently, several groups have pro-
vided evidence that targeting IL-6 or JAKs in the context of co-culturing multiple
myeloma cells with bone stroma cells enhances the efficacy of standard therapy
including melphalan and dexamethasone [40–44]. Thus, in myeloma, it appears
that IL-6 is the predominant cytokine-driving drug resistance in the bone marrow
compartment. In contrast, in CML, experimental evidence indicates that multiple
soluble factors can activate STAT3 and induce resistance to BCR-ABL inhibitors
[20]. Due to the plethora of cytokines and growth factors present in the bone mar-
row milieu capable of activating the JAK/STAT3 pathway, it is likely that target-
ing downstream of the cytokine receptor at the level of JAK or STAT3 will be the
most effective strategy and may circumvent resistance due to the selection of cells
that utilize an alternative receptor for activating the JAK/STAT3 pathway [20] (for
a complete list of drugs targeting JAK/STAT3 pathway see Table 3.2). MSCs are
specifically relevant to tumors which home to the bone such as multiple myeloma
or metastasize to the bone such as lung, breast, and prostate.
Table 3.2 JAK-STAT pathway inhibitors

Drugs Function Indication Reference
Siltuximab Monoclonal antibody Prostrate cancer [227]
against IL-6 Renal cancer [228]
Multiple myeloma [41]
AZD1480 JAK2 inhibition Solid tumors [229]
Myeloproliferative [230]
neoplasma
TG101209 JAK2 inhibition Multiple myeloma [231]
SB1518 JAK2 inhibition Myeloid and lymphoid [232]
cancer
Lestaurtinib JAK2 inhibition Hodgkin’s lymphoma [233]
Myelofibrosis and [234]
polycythemia vera
MS-1020 JAK3 inhibition JAK3-driven cancer [235]
Ruxolitinib JAK1/JAK2 inhibition Myeloproliferative [236]
neoplasma
INCB1656 JAK1/JAK2 inhibition JAK2V617F-driven [237]
neoplasma
Multiple myeloma [43]
CYT387 JAK1/JAK2 inhibition Multiple myeloma [238]
AG490 Pan-JAK inhibition Laryngeal cancer [239]
Acute lymphoblastic [240]
leukemia [241]
Pancreatic cancer
Gö6976 JAK2/FLT3 inhibition Myeloproliferative [242]

neoplasma and acute
myeloid leukemia
TG02 CDKs/JAK2/FLT3 Acute myeloid leukemia [243]
inhibition
Sorafenib JAK/STAT3 signaling Cholangiocarcinoma [244]
inhibition
Trichostatin A JAK/STAT3 signaling Colorectal cancer [245]
inhibition
AUH-6-96 JAK/STAT3 signaling Hodgkin’s lymphoma [246]
inhibition
WP1193 JAK/STAT3 signaling Glioblastoma [247]
inhibition
CEP-33779 IL6/STAT3 signaling Colitis-induced colorectal [248]
inhibition cancer
Quercetin IL6/STAT3 signaling Glioblastoma [249]
inhibition
The origin of cancer-associated fibroblasts (CAFs) found in solid tumors is due

to the active recruitment of bone marrow-derived MSCs by the tumor as well as
recruitment of localized tissue-associated fibroblasts. CAFs are characterized as
expressing “activated” fibroblast markers including fibroblast-specific protein
(FSP) and fibroblast-activated protein (FAP). Experimental evidence indicates

that CAFs are modified by the tumor via epigenetic mechanisms [45]. The end
result is an increased expression of inflammatory cytokines such as IL-6 and IL-8
and growth factor expression such as EGF and SDF-1 which can further promote
tumor progression and drug resistance. Co-culturing cancer cells with activated
fibroblasts has been shown to induce resistance to targeted therapies such as ER
inhibitors for the treatment for breast cancer and EGFR inhibitors for the treatment
for lung cancer [46, 47].
3.2.2 Immune Cells
In cancer, immune cells can participate in surveillance and elimination of tumor

cells. However, paradoxically, immune cells can also participate in conditioning of
the pre-metastatic niche which favors cancer cell initiation and progression of can-
cer as well as contributing to drug resistance. The premalignant niche resembles
chronic inflammatory models and wound healing models, as characterized by sig-
nificant infiltration of leukocytes into the tumor microenvironment. The leukocytes
found in the tumor site include Treg, immature monocytes, and alternative activated
macrophages. Treg cells are a subset of the CD4-positive T-cell population that
expresses lineage-specific Foxp3, IL-2 receptor (CD25), and CTLA-4. Treg cells
are immunosuppressive and critical for curbing autoimmunity but also attenuate
anti-tumor immunity. Increased expression of the Treg population is associated
with poor clinical outcomes for several tumor types and thus inhibiting the traf-
ficking or depleting Treg cells represents a possible strategy for improving clinical
outcomes [48, 49].
Recently, immature myeloid progenitor cells have gained attention for their
immunosuppressive function. In murine models, myeloid-derived suppressor cells
(MDSCs) are identified as Gr1 and CD11b positive [50]. Functionally, MDSCs
are characterized by their ability to suppress T and NK cell proliferation and pro-
mote the growth of Treg cells. The functional phenotype is due to arginase I, induc-
ible nitric oxide synthase expression and subsequent generation of NO [51] and
peroxynitrite [52]. Similar to Treg cells, the abundance of MDSCs is increased in
the tumor and metastatic site. Moreover, MDSCs have been show to confer resist-
ance to anti-VEGF therapies used to target angiogenesis [53]. Certainly, either
selective elimination of MDSCs or blocking trafficking into tumor sites provides
a novel approach for combination therapies. Recently, Schmid et al. [54] showed
that blocking VLA-4 integrin via inhibiting inside-out activation (blocking SDF-1
or IL-1β) of VLA-4 and/or use of VLA-4 blocking antibody reduced the infiltra-
tion of myeloid-derived cells in a murine model of lung cancer and simultaneously
reduced tumor burden. Interestingly, these studies highlight the need to utilize
immune-competent mice when considering strategies for the development of novel
chemotherapeutic agents.
3.2.3 Endothelial Cells
Once tumors have evaded the immune system and established growth that exceeds
a few millimeters, the tumor must quickly establish a blood supply to sustain sur-
vival and growth. This is achieved by the production of stimulatory factors that
attract and activate endothelial cells to form angiogenic vesicles. As the tumor
grows, localized areas of hypoxia arise and results in induced HIF-1α expression
in the tumor, endothelial, and stroma cells. HIF-1α expression is a major player
for driving the expression and secretion of multiple angiogenic factors including
VEGF, PDGF, FGF, angiopoietins, and SDF-1α [55]. Additionally, integrins are
known to augment growth factor receptor signaling and thus may provide an addi-
tional target for inhibiting angiogenesis [56]. One soluble factor that is critical for
the angiogenic switch is vascular endothelial growth factor (VEGF). VEGF stimu-
lates the sprouting and proliferation of endothelial cells. However, it is becoming
clear that targeting VEGF alone will likely not be sufficient for inhibiting angio-
genesis, as resistance has been observed preclinically and clinically [57]. Overall,
due to the ability of tumor cells to quickly adapt to a reduction in blood flow, it
is currently not clear how effective targeting angiogenesis will be in controlling
cancer. Although it is feasible, as we learn more about the bidirectional communi-
cations between the tumor, myeloid population, stroma, and endothelial cells that
such strategies may be fully realized.
3.3 The Tumor Microenvironment Milieu and Drug

Resistance and Tumor Progression
3.3.1 Galectins
Cell surface receptors like cytokine and growth factor receptors are
N-glycosylated transmembrane glycoproteins that can bind glycan-binding pro-
teins called lectins [58, 59]. Galectins are members of the lectin family that has
an affinity for β-galactoside sugars, especially N-acetyllactosamine [60]. Galectins
have been demonstrated to crosslink receptor glycoproteins at the cell surface
and form lattices and thereby dictate the distribution and the retention time of the
receptor on the cell surface [61, 62]. Interestingly, the strength of the interaction
between the galectins and the glycoproteins, and thus the lattice structures, can be
modulated by either regulating the cell surface protein glycosylation or by alter-
ing the expression of galectins [63, 64]. This is important because the distribution
pattern of the cell surface receptors and their residency time on the plasma mem-
brane can modulate the cells response to its microenvironment and is a prevailing
feature of oncogenesis [65]. For example, Mgat5 gene encodes a Golgi-localized
enzyme that initiates N-glycan synthesis on newly formed cell surface receptors
that is recognized by galectins [66]. Granovsky et al. have shown that Mgat5
knockdown inhibits tumor progression and upregulation of Mgat5 increases tumo-
rigenesis in mouse tumor models [67]. Furthermore, Partridge et al. demonstrated
that in Mgat5−/− tumor cells, cell surface density of EGFR and TGF-β receptor
was reduced and activation of these receptors increased their endocytosis, mak-
ing the cells less sensitive to stimulation by EGF, IGF, PDGF, bFGF, and TGF-β
[68]. Also, these receptor glycoproteins were dependent on galectin-mediated lat-
tice formation for surface retention after receptor activation, thereby prolonging
the sensitivity of the tumor cells to EGF and TGF-β [68]. This observation was
later confirmed by Lajoie et al., who showed that EGFR localization, and thus, the
magnitude of EGFR signaling in tumor cells was very much dependent upon the
galectin lattice structures opposing and prevailing over the oligomerized caveo-
lin-1 microdomains that mediate endocytosis [69]. Finally, regulated glycosylation
of cell surface receptors can create or abolish binding domains for galectins and
thereby control important cellular response such as immune responses like T-cell
activation, homing, and survival [70].
Presently, there are 15 members of the galectin family identified that all contain
a conserved globular domain called carbohydrate recognition domain (CRD) that
recognizes and binds to β-galactosidase [71]. The specificity of the galectins for
their substrates is defined by the ligand-binding groove contained within the CRD
of the protein [72]. The 15 family members are divided into three sub-families on
the basis of their molecular structure (see Fig. 3.1): (1) Galectin-1, Galectin-2,
Galectin-5, Galectin-7, Galectin-10, Galectin-11, Galectin-13, Galectin-14, and
Galectin-15 are categorized under the name “proto-type” galectins as they are
comprised of a single polypeptide chain that is able to dimerize; (2) Galectin-3
is categorized under the name “chimera-type” galectin as it consists of one
C-terminal CRD linked to an N-terminal peptide that is able to pentamerize; and
finally, (3) Galectin-4, Galectin-6, Galectin-8, Galectin-9 and Galectin-12 are cat-
egorized under the name “tandem repeat-type” galectins as they are composed of
two CRDs connected by a linker peptide [72–74]. Irrespective of their type, all
galectins have the ability to recognize and bind galactosyl residues without the
requirement for any other catalyst like cations [71]. Additionally, these lectins are
widely localized ranging from within the cytoplasmic and nuclear compartment to
the extracellular compartment where they modulate a myriad of biologic process
(for review see, [75]). Below, we will briefly discuss the role of galectins in tumo-
rigenesis by highlighting the role of each type of the galectin family.
3.3.2 Galectin-1
This protein was first discovered as a low molecular weight, β-galactoside-binding

soluble protein purified from the calf heart and lung [76]. It is a prototype galec-
tin with a single CRD that can form dimers and has been shown to bind to onco-
genic H-Ras to mediate Ras membrane anchorage and cell transformation, thus
CRD
CRD
Tandem repeat-type
galectin
Proto-type
galectin Chimera-type galectin
Fig. 3.1 Model structures of the different types of galectins. Galectins are divided into three
major types according to their structures. The prototype galectin contains one CRD and can exist
as a monomer or a dimer. The chimeric type galectin contains one CRD that is covalently linked
to a non-carbohydrate tail. The chimeric type of galectin, like galectin-3, can exist as a pentamer
and a monomer. Finally, the last type of galectin is the tandem repeat-type galectin that harbors
CRD domains that are covalently linked by a small peptide linker
demonstrating its role in tumor initiation and progression [77]. In addition to

tumor progression, in prostrate and ovarian cancer cells, overexpression of galec-
tin-1 and its resulting accumulation in the extracellular space resulted in increased
adhesion of these tumor cells to the extracellular matrix like fibronectin and
laminin-1 [78, 79]. However, whether this increased cell adhesion led to drug
resistance was not tested in these studies. Furthermore, galectin-1 was shown to be
upregulated in capillaries associated with prostrate tumor cells where it modulates
angiogenesis by mediating the interaction between prostrate carcinoma cells and
endothelial cells [80]. In contrast to above-stated studies, in colon cancer Colo201
cells, overexpression of galectin-1 in the cytoplasm of the cells is associated with
increased cell apoptosis [81]. Due to these opposing effects, depending on the
localization of galectin-1 and the tumor type, it is important to be cautious while
advocating the use of galectin-1-specific inhibitor for the treatment for cancer.
3.3.3 Galectin-3
This protein is a unique chimera-type galectin that has been associated with sev-
eral models of immune disease and cancer [82–84]. Jeon et al. [85] have shown
that galectin-3 can exert cytokine-like regulatory actions by the activation of the
JAK2-STAT1/3/5 pathway in an IFN-γ-independent manner. Indeed, the activa-

tion of the JAK-STAT pathway required that glial cells secrete galectin-3 in the
extracellular compartment under inflammatory conditions, thus demonstrating
galectin-3 to be an endogenous danger signaling molecule leading to inflammatory
events [85]. In addition to inflammation, galectin-3 also enhances proliferation and
angiogenesis of endothelial cells differentiated from MSCs, thereby showing an
ability to promote tumor progression [86]. In MM, inhibiting galectin-3 with the
use of a modified citrus pectin called GCS-100 resulted in specific induction of
cell death in multiple myeloma (MM) cells resistant to conventional and borte-
zomib therapies [87]. The cell death induced by GCS-100 was impressive since it
could overcome growth and survival benefits conferred by the BM microenviron-
ment. Similarly, Streetly et al. have further confirmed the conclusion of the above-
mentioned study by demonstrating that GCS-100 was able to block the activation
of AKT after cytokine stimulation in MM cells and led to caspase-8 and cas-
pase-9 dependent cell death [88]. These preclinical studies provide a rationale for
the clinical evaluation of GCS-100 to kill MM cell and to overcome drug resist-
ance. Galectin-3 has also been shown to protect CML cells from apoptotic stimuli
through stabilization of anti-apoptotic Bcl-2 family proteins [89]. Interestingly,
galectin-3 was shown to be specifically induced in HS-5 BM stromal cells when
these cells were co-cultured with various CML cell lines [90]. Secreted galectin-3
was shown to activate ERK and AKT, induce accumulation of Mcl-1, and promote
cell proliferation and resistance to BCR-ABL inhibitors and genotoxic agents in
CML cells. Also, an in vivo mice transplantation model demonstrated that galec-
tin-3 overexpression promoted long-term BM lodgement of CML, supporting the
hypothesis that galectin-3 may be a viable therapeutic target to help overcome the
BM microenvironment-mediated drug resistance in CML [90].
3.3.4 Galectin-9
This protein is the tandem repeat-type of galectin which, in contrast to galectin-3,

has been shown to have anticancer activity [91, 92]. Kuroda et al. [93] have dem-
onstrated that modified humanized galectin-9 inhibits proliferation of CML cells
in the nM range. Proliferation was inhibited due to apoptosis initiated by the acti-
vation of ATF3 and its downstream effector molecule Noxa, but not Bim [93].
Importantly, this galectin-9-mediated cell death was not influenced by the absence
of p53, overexpression of P-glycoprotein, and the presence of mutant T315I ABL
[93]. On the other hand, Kobayashi et al. [94] have shown that protease-resistant
humanized galectin-9 can induce apoptosis in MM cells by the activation of a cas-
pase-dependent pathway via JNK- and p38-activated proteins. The ability of galec-
tin-9 to induce cell death could be replicated in primary MM cells with very poor
disease prognosis and overt manifestation of drug resistance [94]. The above stud-
ies suggest that galectin-9 is a new therapeutic target for hematological malignan-
cies that may overcome resistance to conventional therapies.
3.4 Hypoxia and Drug Resistance
Hypoxia can occur at the site of the primary tumor due to a decreased oxygen
content found in the center of the tumor or at specialized niches found in meta-
static sites. For example, the bone marrow microenvironment is characteristically
hypoxic; direct measurement of oxygen levels has estimated the oxygen content
to be 1 to 2 % in the bone marrow [95]. Thus, hypoxia represents an initial selec-
tion pressure that contributes to necrotic cell death but also provides pressure for
adaptation and tumor progression. Indeed, exposure to hypoxia can induce resist-
ance in multiple tumor types including leukemia and lung cancer cell line models
[96, 97]. Moreover, in CML, hypoxic conditions that mimic the O2 content of the
bone marrow confer resistance to BCR-ABL inhibitors, suggesting that hypoxia
may contribute to failure to eradicate minimal residual disease [98]. Hypoxia
induces a myriad of effects on tumor cells including selection for p53 mutations
[99] and increased genomic instability due to increases in reactive oxygen spe-
cies and attenuation of DNA repair pathways [100]. Experimental findings suggest
that hypoxia can contribute to angiogenesis, invasiveness, metastasis as well as
immune suppression [101–103]. Many of the phenotypes associated with hypoxia
are the result of the induction of the hypoxia-inducible factor (HIF) family of tran-
scription factors. Three members constitute the family (HIF-1, HIF-2, and HIF-3).
HIF-1α is targeted for degradation by ubiquitination, which is regulated by the
E3 ligase von Hippel–Lindau (VHL) tumor suppressor. Under normal oxygen
levels, HIF-1α is hydroxylated on proline 402 and 564 by prolyl-4-hydroxylases
(PHDs). The reaction requires oxygen and 2-oxoglutarate as substrates [104, 105].
Thus, under normal oxygen concentration, HIF family members are hydroxylated
and subsequently targeted for ubquitination and degradation. In contrast, under
hypoxic conditions, HIF-1α is stabilized and can form a heterodimer with HIF-1β
and drive transcription of target genes [106].
A hypoxic environment allows for two independent targeting strategies: (1)
development of bioreductive prodrugs and (2) use of drugs that inhibits targets that
are specifically expressed under hypoxic conditions. Bioreductive prodrugs typi-
cally take advantage of redox cycling such that the levels of the prodrug radical
are kept low in oxic cells at the expense of generation of superoxide. Common
moieties that have the potential to be metabolized under hypoxic conditions
include nitro groups, quinones, aromatic N-oxides, aliphatic N-oxides, and transi-
tion metals. One example of a hypoxia-activated prodrug is TH-302. TH-302 is
nitrogen mustard based prodrug with promising activity in a phase I clinical trial
for advanced solid tumors [107]. Because the bone marrow is hypoxic and nitro-
gen mustards such as melphalan are a mainstay of therapy for myeloma which
homes to the bone, it would be of interest to test prodrugs in the context of tumors
which home or metastasize to the bone. The identification of pathways that are
activated upon hypoxia that contributes to cellular survival represents an additional
strategy for specifically targeting the hypoxic tumor microenvironment. Three
main pathways activated by hypoxia are the HIF family of transcription factors,
the unfolded protein response (UPR), and autophagy. Severe hypoxia disrupts
the formation of disulfide bond formation leading to misfolded proteins and acti-
vation of the UPR. Thus, targeting the UPR pathway is attractive for selectively
eliminating hypoxic tumors. One promising target in the UPR pathway is the ino-
sitol-requiring enzyme 1 or IRE1 [108]. Inhibitors of IRE1 are currently in devel-
opment and show activity in myeloma models which are tipped toward ER stress
due to the large production of monoclonal antibodies in these cancerous cells
[109, 110]. Another strategy is the use of proteasome inhibitors which also can
trigger additional stress on the UPR response, and correspondingly, bortezomib
shows activity in hypoxic tumor sites [111]. Thus, inhibition of the proteasome
exaggerates the UPR and tips the threshold toward cell death under hypoxic con-
ditions and may represent an attractive strategy for eliminating cells residing in
hypoxic microenvironments.
3.5 Molecules Involved in the Interaction of the Tumor Cell

with the Microenvironment
3.5.1 Integrins
Damiano et al. [112] reported in 1999 that adhesion of myeloma cells via α4β1
integrin was sufficient to cause resistance to structurally and mechanistically
diverse chemotherapeutic agents. Furthermore, they reported that selection for
acquired drug resistance selected for cells with increased expression of β1 and β7
integrin. This report suggested that cell adhesion could contribute to an adaptive
response for cells to evade cytotoxic insult. More recently, α4 integrin expression
was shown to be increased in ex vivo samples collected from relapsed myeloma
patients compared with specimens obtained from newly diagnosed myeloma
patients, albeit only a small sample size was reported [113]. VLA-4 integrin is
known to be important for homing, and more recent evidence indicates that VLA-
4/VCAM-1 interactions between osteoclasts and breast cancer cells drive the
cycle of bone destruction and tumor growth in metastatic models of breast can-
cer [114]. A similar finding was reported for myeloma where adhesion to stroma
induced MIP-1alpha and beta which in turn activated osteoclasts [115] and con-
tributed to further bone destruction and tumor growth. Collectively, these data
indicate that VLA-4 integrin is an attractive target for myeloma and perhaps
solid tumors which metastasize to the bone. Integrins are heterodimeric proteins
comprised of an α and β subunit. Eighteen α and 8 β subunits have been identi-
fied and complex to form 24 known αβ heterodimers. Integrins bind to a diverse
set of extracellular ligands which are found in the extracellular matrix. Integrins
do not contain intrinsic kinase activity; however, integrin ligation allows for the
formation of focal adhesions containing adaptor proteins critical for the activa-
tion of signaling complexes (see Fig. 3.2). Molecules contained within the focal
Fig. 3.2 Signal Transduction events triggered by clustering of integrins. Ligand binding induces

clustering of integrins and causes the aggregation of adaptor proteins like paxillin and talin,
which in turn recruits a large variety of signaling molecules including FAK/Pyk2, Src, vinculin,
and polymerized actin filaments. This complex then forms the platform for signal transduction
and crosstalk events. Some of the main signaling events include Src-JAK-STAT pathway acti-
vation; FAK-RAS-MEK-ERK pathway activation; FAK-RAS-NFKB pathway activation; FAK-
PI3 K-AKT pathway activation; and FAK-ILK pathway activation. All this leads to rapid down-
stream modulation of cell proliferation-inducing proteins, upregulation of anti-apoptotic proteins,
cell adhesion, and cell motility, which ultimately helps the cells to survive, divide, and resist the
actions of anti-neoplastic drugs
adhesions include integrins, FAK/Pyk2, Src, and several scaffold proteins includ-
ing talin, actin, and paxillin. Downstream signaling that is amplified following the
ligation of integrins includes activation of the MAPK-, Src- and AKT-mediated
survival pathways [116, 117]. Finally, integrin signaling is known to augment
growth factor– and cytokine-mediated signaling [27, 118]. Due to the importance
of integrins in sensing and transmitting external cues and contribution to survival,
migration, and growth, integrins remain an attractive target for cancer as well
as autoimmune diseases [32]. Integrins can be effectively targeted by utilizing
blocking antibodies and peptidomimetics which typically compete with consen-
sus ligand sequences such as RGD peptides. The development of small molecules
demonstrating specificity for integrins has been difficult due to inherent difficul-
ties of targeting protein–protein interactions with small molecules.
The structure of integrins has recently been elucidated utilizing crystallogra-
phy to inform structural models [119–121]. The α subunit is comprised of a short
cytoplasmic tail, a single-pass transmembrane domain, and two calf domains

immediately proximal to the transmembrane region which make up the lower
leg region. The upper leg region is followed by 7 repeating units which comprise
the β-propeller region. In nine of the α integrin subunits, an interactive domain
(I) is inserted between two and three of the α-helical repeating units found in the
β-propeller region. The interactive domain modulates the ligand-binding domain
and confers specificity for ligand binding. The β subunit has a longer cytoplas-
mic domain which is critical for recruitment of adaptor molecules and signaling.
The β subunit contains a transmembrane domain, four epidermal growth factor-
like domains which constitute the lower leg region and two hybrid domains
which lie on either side of the β-I domain. The N-terminus folds over exposing
the β-I domain and contributes to the formation of the upper leg region. Integrins
can reside on the cell surface in 3 distinct confirmations. A resting state results
in a bent confirmation and conceals the ligand-binding domain. An intermedi-
ate state has an extended confirmation, while a fully extended molecule is com-
petent to bind ligand. The confirmation of the integrin is regulated by a process
referred to as inside-out signaling (see Fig. 3.3). As the name infers, intracellu-
lar signaling is required to change the conformation of integrins, and thus, inside-
out signaling represents another potential point for pharmacological intervention.
Talin represents a central molecule for regulating integrin activation. Talin binds
to the β integrin subunit resulting in the tail separation of the α and β subunits
leading to conformational changes in the extracellular domain and extension of the
Fig. 3.3 Steps leading to the activation and clustering of integrins. In resting cells, the integrins
are in a bent, low-affinity state. However, once the cell is exposed to an environment with an
abundance of cytokines or growth factors, an inside-out activation pathway is triggered that cul-
minates in an extended, high-affinity state of integrins. The integrins are now receptive to ligand
binding, either in the form of adhesion to the extracellular matrix or adhesion to other cells
within its microenvironment. Ligand binding causes clustering of integrins, with polymerization
of actin, and induction of an outside-in activation cascade
external domain, allowing for ligand binding [122–124]. Talin is comprised of a

50-kDa N-terminal domain and a 220-kDa rod domain structure. Talin binds the
β1 integrin tail via the head domain (THD), resulting in the activation of integ-
rins [125]. Binding of talin to the β1 integrin tail is regulated by phosphorylation
at the NPXY motif. Phosphorylation of this motif results in an increased affinity
for Dok1, which acts as a competitive inhibitor of talin for access to the β1 inte-
grin tail [126]. The C-terminal rod domain binds to vinculin and actin participat-
ing in the cytoskeletal rearrangement that is a hallmark of integrin ligation [127].
In hematopoietic lineages, Rap1 facilitates the targeting of talin to the β1 integrin
tail. Rap1 is a small GTPase and is a member of the RAS superfamily. Rap1 can
be activated by extracellular signals including IL-3- and erythropoietin-mediated
signaling. Moreover, the activation of Rap1 leads to the activation of VLA-4 and
VLA-5 in hematopoietic cells [128]. Both VLA-4- and VLA-5-mediated adhesions
are known to confer a cell adhesion–mediated resistance phenotype or CAM-DR
in hematopoietic lineages [21, 112, 129]. Thus, targeting intracellular pathways,
albeit not specific due to the multitude of downstream signaling that typically are
modulated by GTPases such as Rap1, represents a potential point of intervention.
An attractive strategy for targeting specific integrins is the recent development
of humanized blocking antibodies and peptides which mimic the ligand and thus
represent competitive inhibitors. Natalizumab binds to the α4 subunit and blocks
adhesion of VLA-4 (α4β1)-mediated adhesion to VCAM-1 and fibronectin-medi-
ated and α4β7-mediated adhesion to fibronectin. Natalizumab is approved for the
treatment for multiple sclerosis [130] and Crohn’s disease [131]. However, this
agent is associated in rare instances with progressive multifocal leukoencephalop-
athy, which can be lethal and is thought to be related to the immunosuppressive
effects of the antibody. Moreover, in murine models of myeloma and AML, VLA-4
antibodies have demonstrated good anti-tumor activity [132, 133], making it attrac-
tive for pursuing in cancer indications for diseases that home or metastasize to the
bone. Peptides represent another potential strategy for blocking integrin–matrix
interactions. The RGD sequence is a consensus recognition site found in multiple
extracellular matrix ligands [134]. Disintegrins are RGD peptides initially found in
snake venom [135]. RGD-containing disintegrins have been found to inhibit tumor
growth and angiogenesis [136]. However, natural product–derived peptides are lim-
ited by their relatively large size and poor bioavailability. Efforts to design more
drug-like candidates have included incorporation of D-amino acids to decrease
proteolytic cleavage of the peptide. To this end, Cress and colleagues screened all
D-amino acid peptide library using cell adhesion of prostate cells overexpressing
α6β1 integrin. These investigators identified the sequence KIKMVISWKG and
showed that this peptide allows for adhesion, binds α3 and α6 integrin, and blocked
migration and invasion [137–139]. More recently, Nair et al. showed that this same
peptide induced necrotic cell death in myeloma using both in vitro and in vivo
models [140]. Another strategy for increasing the drug-like properties of peptides
is via cyclization strategies. Linear peptides can exist in multiple conformations in
solution, and cyclization allows for constraining the peptide in one conformation.
Cilengitide is an RGD cyclic peptide c(RGDf(nMe)V) which binds αvβ3/αvβ5
integrin and competes with RGD sequences found in components of the matrix
[141]. RGD sequences are contained in multiple ligands, yet integrins demon-
strate specificity which cannot be entirely explained by flanking sequences. Thus,
it was postulated that secondary structure was critical for sequence recognition.
Cyclization represents a strategy to constrain the recognition sequence in a well-
defined secondary structure. Kessler and colleagues performed extensive structure
activity relationship prior to knowledge of crystal structures, and thus, they applied
the following strategies for optimizing RGD sequences for inhibition of αVβ3-
mediated adhesion. They reduced the conformation space using cyclization strat-
egies, spatial screening of peptides, and N-methyl scanning [141]. N-methylation
of peptide bonds often increases their metabolic stability and bioavailability [142].
Merck-Serona has published on the phase II trial and just completed the accrual of
a phase III clinical trial with cilengitide for the treatment for glioblastoma [143].
Preclinically, cilengitide was shown to have activity by inhibiting growth directly,
as well as inhibiting invasion and angiogenesis [144]. These studies will provide
proof-of-principle whether targeting cell adhesion receptors with cyclized peptides
is a viable strategy for drug discovery and development in oncology.
3.5.2 CD44
CD44 is a family of cell surface receptors that similar to integrins mediate cell–
matrix adhesion. One gene encodes the CD44 family, but alternative splicing leads
to multiple variants which are thought to be, in part, responsible for the apparent
diverse functions attributed to this single-pass membrane-spanning cell surface pro-
tein. CD44 facilitates cell adhesion and metastasis, augments growth factor signal-
ing, and expression of CD44 protects against hypoxia-induced lung injury
[145–155]. CD44 can be shed from the cell membrane and soluble CD44 is associ-
ated with advanced disease in chronic lymphocytic leukemia [156–160].
Hyaluronic acid (HA) is the most predominant ligand for CD44. HA is an abundant
polysaccharide found in extracellular matrixes. However, CD44 can also bind
fibronectin and osteopontin [161]. The structure of CD44 consists of a constant
region spanning the first five exons which defines the HA-binding domain and is
found in all splice variants. Exon 7 through 15 (corresponding to v2–v10) is the
variable region of the molecule. This region is localized on the external domain and
adds to the stem region of CD44. The highly conserved cytoplasmic region has part
of exon 18 combined with exon 19 and 20 [162]. CD44 variant expression associ-
ates with markers of cancer stem cells and is a poor prognostic indicator for many
cancer types including multiple myeloma [163–165]. Paradoxically, the activation
of CD44, depending on the cell context, is associated with both cell death and sur-
vival [64, 166–168]. Moreover, CD44v associates with α4β1 integrin in CLL and
positively regulates adhesion of myeloma cells to stroma and extracellular matrices
[148, 165, 169]. Finally, it is known that CD44 associates with VLA-4 integrin in
T cells and CLL [169, 170]. Along with VLA-4, CD44 is considered the major
homing receptor for hematopoietic cells for trafficking to the bone marrow com-
partment [171] and thus, similar to VLA-4, may play a role in the recruitment of
accessory cells to the tumor site as well as metastasis of the tumor. CD44 has an
N-terminal ligand-binding ectodomain containing the Link module; comprised of
three intradisulfide bonds, a stalk region containing one or multiple variant exons, a
transmembrane domain, and a short C-terminal tail which is important for cytoskel-
etal attachment and signal transduction. CD44 has a highly conserved amino acid
sequence among mammalian and avian species sharing between 47–93 % sequence
homology with humans and an almost identical homology in the Link module,
transmembrane, and cytoplasmic tail regions [172]. The CD44 receptor also con-
tains many posttranslational modifications. It has been estimated that 25–40 % of
CD44 molecules are phosphorylated at Ser325 and that CD44 is constitutively
phosphorylated at this site in cultured cells [172]. While the exact role of Ser325
phosphorylation is unclear, mutations at this site have been shown to inhibit HA
binding and cell migration, as well as modulating the interaction of CD44 with the
ERM (ezrin, radixin, moesin) family of proteins [173]. CD44 is also palmitoylated
at Cys286 and Cys295, and while the exact roles these modifications play are not
yet fully understood, it appears that palmitoylation enhances the association
between CD44 and ankyrin and may also induce clustering of CD44 into mem-
brane subdomains [172]. The CD44 receptor is also highly glycosylated and linked
to chondroitin sulfate; with at least 5 conserved N-glycosylation sites in the ectodo-
main as well as being highly O-glycosylated in the extracellular region, both of
which have been shown to affect ligand-binding affinity [173]. The crystal structure
of HA bound to murine CD44 has been solved, and it has been observed that CD44
adopts two conformational binding states and this binding is the result of hydrogen
bonding [174]. Further studies by nuclear magnetic resonance (NMR) and surface
plasmon resonance (SPR) have further elucidated the two binding configurations of
the CD44 receptor, designated as ordered (O) and partial disordered (PO) [175,
176]. It has been shown that the binding of HA oligomers at an allosteric site on the
ectodomain leads to conformational change in the Link module and switches the
module from the O to PO configuration. Furthermore, while the minimum binding
requires an HA hexamer, recent studies have demonstrated that there is a positive
correlation with the length of HA and binding affinity. It has long been speculated
that this is the result of multiple CD44 molecules binding to one HA polysaccha-
ride. This was recently confirmed by Wolny et al. using an artificial membrane sys-
tem in which they demonstrated that multiple CD44 molecules can bind a single
HA polysaccharide through a multivalent nature [176]. While the crystal structures
of CD44 bound to other ligands have yet to be solved, it is postulated that other
ligands bind in the ectodomain adjacent to the Link module. The CD44 transcript
contains twenty exons that give rise to seventeen known isoforms through alterna-
tive splicing and posttranslational modifications. The most ubiquitously expressed
isoform is the standard form (CD44s), which is comprised of exons 1–5 and 16–20
[172]. CD44 expression is regulated through receptor-mediated endocytosis as well
as the proteolytic cleavage of the extracellular domain by various matrix metallo-
proteinases (MMPs). CD44 activation through ligand binding has been shown to
have diverse cellular effects. CD44s activation has been shown to activate apoptosis
in Jurkat cell lines, whereas CD44v induction has been suggested to do the con-
verse via sequestering FasR [147]. Similar to integrins, CD44 has no intrinsic
kinase activity; however, it gains access to kinases such as focal adhesion kinase
(FAK), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3K) through
adaptor proteins such as the ERM family [177]. In addition to CD44 gaining access
to kinases through adapter proteins, it has been demonstrated that CD44 can also
gain access to other signaling complexes through association with other adhesion
receptors, most notably α4 integrin in lymphocyte extravasation as well as cell
growth and proliferation signaling through the human epidermal growth factor
receptor 2 (HER2), c-Met, and VEGF [178–180]. CD44-deficient mice exhibit nor-
mal development; however, the mice are resistant to T-cell activation-induced cell
death (AICD) [181]. Recently, several in vitro studies targeting CD44 with mono-
clonal antibodies alone and in combination with low molecular weight HA have
shown induction of apoptosis in myeloid cells as well as a novel caspase-independ-
ent pathway in erythroleukemia cells. Together, these data suggest that agonistic
signals may actually activate cell death in tumor cells. More specifically, one study
was able to eradicate human acute myeloid leukemia stem cells in non-diabetic
severe combined immune-deficient (SCID) mice by targeting CD44 receptor with a
CD44 monoclonal antibody that recognized all isoforms and was characterized as
an activating antibody [64]. There has also been an interest in targeting CD44 via
peptidomimetics. In one study, a group using a CD44v6-derived peptide has indi-
cated a role of CD44 in VEGF signaling and angiogenesis [182]. A second peptide
named A6 (acetyl-KPSSPPEE-amino) has been shown to activate CD44 and inhibit
migration and metastasis in CD44-expressing cells [183]. Furthermore, this peptide
has shown promise in phase I and phase II clinical trials in patients with ovarian
cancer. Another molecule that has shown promise in early stage clinical trials is the
immunoconjugate bivatuzumab, a human anti-CD44v6 monoclonal antibody cou-
pled with a benzoansamacrolide moiety, which has been tested in head and neck
squamous cell carcinomas [184]. While these molecules have shown promise in the
clinic, targeting CD44 will remain a difficult task until the diverse expression and
signaling of the many CD44 isoforms is more fully understood in both healthy and
diseased cell lineages.
3.6 Signaling Pathways Activated Due to the Interaction

of the Tumor Cell with the Microenvironment
3.6.1 FAK/Pyk2
Due to the existence of multiple cell adhesion receptors and potential r edundancy, it
is attractive to speculate that downstream targets will be more efficacious for target-
ing cell adhesion mediated drug resistance or CAM-DR. Immediately, downstream
of multiple cell adhesion receptors, as well as cytokine and growth factor recep-
tors, is FAK and/or Pyk2 which is a FAK homolog. FAK is a non-receptor tyrosine
kinase, is ubiquitously expressed, and localizes to focal adhesions. FAK contains
three major structural domains: (1) an N-terminal FERM domain, (2) a tyrosine
kinase catalytic domain, and (3) a C-terminal focal adhesion–targeting domain
[185]. The activation of FAK requires autophosphorylation at Y397 following the
integrin ligation [186]. Phosphorylation of FAK leads to the recruitment and acti-
vation of Src family members as well as other components of the focal adhesion
including p130Cas and paxillin [187–189]. The activation of FAK leads to the inte-
gration and spatial organization of signaling pathways including activation of AKT,
Src, and the MAPK pathway. Pyk2 is a closely related homolog to FAK, with a
more restricted tissue distribution as expression is limited predominately to hemat-
opoietic cells, vascular smooth muscle, endothelium, spleen, kidney, and the cen-
tral nervous system [190]. Although Pyk2 and FAK are functionally related, they
are not redundant with respect to the phenotype observed in the respective knock-
out mice. FAK knockout mice are embryonic lethal due to defects in the axial
mesodermal tissue and cardiovascular system [191]. In contrast, Pyk2 knockout
mice develop normally, but do exhibit defective macrophage migration and have
increased bone density [192, 193]. PF-562,271 is a promising dual FAK/Pyk2
inhibitor being developed by Pfizer. This compound competes for the ATP-binding
domain and is a reversible inhibitor of FAK and Pyk2 catalytic activity. This com-
pound has shown anti-tumor activity in multiple in vivo xenograft tumor models
[194]. Moreover, in models of lytic bone disease involving implantation of breast
cancer cells into the tibia, investigators have shown that the FAK/Pyk2 inhibitor
reduced the tumor burden and lytic lesions [195]. This is consistent with data show-
ing that Pyk2−/− knockout mice show increased bone mass, indicating that the inhi-
bition of Pyk2 may be helpful for diseases such as myeloma, breast, and lung that
either home or metastasize to the bone and induce lytic lesions [193]. Recently, a
phase 1 clinical trial was reported that showed the compound was well tolerated,
and 12 % of patients showed prolonged stable disease for six or more cycles [196].
3.7 Conclusion
Historically, drug development has focused on developing strategies that will tar-
get the Achilles heel of tumors. Strategies with a track record of success in the
clinic include targeting rapidly dividing cells with DNA-damaging agents or radia-
tion. More recently, focus has centered on targeting oncogene addiction intrinsic to
the tumor cells. However, emerging evidence indicates tumors are very efficient at
using opportunistic signals provided by the microenvironment, and in fact, current
therapies may select for cells more fit to utilize survival signals contained within
the microenvironment allowing for survival during exposure to therapy. The chal-
lenge with targeting the microenvironment will be to a) determine redundancy
and delineate whether multiple targets will need to be inhibited and b) determine
patient variability contained within the tumor microenvironment and whether a
personalized medicine approach will be required for targeting the tumor micro-
environment. Targeting the microenvironment will present challenges due to the
complexity and the inherent ability of tumors to provide adaptive signals that con-
tinue to inform the surrounding niche to favor tumor progression in a dynamic
rather than static fashion. However, it is clear that target validation and drug dis-
covery will need to consider both the intrinsic oncogenic signals and the tumor
microenvironment for increasing the efficacy of standard therapy in cancer indica-
tions that are currently incurable.
Acknowledgments We are grateful to Deepa G Rathod for her assistance in preparation of the
tables & figures.
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Chapter 4
The Role of Autophagy in Drug Resistance
and Potential for Therapeutic Targeting
Reshma Rangwala and Ravi Amaravadi
Abstract Autophagy is a cellular survival mechanism influenced by a wide vari-

ety of intracellular and extracellular stresses including energy and oxygen depri-
vation, signaling aberrancies, ER stress, DNA damage, systemic cancer therapies,
and radiotherapies. There is growing evidence that it may potentiate cancer sur-
vival. A number of clinical trials have been launched using autophagy inhibition
in combination with standard cancer therapeutics. The information gleaned from
these as well as ongoing in vitro and in vivo studies will allow us to better under-
stand the role of autophagy in cancer.
4.1 Introduction
Cancer cells are faced with multiple metabolic and therapeutic stresses and rely
on intracellular stress response systems for survival. One such stress response that
can be activated by nutrient deprivation, hypoxia, intracellular pathogens, chemo-
therapy, targeted therapies, and/or radiotherapy is autophagy. Autophagy is a term
derived from the Greek word, “to eat” (-phag, y) “oneself” (auto). The term was
first coined to describe vesicular structures noted on electron microscopy (EM)
that contained cytoplasmic contents. Subsequent studies demonstrated that these
contents consisted of organelles and proteins that were in various stages of catabo-
lism [1, 2]. Autophagy is the only mechanism that eukaryotic cells possess that
allows for the bulk degradation of intracellular organelles, which are often dam-
aged and serve as a liability in stressed cells. Autophagy consists of multiple
related vesicular trafficking programs within the cancer cell and is coordinated by
a complex interplay between dedicated enzymes, cellular membranes, cytoskele-
ton, and motor proteins.
Initially, autophagy was described as type II programmed cell death, and “self-
eating”, and, if persistent, can result in exhaustion of all intracellular resources,
R. Rangwala · R. Amaravadi (*)

Abramson Cancer Center and Department of Medicine, Perelman School of Medicine, University
of Pennsylvania, 16 Penn Tower, 3400 Spruce Street, Philadelphia, PA 19104, USA

88 R. Rangwala and R. Amaravadi
and terminal starvation. However, unlike apoptosis and necrosis, autophagy is

reversible once initiated and occurs at a basal level in all eukaryotic cells. In can-
cer cells, autophagy can be induced to even higher levels than in surrounding nor-
mal tissue. Autophagic catabolism serves a functional role in the stressed cell by
producing energy sources that can be used by the cell to generate ATP and build-
ing blocks that can be recycled to fuel further growth [3–6]. These characteris-
tics may explain how autophagy serves as a key survival mechanism in cancer
cells. Autophagy is controlled by growth factor–kinase signaling, the ER stress
response, the DNA damage response, and the immune system: systems that are
modulated by cancer therapies.
This chapter will first define autophagy, catalog the molecular machinery
required for functional autophagy, and detail the current tools used to measure
autophagy and to characterize the known molecular links between autophagy and
signaling pathways targeted by cancer therapies. The remainder of this chapter
will review the current understanding of how metabolic and therapeutic stresses
can induce autophagy, the evidence that supports autophagy’s role as a tumor sup-
pressor mechanism, and the evidence that supports autophagy’s role as a tumor
survival mechanism. Finally, this chapter will survey the current efforts to block
autophagy in cancer therapy and possible interactions between autophagy and
immune system.
4.2 Defining Autophagy
Three forms of autophagy have been identified, each defined on the basis by which
the lysosome obtains the material targeted for recycling. In “macroautophagy,”
(hereafter referred to as autophagy) a double-membrane structure defined as the
autophagosome, or autophagic vesicle, envelopes the cargo and then fuses with
the lysosome. In “microautophagy,” an invaginated lysosomal membrane engulfs
the cargo [7]. In “chaperone-mediated autophagy,” a chaperone protein deliv-
ers protein cargo directly to the LAMP2 receptor on lysosomal membranes [8].
Unlike the other two forms of autophagy, chaperone-mediated autophagy has been
characterized in higher eukaryotes but not in yeast.
In addition to these described forms of autophagy, there is an increasing aware-
ness of the distinct roles of “basal autophagy” and “stress-induced autophagy”.
Basal autophagy likely plays an essential homeostatic role in removing and recy-
cling damaged parts, cellular metabolism and may also play a role in atypical
protein secretion processes [9]. Basal autophagy may be regulated entirely at the
posttranslational level in order to maintain the integrity of cellular constituents,
while stress-induced autophagy may involve the regulatory input from pathways
described below.
While autophagy has been generally considered a non-selective degradative
process, selective autophagy has been described and includes mitophagy (mito-
chondria), ribophagy (ribosomes), pexophagy (peroxisomes), and reticulophagy
4 The Role of Autophagy in Drug Resistance 89
(endoplasmic reticulum) [10–13]. The role of these multiple forms of autophagy in

benign and malignant conditions is currently being explored.
Despite the emerging appreciation for the heterogeneity of autophagy, the com-
ponents of autophagy are evolutionarily conserved and have been found to play
key roles in physiological and pathologic conditions [14], including cancer [15]. In
yeast, autophagy together with the CVT pathway, maintains survival when extra-
cellular nutrients are sparse [16]. Much of our understanding of autophagy comes
from studying deletion mutants in yeast (see Table 4.1). Key studies performed in
Caenorhabditis elegans [17] and drosophila [18] have added to our understanding
of autophagy’s role in multicellular organisms.
Mechanistically, autophagy is initiated by the enclosure of cytosolic proteins
and organelles. Closure of the phagophore isolates the cytosolic contents result-
ing in the formation of double-membrane-bound autophagosomes [15]. The later
stages of autophagy are defined by the fusion of the autophagosomes with the lys-
osomes to form autolysosomes. The engulfed contents are digested by acid hydro-
lases, and the resultant breakdown products, including amino acids, sugars, and
lipids, are shuttled back to the cytoplasm via lysosomal membrane permeases for
reuse [15, 19].
4.3 The Core Machinery Required for Autophagy
A complex set of autophagy proteins and autophagy-associated proteins regulate

the formation of the autophagosome, its subsequent fusion with the lysosome,
and the degradation and recycling of cellular components [4–6] including class
III phosphatidylinositol-3-kinase (PI3K). The core autophagy protein complexes
consist largely of ATG genes. Currently, there are a number of mammalian ATG
genes that have been characterized, with active, ongoing research to identify oth-
ers. These genes include positive and negative regulators of autophagy and have
many functions including kinase activity, ubiquitination, lipidation, conjugation,
recycling, and release [20–22]. Many of these genes are part of “core” complexes
that contribute to the autophagic process. This process can be further classified
into five steps: induction, vesicle nucleation, vesicle expansion, cargo recruitment,
and autolysosome formation (see Table 4.1).
4.3.1 The Five Steps in the Autophagic Process
4.3.1.1 Step 1: Induction
In yeast, induction is initiated by the activation of the Atg1 complex, which

includes Atg1, Atg13, and Atg17-Atg31-Atg29 subcomplex [23]. The mammalian
Atg1 complex, also known as the ULK complex, is composed of the mammalian
Atg1 homolog Unc-51-like kinases 1 or 2 (ULK1 and ULK2, respectively), the

mammalian autophagy-related 13 homolog (Atg13), which is a putative coun-
terpart of yeast Atg17, RB1-inducible coiled-coil 1 (RB1CC1, also known as
FIP200), and Atg101, an Atg13-binding protein. ULK1 is phosphorylated at
specific serine residues by both mTORC1 [24, 25] and AMPK1 (AMP-activated
protein kinase) (see below), resulting in the disinhibition of the ULK com-
plex activity. It is still unclear what the specific substrate of the ULK complex
is that gives rise to vesicle nucleation. In addition, there is some evidence that
autophagy can be activated in the absence of ULK1 and ULK2 in response to cer-
tain stimuli [26].
4.3.1.2 Step 2: Vesicle Nucleation
Vesicle nucleation is the initial process by which proteins and lipids are recruited
for autophagosome formation. Studies indicate that the lipid membrane compo-
nent of the autophagosome may be derived in whole or from parts of the mito-
chondria, the endoplasmic reticulum, the plasma membrane or the nuclear
membrane [27–29]. As further evidence of this, Atg9 has been shown to traffic
between the trans-Golgi network, endosomes, and autophagosome precursors
[30], implying that the membrane may be derived from the above precursors.
Finally, electron tomography has demonstrated connections between the ER and
the autophagosomal membranes indicating that the ER cisternae associate with the
developing autophagosomes [31, 32].
Vesicle nucleation starts with the recruitment of Atg proteins to the phagophore
assembly site (PAS). While the exact mechanism of this step is unclear, activation
of a phosphatidylinositol 3-kinase (PtdIns3K) complex is necessary. Generation
of phosphoinositide signals on the surface of source membranes is accomplished
by various protein complexes that include the class III phosphoinositide 3-kinase
(PI3K), Vps34, and Beclin1 [33], which further complexes with UVRAG and
Bif-1 and thus activates autophagy [34]. Association with Rubicon inhibits traf-
ficking of autophagic vesicles [35]. These findings implicate Vps34/Beclin1 com-
plexes in early and late roles in autophagic flux.
4.3.1.3 Step 3: Vesicle Expansion
Formation of the autophagosome is a de novo process. Membranes at the PAS

expand and subsequently close to isolate the cytosolic cargo. The two ubiquitin-
like conjugation systems, Atg8 and Atg12, are involved in vesicle expansion and
completion. Atg8 is conjugated to the lipid phosphatidylethanolamine (PE) to form
Atg8-PE, also known as LC3, while Atg12 is conjugated to Atg5. MAP1LC3 (LC3),
the mammalian Atg8 homolog, is a key molecule used in assessing the autophagic
process. Four mammalian Atg8 homologs have been identified: microtubule-associ-
ated protein 1 light chain 3 (LC3/MAP1–LC3/LC3B), GABAA receptor-associated
Table 4.1 Yeast autophagy genes and mammalian homologs and their respective functions
Yeast Mammal Function References
Atg1 complex Induction
Atg1 Ulk1 and Ulk 2 Serine/threonine [24, 25]
protein kinase
Atg13 Atg13 Unknown [24, 25]
Atg101 Atg13-binding protein [15]
Atg17 FIP200 (RB1CC1) Modulates response of [24]
autophagy
Atg24 (Snx4) PtdIns(3)P-binding protein [200]
PtdIns3K complex Vesicle nucleation

Atg6 (Vps30) Beclin1 Involved in activating [33]
autophagy
Atg14 Atg14L Component of the class III [34, 35]
PtdIns3K complex
Vps34 PIK3C3 Kinase [33]
Vps15 PIK3R4 Component of the class III [33]
PtdIns3K complex
Vps38 UVRAG Multiple; involved in [34]
activation and maturation
steps of autophagy
Atg18 WIPI proteins Binds PtdIns(3)P and [201]
PtdIns(3,5)P2
Atg21 WIPI proteins Binds PtdIns(3)P and [202]
PtdIns(3,5)P2
Atg8 MAPLC3, Ubiquitin-like conjugation [36, 53]
GABARAP, system, involved in
GATE-16, vesicle expansion
mATG8L
Atg3 Atg3 Ubiquitin-conjugating [40]
enzyme (E2) analog,
conjugates Atg8 to/LC3
to PE
Atg4 Atg4A Cysteine protease that [203, 204]
Atg4B processes Atg8/LC3,
Atg4C also removes PE from
Atg4D Atg8/LC3
Atg5 Atg5 Component of the Atg12- [205]
Atg5-Atg16 complex,
serves as a E3 ligase for
Atg8/LC3 conjugation
Atg7 Atg7 Ubiquitin-activating (E1) [206]
enzyme homes, activates
Atg8/LC3 and Atg12
Atg12 Atg12 Ubiquitin-like protein that [205]
modifies an internal lysine
of Atg5
Atg16 Atg16L1 Component of the Atg12- [207]
Atg16L2 Atg5-Atg16 complex
(Continued)
Table 4.1 (Continued)
Yeast Mammal Function References
Atg9 mAtg9A Transmembrane protein, [30]
serves as a lipid carrier
Ypt1 Rab1 Required for correct [208]
localization of Atg8 to
the PAS; in mammals,
Rab1 is also required for
autophagosome formation
Ypt7 Rab7 Small GTP-binding protein, [209]
facilitates transport from
early to late endosomes
and from late endosomes
to lysosomes, facilitates
clearance of autophagic
compartments
Sec18 NSF ATPase responsible for [42]
SNARE disassembly
Atg Autophagy related, ULK Unc51-like kinase, FIP200 focal adhesion kinase (FAK) family–
interacting protein of 200 kDa, RB1CC1 retinoblastoma 1-inducible coiled-coil 1, Beclin1 Bcl-2
interacting myosin/moesin-like coiled-coil protein 1, Vps vacuolar protein sorting, UVRAG UV
irradiation resistance-associated gene, WIPI WD repeat protein interacting with phosphoinositides,
Barkor Beclin1-associated autophagy-related key regulator, GATE-16 Golgi-associated ATPase
enhancer of 16 kDa/GABARAPL2, GABARAP gamma-aminobutyric acid receptor-associated
protein, LC3 microtubule-associated protein 1 light chain 3, TSC1/2 tuberous sclerosis complex ½,
Ypt yeast protein, PE phosphatidylethanolamine
protein (GABARAP), Golgi-associated ATPase enhancer of 16 kDa (GATE-16),

and mAtg8L [36]. During the formation of the autophagosome, LC3 is conjugated
to PE [36, 37]. Cleavage of proLC3 by the cysteine protease and redox sensor, Atg4
[38], leads to the formation of cytoplasmic LC3-I. A ubiquitin-like protein conju-
gation cascade involving an E1-like enzyme (Atg7) and E2-like enzyme (Atg3)
is responsible for conjugation of LC3 to PE. On gel electrophoresis, this lipidated
form migrates differently than the cytosolic form of LC3 (LC3-I) and is referred to
as LC3-II. Once AV are formed and marked by lipidated LC3, LC3 is cleaved from
PE by Atg4 and LC3 is recycled, a process which has been shown to be necessary
for effective autophagy [36, 39–41]. There are emerging data that SNARE proteins
are involved in the expansion of lipid membrane during autophagosome formation
by recruiting key autophagy components to the site of construction [42].
4.3.1.4 Step 4: Cargo Recruitment
LC3 not only plays a direct role in vesicle nucleation, but also plays a role in the
recruitment of cargo into the developing autophagic vesicle. p62/SQSTM1 has been
shown to recruit aggregated proteins to autophagic vesicles [43], while NBR and
NIX are responsible for the recruitments of organelles [44]. NBR and p62 contain
ubiquitin-binding domains and LC3-binding domains. These domains allow for the
tight sequestration of cargo by the surrounding LC3-containing membranes, while

limiting the amount of cytosol included [45]. The adaptor protein NIX recruits
mitochondria in a similar fashion to LC3-containing membranes [46]. Evidence
indicates that active recruitment of cargo into the AV promotes AV closure [47].
4.3.1.5 Step 5: Fusion with Lysosome, Lysosomal

Degradation, and Recycling
When the autophagosome is formed, it is transported on microtubules and fuses with

the lysosome to form an autolysosome. Rab GTPases play a role in vesicle matura-
tion and fusion with the lysosomes [48]. The endosomal sorting complexes required
for transport (ESCRT) machinery [49] and multi-vesicular bodies [50] contrib-
ute to vesicle-lysosomal fusion and provide cross talk between the core autophagy
machinery and the other components of endovesicular trafficking. Within the acidic
environment of the autophagolysosome, the cytosolic cargo is degraded via pH-
dependent hydrolases. The breakdown products of this degradation are released
back into the cytosol by permeases [22]. AV components not exposed to lysosomal
hydrolases are then recycled involving components of the outer membrane including
Atg9, Atg2, Atg18, and Atg21 [51]. An alternative path of release occurs when the
autophagosomes fuse with the plasma membrane and release their contents [52].
4.3.2 Redundancy, Non-Specificity, and the Non-Canonical

Autophagy Program
There is tremendous redundancy in the autophagic machinery, including five

human ULK homologs, six mammalian Atg8 homologs, and four mammalian
Atg4 homologs, some of which are likely to be functional [53–57]. In addition,
metabolic stress–induced autophagy can proceed in the absence of ULK1 and
ULK2 [26]. Furthermore, while the components described in each of the steps
above have been characterized in terms of discrete complexes critical to the
autophagic process, the individual proteins have also been implicated in other
protein–protein interactions [58], and these proteins have been implicated in pro-
cesses irrespective of their role in autophagy. For example, Beclin1, a component
of the mammalian class III PtdIns3K complex, has been implicated in angiogen-
esis [59], adaptation to stress, development, endocytosis, cytokinesis, immunity,
tumorigenesis, aging, and cell death independent of autophagy [60].
Evidence for a non-canonical autophagy program comes from the study of MEFs
deficient in Atg5 or Atg7. LC3-null vesicular structures formed in response to stress
are thought to be functional autophagosomes derived from the trans-Golgi network
and require the functional Rho-like GTPases, Rab9 [61]. These findings indicate that
multiple layers exist in the autophagic machinery that provide for parallel “autophagy-
like” pathways that may be recruited when canonical components are missing.
4.3.3 Unanswered Questions for the Autophagic Process
The significance of membrane composition and the fate of the membrane follow-
ing autolysosome formation are unanswered questions. Furthermore, studies are
needed to identify the mechanisms by which the non-selective versus selective
autophagy pathways are discriminated, the mechanism(s) by which organelles
and/or cytosolic debris are identified and marked for autophagy, and the regulatory
components that limit degradation.
4.4 Measuring Autophagy in Cancer Cells
There are multiple in vitro tools to measure autophagy [62]. LC3-I is localized in
the cytosol and LC3-II on the autophagosome surface; assessment of the ratios of
LC3-I to LC3-II using protein-based assays can be employed as a surrogate for
autophagosome induction and flux. Use of inhibitors to cathepsin B, H and L and
pepstatin A can inhibit lysosomal function [63] and further define these processes.
Lysosomal inhibition causes accumulation of autolysosomes and therefore LC3-II.
This finding can be recapitulated by using other lysosomotropic agents like chloro-
quine derivatives or bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Because
V-ATPase contributes to the acidification of other organelles, including endosomes,
bafilomycin A1 may show multiple off-target effects.
In situ imaging of LC3 dynamics can be visualized by fusing LC3 to a green
fluorescent probe (GFP). In the absence of autophagy induction, GFP-LC3 fluo-
rescence is diffuse, while discrete puncta are formed upon induction or blockade
of autophagic flux. It is important to note that in certain circumstances, GFP-LC3
can form puncta in cells independent of autophagy [64, 65] and that GFP fluores-
cence in lysosomes may occur even after degradation of the LC3 moiety. As such,
this method may overestimate the number of autophagosomes. A recent iteration of
the GFP-LC3 assay involves the mRFP-GFP-LC3 color change assay. This assay
capitalizes on differential pH stability between GFP and mRFP, and differential
pH of autophagosomes and lysosomes, the latter which has an acidic pH. In acidic
environments, the fluorescence of mRFP is stable, while GFP is decreased. Merged
mRFP-GFP-LC3 in autophagosomes is yellow, whereas it is red in autolysosomes
[66]. Cells lacking Atg3 continue to have impaired but present autophagosome for-
mation [67], indicating that LC3 lipidation is not the only process required for mem-
brane maturation and should not be the only marker used to assess induction/flux.
Adaptor molecules such as p62/SQSTM1 and NBR1 bind to ubiquitin-labeled
structures and recruit them inside autophagosomal. P62/SQSTM, which facilitates
protein aggregate clearance by autophagy, is degraded by autophagy and can be
found in cellular inclusion bodies that are presumably remnants of autophagic diges-
tion [43, 68]. Inhibition of autophagy leads to the accumulation of p62/SQSTMI.
EM is often viewed as the gold standard for qualitatively assessing autophagy.
Although EM measurements can be subjective, criteria have been defined for
assessing autophagic vesicles [69]. Thus, meaningful interpretations of AV accu-

mulation in peripheral blood cells and mouse and human tumor tissue can be
made.
4.5 Molecular Links Between Autophagy and Signaling

and Other Stress Pathways
4.5.1 PI3K/Akt/mTOR
The PI3K/AKT/mTOR signaling pathway is a major regulator of cell growth [70],

metabolism [71], survival [3], and autophagy (Fig. 4.1). The mTOR kinase activity
is divided among two protein complexes: mTORC1, which is responsible for con-
trol of protein translation and nutrient uptake and mTORC2, a regulator of AKT
signaling and the cytoskeleton. mTORC1 is an upstream negative regulator of the
ULK1-Atg13-FIP200-Atg101 kinase complex [10, 18]. Activation of mTORC1
complex is controlled by Rheb GTPase and the tuberous sclerosis complex (TSC)
TSC1/2. Growth factor signaling through the PI3K/AKT signaling controls
TSC1/2- dependent mTOR signaling.
Rapamycin, an inhibitor of mTOR, induces autophagy in an mTOR-dependent
manner in rat hepatocytes while relieving the inhibitory effect by amino acids
[72]. In response to nutrients, mTOR binds to the ULK1-Atg13-FIP200 complex
and phosphorylates ULK1 and Atg13. Binding and subsequent phosphorylation of
ULK1 by mTOR inhibit the kinase activity of ULK1 [24, 73]. Precise regulation
of autophagic degradation and flux by mTORC1 kinase activity occurs as a result
of the close proximity of these two pathways. mTORC1 is bound to the surface of
lysosomes and likely autophagolysosomes through the regulator complex [74].
Other nodes of the PI3K/AKT/mTOR pathway are critical too. Overexpression
of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K-dependent
lipid kinase activity, promotes autophagy [75], whereas targeted deletion of PTEN
in mice strongly inhibits autophagy [76]. AKT inhibition promotes autophagy; con-
stitutively, active AKT inhibits it [77]. Stabilization of TSC2, an inhibitor of mTOR
signaling, promotes autophagy and suppresses tumorigenesis [78]. Stimulation of
autophagy by mTOR has been described: mTOR and its downstream mediator S6
kinase 1 may positively regulate autophagy in 6-thioguanine-treated cells, possibly
through the negative feedback inhibition of Akt [79].
4.5.2 MAPK Signaling
Abnormal activation of oncogenic Ras is frequently noted in cancers and activates

autophagy. Expression of active Ras alone may induce a cellular senescence and
is insufficient to transform cells [80, 81]. Human ovarian surface epithelial cells
Fig. 4.1 Regulation of autophagy. Autophagy induction is influenced by a wide variety of inter-

secting signals internal and external to the cell comprising signaling transduction pathways;
nutrient and energy deprivation; systemic cancer therapies including DNA-damaging agents, hor-
monal therapies, and proteasomal inhibitors; radiotherapy; and ER stress. Emerging cancer thera-
pies including BRAF and PI3K/mTOR inhibition also induce autophagy
expressing HRASV12 undergo a caspase-independent cell death consistent with

autophagy [82]. This is prevented by knockdown of Beclin1 or Atg5. This occurs
because of upregulation of Beclin1 through the mitogen-activated protein kinase
kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade and by induc-
tion of Noxa (a BH3-only protein that displaces Beclin1 from Mcl-1). The unop-
posed Beclin1 promotes autophagy and leads to a type II cell death. However,
HRAS-mediated autophagy is cytoprotective under certain conditions. BNIP3
(Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a downstream effector of
the Ras/Raf/ERK pathway and inducer of autophagy. Overexpression of BNIP3 by
H-Ras(val12) competes with Beclin1 for binding to Bcl-2, induces autophagy, and
reduces cell proliferation in the first 48 h. But overexpression of Ras for two weeks
leads to enhanced cell proliferation and autophagy-mediated cell survival [83], indi-
cating that autophagy’s role in cell death or cell proliferation is context dependent
and influenced by the degree of autophagy, stage of tumor, and oncogenic mediators.
Autophagy has also been shown to promote survival in Ras-transformed cells
by induction of basal autophagy [84], potentially promoting the accumulation of
abnormal mitochondria, depleting metabolites for the Krebs cycle, and decreas-
ing oxygen consumption [85]. Data also indicate that hyperactivation of oncogenic
BRAF induces autophagy; subsequent inhibition and/or depletion of its down-
stream effectors, MEK or ERK, inhibits it [86].
4.5.3 Bcl-2
Anti-apoptotic members of the B-cell lymphoma (Bcl-2) family are BH domain–

containing proteins, which antagonize proapoptotic members including Bak and
Bax. In addition to the regulation of apoptosis, Bcl-2 also inhibits autophagy [87].
Bcl-2 complexes with Beclin1, which serves as a platform for the recruitment of
other proteins including UVRAG, Bif-1, and PI3KC3. Binding of Beclin1 to the
anti-apoptotic protein Bcl-2 decreases Beclin1 association with Vps34 PtdIns3K
activity, thus inhibiting autophagy [88]. The multiple resultant complexes are
involved in different stages of autophagosome formation and maturation [89].
4.5.4 AMPK
The liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) energy sensor

pathway is a critical direct and indirect regulator of autophagy by effecting prolif-
eration and apoptosis during metabolic stress [90]. Activation of this pathway fur-
ther stimulates autophagy through multiple pathways including stabilization of p27
[91] and inhibition of mTOR signaling [92–94]. AMPK signaling also integrates
the pro-autophagic effect of reactive oxygen species (ROS)-dependent activation
of ataxia telangiectasia mutated (ATM) [94]. Furthermore, TAK1 (transform-
ing growth factor—activated kinase 1) activates AMPK independently of LKB1,
which leads to the induction of autophagy in TRAIL (tumor necrosis factor–related
apoptosis-inducing ligand) treated epithelial cells [95]. Oxidative stress leads to a
calmodulin-dependent activation of the LKB1–AMPK signaling pathway [96].
4.6 Induction of Autophagy by Metabolic and Therapeutic

Stresses
Autophagy is required for and/or significantly impacts a wide variety of other cel-
lular processes including but not limited to apoptosis [97, 98], necrosis [99], cell
cycle control [100], immune modulation [101], angiogenesis [102], cell metabo-
lism [103], protein and organelle turnover [104], and cell survival [104]. These
internal and external processes are, in turn, mediated by nutrient, energy, oxygen,
and hormonal demands. Processing these diverse cellular inputs is critical for
maintaining cellular homeostasis. The primary role of autophagy is to protect cells
under stress conditions, such as starvation and hypoxia. During periods of nutri-
ent deprivation, autophagy degrades cytoplasmic materials to produce amino acids
and fatty acids that can be used to synthesize new proteins or ATP [105]. When
induction of autophagy exceeds homeostatic control, autophagic cell death, also
known as type II programmed cell death, is induced [105–108]. Understanding
how autophagic regulation is effected by glucose, oxygen, and amino acid stores
may provide further insight that will ultimately allow us to leverage autophagy to
our therapeutic advantage in treating disease states such as cancer.
4.6.1 The ER Stress Response and Autophagy
The ER stress response is a cellular homeostatic program initiated by an excess of

unfolded or misfolded client proteins in the ER lumen. Protein kinase RNA-like endo-
plasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6α), and ino-
sitol requiring transmembrane kinase and endonuclease 1 (IRE1) [109, 110] act as ER
stress sensors. Accumulation of excessive unfolded proteins and calcium depletion in
the ER results in their activation [111]. Previous work in yeast [112] and mammalian
cells [113, 114] has established that ER stress can activate autophagy. The contribu-
tion of IRE1α and ATF6α in the activation of autophagy has been less well studied but
may occur through the IRE1α-JNK/p38-p53-PUMA pathway [115]. The transcription
factor, ATF6α, has unknown influences on the activation of cytoprotective autophagy.
4.6.2 DNA Damage Response and Autophagy
The DNA damage response is a cellular stress response to double-strand DNA

breaks [116]. Although the mechanism that links DNA damage with autophagy
is not completely understood, the DNA damage response results in alterations in
other pathways including Bcl-2 family members [117], mTOR [118], and the ER
stress response [119]. Activation of p53 is a common event during DNA damage.
If the DNA damage is significant, p53 induces apoptosis. Nuclear p53 serves as
a transcriptional factor and stimulates autophagy through upregulation of DRAM
(damage-associated autophagy modulator), a lysosomal protein that may be criti-
cal for functional autophagy [120] and by promoting the dissociation of Beclin1-
Bcl-2 and inhibiting mTOR signaling [121, 122].
4.6.3 Effect of Glucose Deprivation on Autophagy
Incubation of murine breast cancer cells in media containing less than 5 mM of
glucose induces autophagy [123]. Glucose deprivation activates the AMP kinase
(AMPK) and TSC2, which, in turn, inhibits the GTPase Rheb and its downstream
effector mTORC1 [124, 125] by direct activation of the ULK1 complex [126]. Also,
oncogenic Ras and Myc can shunt energy production from oxidative phosphoryla-
tion to glycolysis [127] as a result of PI3K/Akt/mTOR pathway activation. The loss
of the tumor suppressor p53 promotes glycolysis through enhanced glucose trans-
port [128], enhanced phosphoglycerate mutase expression [129], and suppression of
the mitochondrial enzyme synthesis of cytochrome c oxidase 2 (SCO2) [130].
4.6.4 Hypoxia and Autophagy
Autophagy is induced in cells grown in hypoxic (1 % pO2) conditions (normoxia

corresponds to 21 % pO2) [131]. Furthermore, hypoxic tumors show markers
of increased autophagic activity [132] as well as increased generation of reac-
tive oxidation species (ROS) [133] a direct inducer of autophagy [134, 135]. The
effect of oxygen on the cell is mediated through the hypoxia-inducible factors
(HIF), which function as heterodimers. Expression of these heterodimers is con-
trolled by prolyl hydrolases. In the presence of oxygen, the HIF 1/2α subunits are
targeted for degradation; in low oxygen states or in the presence of reactive oxi-
dation species, HIF 1/2α subunits are stabilized and partner with a HIF-1β subu-
nit, which then enter the nucleus and regulate hypoxia-induced gene expression
[133]. Inhibition of autophagy sensitizes cells to hypoxia and ROS-induced cell
death [132, 136].
4.6.5 Amino Acid Stores and Autophagy
Amino acid deprivation is one of the most potent inducers of autophagy [137].
mTORC1 regulates both protein and cytoplasmic amino acid levels by regulating
the amino acid transporter expression and autophagy [138]. Furthermore, mTORC1
facilitates the transportation of leucine well as its degradation by autophagy and the
ubiquitin-proteasomal system [139, 140]. Additional regulation occurs as a result
of convergence of AMPK onto mTORC1. Ammonia, a glutaminolysis byproduct,
stimulates autophagy by a mechanism dependent on Atg5 [141, 142].
4.6.6 DNA-Damaging Agents: DNA Alkylating Agents

and Topoisomerase Inhibitors
Temozolomide, a DNA alkylating agent, is a potent inducer of autophagy [143] and

mitochondrial-induced apoptosis [144]. Other DNA alkylators including the nitro-
gen mustards, methylnitrosourea (MNU), or N-methyl-N-nitroso-N′-nitroguanidine
(MNNG) also induce autophagy. Reseau and colleagues demonstrated that pancreatic
organ explants treated with either agent had an increased number of autophagic vesi-
cles as compared to controls [145]. Autophagy induction has also been implicated as
a resistance mechanism to topoisomerase I and topoisomerase II inhibitors [146].
4.6.7 Hormonal Therapies: Tamoxifen
Modulation of estrogen receptor signaling is a common therapeutic maneuver in

the treatment for ER+ (estrogen receptor) breast cancer; however, de novo and
acquired resistance is common. It competes with endogenous estrogen in bind-
ing to the estrogen receptor and thus modulates the effects of ER complexes
and downstream gene transcription. Recent studies indicate that tamoxifen-
sensitive cells induce autophagy [147]. Although the exact mechanism is not
known, tamoxifen binds with high affinity to microsomal antiestrogen-binding
site (AEBS), a hetero-oligomeric complex involved in cholesterol metabolism.
Binding of tamoxifen with AEBS induces autophagy through resultant sterol accu-
mulation [148].
4.6.8 Proteasomal Inhibitors
Autophagy and the ubiquitin-proteasome system (UPS) are the two intracellu-
lar mechanisms by which proteins are degraded. Autophagy degrades long-lived,
cytosolic proteins and damaged organelles; while shorter-lived proteins are tar-
geted for catabolism by the UPS. Inhibition of the UPS by proteasomal inhibitors,
like bortezomib, induces autophagy by multiple pathways including activation
of HDAC6, activation of IRE1-JNK, stabilization of ATF4, inhibition of mTOR
signaling, and decreased proteasomal degradation of LC3. Not surprisingly, these
pathways show commonality with the regulatory mechanisms of ER stress. The
induction of autophagy has also been shown to mitigate the anti-tumor effects of
proteasome inhibition, including in multiple myeloma [149].
4.6.9 Histone Deacetylase Inhibitors
Acetylation and deacetylation of lysine residues are common epigenetic modula-

tions. Aberrant deacetylation of non-histone proteins has also been implicated in
a variety of pathologic states, including cancer [150]. Given its role in oncogen-
esis and tumor maintenance, HDAC inhibitors such as vorinostat (rINN) or suber-
oylanilide hydroxamic acid (SAHA) have been used clinically in the treatment
for cancers including cutaneous T-cell lymphomas. Growing evidence indicates
that the HDAC inhibitors induce autophagy dependent and independent of other
intersecting pathways including mTOR, tyrosine kinase induction as a result of
BCR-ABL fusion, and estrogen receptor–activated signaling [151–154] (see
Fig. 4.1).
4.7 Autophagy and the Immune System: Role as Both

Inducer and Repressor
Mutation in autophagy genes increases susceptibility to infection by intracel-

lular pathogens. Autophagic machinery interfaces with pathways including
immune responses and inflammation and involves direct interactions between
autophagic proteins and immune signaling molecules [155]. Cross talk between
these two seemingly disparate components has been identified in which
autophagic proteins and immune and inflammatory response can both induce
and suppress each other.
In mice, knockout of Atg5 in macrophages and neutrophils increases suscep-
tibility to infection with Listeria monocytogenes and the protozoan Toxoplasma
gondii [156]. Association between infection and tumorigenesis has been well
established especially in the cases of viruses. These include HPV which has been
associated with squamous cell carcinoma of the head and neck, esophagus, and
cervix; HHV-6 which has been associated with Kaposi’s sarcoma; hepatitis B
and C have been implicated in hepatocellular carcinoma; and EBV which has
been implicated in aggressive non-Hodgkin’s lymphoma. The association between
these infections and tumorigenesis may involve a viral strategy that blocks host
autophagy, including IFN-inducible RNA-activated eIF2α and AKT/TOR [157]. In
addition, several viral proteins target the core autophagy protein Beclin1 and its
association with herpes simplex virus 1 (HSV-1) neurovirulence factor ICP34.5,
the oncogenic herpesvirus-encoded viral BCL2-like proteins, the HIV accessory
protein Nef, and the influenza virus matrix protein 2 [157]. Tumorigenesis initi-
ated by these viruses is likely due to more than one mechanism; one can propose a
multi-pronged approach by which the virus affects the host autophagic process and
directly or indirectly induces tumorigenesis [158]. Michaud and colleagues report
that autophagy is necessary to elicit an immune response to chemotherapy through
the release of immunostimulatory extracellular ATP [159]. In contrast, Noman and
colleagues found that in human lung cancer cells hypoxia-induced autophagy pre-
vented T-cell-mediated cytotoxicity. Knockdown of autophagy genes restored sen-
sitivity to cytotoxic T cells [131]. In an immunocompetent model of melanoma,
the combination of genetic or pharmacological autophagy inhibition in combina-
tion with melanoma peptide vaccination resulted in synergistic tumor regression
compared with either treatment alone [131]. These results suggest the complex
interactions between tumor cell autophagy and the immune system may be context
dependent.
4.8 Arguments for a Tumor Suppressor and Tumor

Promoter Role for Autophagy in Cancer
4.8.1 Role as a Tumor Suppressor
Autophagy was initially considered a tumor suppressor mechanism. Beclin1 is a

phylogenetically conserved protein essential for autophagy and homolog of the
yeast gene apg6/vps30 [160]. Monoallelic deletion of beclin1 on chromosome
17q21 occurs in 40–75 % of ovarian, breast, and prostate cancers [161]. Mice
with monoallelic loss of beclin1 show accelerated development of spontaneous
malignant tumors [162, 163]. Similarly, a mouse with a mosaic deletion of Atg5
in all tissues was predisposed to the development of benign liver tumors, with no
detection of tumors in any other organs [164]. Multiple groups have demonstrated
that autophagy defects lead to increased DNA damage, genomic instability, and
tumor progression [165–167]. Defects in autophagy can lead to the elevation of
p62/SQSTMI, which has been shown to promote tumorigenesis through multiple
mechanisms including alterations in NF-κB signaling [68]. There is also growing
evidence that p62 binds to Keap1 with subsequent upregulation of the transcrip-
tion factor NRF2 whose function is to coordinate antioxidant defense [168, 169].
Furthermore, persistent activation of NRF2 is critical for anchorage-independent
growth of hepatocellular carcinoma cells in the context of p62/SQSTMI overex-
pression [170]. The tumor suppressor function of autophagy may also be attributed
to its role in cellular senescence. Autophagy is activated during oncogene-induced
senescence by oncogenic Ras. Inhibition of autophagy relieves this senescence
[171]. Given that cancer is associated commonly with activating mutations in
growth factor signaling pathways (such as PI3K/Akt/mTOR), overexpression
of anti-apoptotic proteins (such as BCL2), it would be predicted that autophagy
would be suppressed in most cancers [172–175]. However, recent evidence indi-
cates that constitutive Ras activation actually provokes elevated levels of basal
autophagy [84].
4.8.2 Role of Autophagy as a Tumor Promoter
The above data implicate autophagy as a guardian against genomic instability and
resultant tumorigenesis and therefore support a role for autophagy as a tumor sup-
pressor mechanism. However, the critical role of autophagy as a stress response in
tumor cells, suggests that human tumors growing within the harsh conditions of
the tumor microenvironment may in fact have high levels of autophagy. The ear-
liest data supporting this view come from experiments characterizing autophagy
in mouse models of Akt-driven solid tumors that are deficient in apoptosis [165].
Furthermore, these studies demonstrated that autophagy was induced in tumors
grown in hypoxic environments.
Additional evidence to support this tumor potentiating role is the high levels of
autophagy, as assessed by EM and LC3 immunohistochemistry, in both metastatic
melanoma and pancreas cancer [85]. LC3B was elevated in 84 % of cases of 20
advanced cancer histologies, and elevated levels of autophagy in breast cancer and
melanoma primary tumors correlated with lymph node metastases and poor sur-
vival [131].
Although monoallelic deletion of beclin has been found in a large subset of
common tumors such as breast and prostate cancer, the retained allele of beclin
in these tumors is always wild type. Beclin1 “deficient” cells usually have simi-
lar levels of autophagy induced by stresses as compared to beclin WT cells.
Beclin1 is bound tightly in a complex involving cytosolic p53. In cells derived
from beclin+/− mice, p53 levels were found to be lower than in cells derived from
beclin WT mice. This finding suggests that p53 deficiency and not autophagy
deficiency may explain why beclin-deficient mice develop malignancies, with
the caveat that Liu and colleagues did not differentiate cytosolic from nuclear
p53 [176]. Furthermore, no other mouse model of genetic autophagy deficiency
has produced true spontaneous malignancies. For example, FIP200−/− mice are
found to have impaired tumorigenesis [177]. In addition, inactivating somatic
mutations have yet to be reported in any other autophagy genes, save for poly-
morphisms in the ATG16L1 gene, which predispose patients to Crohn’s disease
[178] and not cancer. Finally, there is compelling evidence that Ras transforma-
tion produces a massive derangement in cancer cell metabolism that necessitates
high levels of autophagy to avoid oncogene-induced senescence or cell death [84].
Therefore, autophagy may be essential for tumorigenesis and cancer maintenance
and autophagy-deficient tumors may be rare.
4.8.3 Autophagy as Both Tumor Suppressor and Tumor

Potentiator
How can these dual, opposing roles be reconciled? As detailed above, autophagy
is regulated by multiple, intersecting pathways. Emerging evidence supports the
concept that its tumor suppressor versus tumor potentiator role may be pathway
specific and not necessarily mutually exclusive. Given the multiple mechanisms
by which autophagy is regulated, mutations in any of these signaling nodes will
affect cellular homeostasis and therefore the role of autophagy within that cel-
lular environment. This, in turn, effects whether autophagy modulates pro-sur-
vival versus pro-death pathways. Both the degree and duration of autophagy has
been proposed as an explanation as to the role in which autophagy functions. In
C. elegans, physiological levels of autophagy promote survival during nutri-
ent poor conditions, while either insufficient or excessive autophagy under these
same nutrient poor conditions leads to cell death [179]. In addition, excessive
autophagy likely compromises the integrity of the lysosomal membranes result-
ing in the release of cathepsins into the cytosol [180–182] and may underlie the
mechanism of such autophagic inducing drugs like temozolomide [183]. These

data implicate LMP as an effector pathway for autophagic-induced cell death, or
type II cell death [184]. However, the same stressors that activate autophagic cell
death in vitro can promote autophagic cell survival in vivo as a result of cytokines
and components of the extracellular matrix that are responsible for this switch in
cell fates [185]. Because of growing evidence in vivo that autophagy serves more
as a survival mechanism than a pathway for cell death, it has become a target for
novel therapeutic inhibitors.
4.9 Preclinical Evidence Supporting Autophagy Inhibition

as a Therapeutic Strategy in Cancer
The cytotoxic effects of blocking autophagy with chloroquine in cells that are
reliant on autophagy for survival was first demonstrated in apoptosis-defective
cells exposed to the stress of growth factor withdrawal [186]. Chloroquine also
augmented chemotherapy-induced tumor impairment in a model of Myc-induced
lymphoma [3]. Since these papers, numerous investigators have demonstrated that
combining chloroquine or siRNA against essential autophagy genes can augment
the efficacy of many existing and emerging cancer therapies in many different dis-
ease models (see selected examples in Table 4.2). Because effective autophagy
inhibition can be achieved in vivo with the anti-malarial drug chloroquine (CQ)
[3], and there is extensive experience with CQ derivatives for the treatment for
malaria [187], rheumatoid arthritis [188], and HIV [189], multiple trials have been
launched across a wide variety of tumor types.
4.10 Clinical Trials Involving Hydroxychloroquine

as a First-Generation Autophagy Inhibitor
A phase III trial in glioblastoma patients treated with radiation and carmustine with
or without daily CQ found a median overall survival of 24 and 11 months in CQ-
and placebo-treated patients, respectively [190]. While it was not adequately pow-
ered to detect a significant difference in survival, it established the safety of adding
low dose CQ to DNA alkylators. Its long half-life and low potency may limit its effi-
cacy as an autophagy inhibitor in patients [191]. To address these concerns, a phase
I/II trial of HCQ with temozolomide and radiation for glioblastoma patients was
launched and included pharmacodynamic (PD) and pharmacokinetic (PK) analyses.
PD evidence of HCQ dose–dependent autophagy inhibition was observed using an
EM assay on serial blood mononuclear cells [192]. The implications of autophagy
as a mechanism by which tumor growth is potentiated have significant clinical
implications: clinical effect observed with standard therapies may be improved
upon by the addition of drugs that inhibit autophagy. To date, only the chloroquine
Table 4.2 Preclinical studies that demonstrate augmented anti-tumor activity with autophagy

inhibition
Metabolic or therapeutic
stress Therapeutic class In vivo malignancy Reference
Growth factor limitation Metabolic stress Bax/bak-deficient cells [186]
P53 activation or MNNG DNA-damaging agent Lymphoma [3]
SAHA Histone deacetylase inhibitor CML [151]
Akt-1 Akt inhibitor Prostate [210]
Imatinib CKIT, Abl kinase inhibitor CML [211]
GIST [212]
Bortezomib Proteasome inhibitor Myeloma [213]
Hypoxia Metabolic stress Colon [214]
BEZ235 PI3 K/mTOR inhibitor Glioma [215]
Doxorubicin melphalan DNA-damaging agent Myeloma [216]
Leucine deprivation Metabolic Stress Melanoma [217]
derivatives (chloroquine and hydroxychloroquine) have been used in the clinical set-
ting. Since 2007, more than 20 trials have been launched involving HCQ. A com-
plete listing of ongoing and/or completed trials can be found at clinicaltrials.gov.
The knowledge gained from the PD, PK, and predictive biomarkers in these studies
will guide the development of more potent and specific autophagy inhibitors.
4.11 Emerging Inhibitors of Autophagy
Chemical autophagy inhibition has become an important tool to understand the

role of autophagy in various disease states including cancer. A common concern
is that most of the autophagy inhibitors described below are not pharmacological
compounds and lack specificity (see Fig. 4.2).
1. Pan phosphatidylinositol 3-kinase (PtdIns3K) inhibitors, including wortman-
nin and LY294002: inhibit both class I and class III PtdIns3K. The implica-
tions of this lack of specificity are significant since the class III PtdIns3K
product, phosphatidylinositol 3-phosphate (PtdIns(3)P) is essential for
autophagy, whereas the class I PtdIns3K products, phosphatidylinositol
(3,4)-bisphosphate (PtdIns(3,4,5)P3) have inhibitory effects [193]. Because
the pan PtdIns3K inhibitors inhibit both classes of PtdIns3K enzymes, both
autophagy and S6 phosphorylation are downregulated. These two compounds
serve as a proximal inhibitors of autophagy.
2. 3-methyladenine (3-MA): a pan PtdIns3K inhibitor. Unlike wortmannin and
LY294002, 3-MA promotes autophagic flux under nutrient-rich conditions,
but suppresses autophagy during periods of nutrient deprivation. These oppos-
ing effects are due to 3-MAs differing temporal effects on class I and class III
PtdIns3K; 3-MA blocks class I PtdIns3K persistently, whereas its suppressive
Fig. 4.2 Inhibitors of autophagy. Spautin, wortmannin, LY294002, and 3-MA are pan inhibitors
of PtdIns3K. Lucanthone, Lys05, and chloroquine inhibit lysosomal function. Bafilomycin A1
prevents the maturation of autophagic vacuoles
effect on class III PtdIns3K is transient [194]. 3-MA serves as a proximal

inhibitor of autophagy.
3. specific and potent autophagy inhibitor-1 (Spautin-1): a small molecule
inhibitor of autophagy that promotes the degradation of Vps34/PtdIns3
kinase complexes by inhibiting two ubiquitin-specific peptidases, USP10 and
USP13, which target Beclin1. Because USP10 also mediates the deubiquitina-
tion of p53, Beclin1 indirectly regulates p53 levels [176]. It serves as a proxi-
mal inhibitor of autophagy.
4. Bafilomycin A1: a vacuolar ATPase. This compound prevents maturation of
autophagic vacuoles by inhibiting fusion between autophagosomes and lys-
osomes [195]. Bafilomycin A1 serves as a distal inhibitor of autophagy.
5. Lucanthone: an anti-schistosome agent that blocks topoisomerase II activity
and inhibits AP endonuclease (APE1), a DNA base excision repair enzyme
[196, 197]. It disrupts lysosomal function and serves as a distal inhibitor of
autophagy. Autophagy inhibition by lucanthone induces apoptosis by a cath-
epsin D-mediated process [198].
6. Phenylethynesulfonamide (PES): a sulfonamide that binds to and inhibits heat
shock protein (HSP)70 function, which leads to misfolding of critical lysoso-
mal proteins. PES treatment of cell lines produces striking accumulation of
autophagic vesicles similar to chloroquine treatment [199].
7. Chloroquine derivatives: an anti-malarial agent that also has been used as a
lysosomal trafficking inhibitor. It inhibits lysosomal acidification and prevents
the fusion of autophagosomes with lysosomes. It serves as a distal inhibitor of
autophagy and is the only clinically applicable inhibitor of autophagy.
8. Lys05: a dimeric chloroquine found to be a 10-fold more potent autophagy

inhibitor. This compound has single-agent anti-tumor activity in multiple
xenograft models and, when given at doses above the maximal tolerated dose,
reproduced an intestinal phenotype similar to patients and mice deficient in
ATG16L1. These results suggest chloroquine derivatives can phenocopy a
genetic autophagy deficiency (Mcafee et al. PNAS 2012 in press)
4.12 Summary and Future Directions
Providing for cellular homeostasis in the face of rapid proliferation can be a challenge
as the cell integrates signals generated from intracellular alterations in glucose, oxygen,
energy, and amino acid stores. An additional layer of complexity lies in the fact that the
cell is also responsible for assimilating extracellular signals arising from the immune
system, pathogens, radio-, targeted-, hormonal-, and/or chemotherapies. These diverse,
sometimes diametrically opposed signals can each stimulate autophagy. In this capac-
ity, autophagy can serve as a stress mechanism that can maintain survival despite limi-
tations in essential nutrients and has been described as such in both physiological and
pathophysiological settings. Interestingly, though, unchecked autophagy can also lead
to cell death. Predicting these diametrically opposed roles is at the heart of leveraging
autophagy as a novel target for cancer therapy. Currently, the therapeutic strategy tar-
geting autophagy as a means to enhance clinical cancer benefit is by inhibiting it. An
abundance of preclinical evidence exists supporting autophagy as a mechanism by
which a cancerous cell protects itself from reactive oxygen and reactive nitrogen spe-
cies accumulation, genetic instability, hypoxia, and diminished glucose stores. Multiple
clinical trials have been launched utilizing chloroquine and the chloroquine derivative,
hydroxychloroquine. Despite their extensive clinical use for the treatment for malaria,
rheumatoid arthritis, and HIV, their oncology use may be limited by their narrow thera-
peutic window, their long half-life, and accumulating toxicities. Development of new
autophagy inhibitors is being pursued and will likely advance this target as a cancer
therapy adjunct.
Hand in hand with the development of new autophagy inhibitors is our ability
to prospectively identify cancer patients who will benefit from autophagy inhibi-
tion. Currently, data from ongoing and completed trials that couple standard thera-
peutics with autophagy inhibition show mixed response. Further work needs to be
pursued concerning pre-identification of sensitive tumor types, recognizing appro-
priate therapeutic regimens that synergistically act with autophagy inhibition to
enhance clinical benefit, and development of more robust pharmacodynamic mark-
ers to assess autophagy inhibition in vivo.
A yet theoretical application for autophagy lies in the use of pharmacologic
autophagic inducers in those cells or tumors in which autophagy stimulates cell
death. The challenge for this application lies in the development of such drugs as
well as our ability to prospectively identify those cells in which autophagy induces
cell death, not cell survival.
Regardless of the above challenges, research into the metabolic drivers, iden-
tification of the delicate interplay between the immune system and autophagy as
well as development of mouse models is being actively pursued. These insights
will improve our understanding of the role of autophagy in normal, dysplastic,
and cancerous cells and therefore will allow us to better target autophagy and ulti-
mately improve cancer therapy.
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Chapter 5
MicroRNAs in Cell Death and Cancer
Jong Kook Park and Thomas D. Schmittgen
Abstract MicroRNAs (miRNAs) are small, non-coding RNAs (ncRNAs) that

post transcriptionally regulate protein levels by binding to the 3′ UTR of the
mRNA. miRNAs are differentially expressed in many solid tumors and often cre-
ate a unique signature for each tumor type. This chapter explains the function of
miRNAs in cancer based on their potential target genes.
5.1 Introduction
Approximately 97 % of the human genome is non-coding sequences. Over the

past two decades, it has been considered that non-coding RNAs (ncRNAs) are
important to fundamental biologic processes, contributing significantly to patho-
physiological cell mechanisms. A growing number of non-coding transcripts
have been found to play important roles in several gene regulation mechanisms
[1–3]. ncRNAs are traditionally classified into long ncRNAs and shorter spe-
cies of ncRNAs. Three main categories of small ncRNAs are short-interfering
RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs).
It is increasingly difficult to discern the boundaries between the various small
ncRNAs, however, the double-stranded structure of precursors is the feature of
both siRNA and miRNA [4]. piRNAs utilize their function in the germline [5].
miRNAs are single-stranded 21–25 nucleotide ncRNAs that regulate gene expres-
sion posttranscriptionally by base-pairing with the 3′ untranslated region (3′ UTR)
of target messenger RNA (mRNA) [6]. Since the discovery of the first miRNA,
Lin-4, in Caenorhabditis elegans by Victor Ambros and Gary Ruvkun [7, 8], over
1,500 native miRNAs have been identified in vertebrates.
Given that multiple mRNAs can be targeted by a single miRNA and that indi-
vidual mRNA can be modulated by numerous miRNAs, interactions of miRNA
with mRNA provide significant insights into highly complicated cell signaling
J. K. Park · T. D. Schmittgen (*)

Division of Pharmaceutics, College of Pharmacy,
Ohio State University, Columbus, OH, USA
118 J. K. Park and T. D. Schmittgen
networks. Indeed, alterations of miRNA expression contribute to the pathogenesis

of various human malignancies such as cancer, cardiovascular, neurodegenerative,
and autoimmune diseases [9–12].
5.2 Location and Biogenesis of miRNAs
Genome location analysis of miRNAs has suggested that over 70 % of miRNAs are
transcribed from intronic regions of both protein-coding and long non-protein-cod-
ing genes [13, 14]. Expression of these miRNAs shows the remarkable coincidence
with the expression of their host gene transcripts. Otherwise, miRNAs are located in
intergenic regions and apparently possess independent transcription units. Long pri-
mary transcripts containing multiple miRNAs are commonly located as clusters of
polycistronic units. The first step of miRNA biogenesis is the transcription of the nas-
cent primary miRNA precursors (pri-miRNAs), which is mainly mediated by RNA
polymerase II [15] (Fig. 5.1). The pri-miRNAs have a hairpin structure containing
the mature miRNA sequences. Pri-miRNAs also have a 5’ cap structure and poly-
A tails, similar to mRNAs [16]. Binding of DiGeorge syndrome critical region gene
8 (DGCR8) to the pri-miRNAs recruits the class 2 RNase III enzyme, Drosha, fol-
lowing the formation of a multi-protein complex called the microprocessor [17, 18].
Co-factors including the DEAD box RNA helicases p68 (DDX5), p72 (DDX17), and
heterogeneous nuclear ribonucleoproteins (hnRNPs) also compose the microproces-
sor complex [18]. DGCR8 contains two double-stranded RNA-binding domains
(dsRBDs) and is able to bind to a single-stranded portion of the pri-miRNAs for
the appropriate processing [19]. Cleavage of the pri-miRNAs by Drosha produces
~60–70-nt precursor miRNAs (pre-miRNAs) (Fig. 5.1). Following the initial cleav-
age by Drosha, the pre-miRNAs are then recognized by the nuclear export protein,
Exportin-5, and actively transported to the cytoplasm in a Ran-GTP-dependent man-
ner [20]. Recently, it was proposed that other factors such as nuclear export recep-
tor Exportin 1 (XPO1), cap-binding complex (CBC), and arsenite resistance protein 2
(ARS2) act a part of the pri-miRNAs processing [21, 22]. Once inside the cytoplasm,
the endoribonuclease Dicer cleaves the terminal loop of the pre-miRNA. A part-
ner protein, HIV-1 transactivating response (TAR) RNA-binding protein (TRBP) in
humans (R2D2 and loquacious (Loqs), in Drosophila), assists the cleavage resulting in
22–23-nt mature miRNA duplexes (miRNA/miRNA*) (Fig. 5.1) [23–25]. Following
Dicer processing, the guide strand is preferentially incorporated into miRNA-induced
silencing complex (miRISC) and guides Argonaute (AGO) proteins to target genes
(Fig. 5.1). miRNA*, the passenger strand, is commonly degraded [26, 27].
5.3 miRNA Decay
Expression of miRNAs is coordinated by transcription rates, processing, and

decay. Treatment of RNA polymerase II inhibitors or interruption of miRNA pro-
cessing demonstrated that miRNAs are highly stable and have prolonged half-lives
5 MicroRNAs in Cell Death and Cancer 119
Intronic/exonic Intergenic Polycistronic
PNAPII PNAPII
PNAPII
PNAPII
pri-miRNA
m7G AAAA
m7G AAAA
DGCR8
Drosha
pre-miRNA
Exportin-5
AGO
AGO
TRBP
Dicer miRNA/miRNA* duplex
miRISC
m7G polyA
3 UTR of a target gene
Fig. 5.1 Biogenesis of miRNAs. Primary transcripts (pri-miRNAs) are encoded from individual

miRNA genes. Pri-miRNAs are processed into hairpin-structured pre-miRNAs by Drosha and a
co-factor, DGCR8, in the nucleus. Pre-miRNAs are exported from the nucleus via exportin-5
from several hours to days in cell lines and organs [28]. Since miRNAs can act
as on and off switches and play critical roles in developmental transitions, it is
required that expression of miRNAs is actively regulated in cells. In mouse reti-
nal neurons, turnover of several miRNAs due to rapid decay can be prevented by
blocking the action potential or glutamate receptors. This suggests that turnover-
mediated transition of miRNA expression plays an important role for neuronal
function [29]. miR-29b, which is primarily confined in the nucleus, is an exam-
ple of sequence-specific decomposition of miRNA [30]. miR-29b rapidly decays
due to the uracils at position 9–11. Uracil-rich sequence affects the dynamics
of miRNA decay depending on the sequence context [31]. m169, a transcript of
murine cytomegalovirus (MCMV), inhibits the function of miR-27 through deg-
radation. Its expression kinetics correlate with miR-27 degradation which implies
that miRNA degradation mediated by viral RNA may be a manipulation strategy

of virus against host cells [32].
Moreover, recent progress demonstrated the effect of enzymes on miRNA
stability. PAP-associated domain containing 4 (PAPD4, alternative syno-
nyms are GLD-2 and TUTase2) is the cytoplasmic poly (A) RNA polymerase.
A single adenosine monophosphate monomer is added to the 3′ end of mature
miR-122 by PAPD4 leading to the stabilization of miR-122. Mature miR-122
levels are especially lower in liver tissues of PAPD4-null mice than in those
of heterozygous mice [33]. Stability of miRNAs is also affected by incorpora-
tion into AGO proteins. 5′–3′ exoribonuclease 2 (XRN-2) is an exoribonuclease
enzyme which catalyzes the degradation of single-stranded mature miRNAs.
Complexation of miRNAs and target genes with AGO proteins prevents miRNA
release resulting in the protection of vulnerable miRNA substrates from degra-
dation. Therefore, expression levels of AGO proteins affect the abundance of
mature miRNA. Suppression of AGO proteins leads to diminished expression of
mature miRNAs [34].
5.4 Principles of miRNA–mRNA Interactions and miRNA’s

Functions
A principal function of miRNAs is the inhibition of protein synthesis of pro-

tein-coding genes through suppression of translation and/or mRNA degrada-
tion. AGO family proteins are crucial elements of miRISC and directly associate
with miRNAs. AGO proteins orientate miRNAs for association with their target
mRNAs. There are four AGO proteins (AGO1–AGO4) in mammals. Among
them, AGO2 has an RNase H-like P-element-induced wimpy testis (PIWI)
domain which can endonucleolytically cleave target mRNAs at the miRNA–
mRNA duplex region [35].
miRNAs interact with the 3′ UTR of their target mRNAs by means of
Watson–Crick complementary for direct posttranscriptional repression. The
most rigorous condition for target recognition is perfect complementary and
uninterrupted base-pairing of miRNA nucleotides 2–8 (seed sequence) with tar-
get gene sequences. A residue on the opposite side at position 1 of the miRNA
enhances the site efficiency even though there is no miRNA–mRNA association.
In addition, additional complementarity 3′ of miRNAs stabilizes the miRNA–
mRNA interactions. Furthermore, multiple targeting sites of the identical or
distinct miRNAs are typically claimed for potent repression of gene expression
[36]. Processing bodies (P-bodies) consist of various proteins which are nec-
essary for miRNA-mediated gene silencing. GW182 (also known as trinucleo-
tide repeat containing 6A), AGO proteins, and RNA helicases are enriched in
P-bodies, and disruption of P-bodies can result from lack of GW182 proteins
[37]. There is a positive correlation between miRNA-mediated gene silencing
and the cumulation of target genes in P-bodies. For example, cationic amino acid
transporter-1 (CAT-1) mRNA is localized to P-bodies when it is repressed by
miR-122. Upon stress, the interaction of CAT1 with HuR results in the release
of CAT-1 from P-bodies followed by re-initiation of translation activation [38].
Disappearance of P-bodies in developing mouse oocytes shows the coincidence
with global suppression of miRNA functions even though there are a plentiful
amount of miRNA [39, 40].
In addition to the inhibitory function of protein synthesis, miRNA can have
a decoy activity. Posttranscriptional CCAAT/enhancer-binding protein (C/EBP),
alpha (CEBPα) suppression is mediated by interaction with a heterogeneous ribo-
nucleoprotein hnRNP-E2 (also known as poly(rC) binding protein 2). miR-328
interacts with hnRNP-E2 independently of seed sequence, resulting in the release
of CEBPα mRNA from the translation inhibitory effect of hnRNP-E2 [41].
Furthermore, miRNAs can also paradoxically activate the target mRNA’s transla-
tion. Under serum deprivation conditions, both AGO and fragile X mental retarda-
tion–related protein 1 (FXR1) are assembled into an AU-rich element (ARE) in
tumor necrosis factor alpha (TNFα) mRNA, which is mediated by base-pairing of
miR-369-3 with ARE to activate translation [42]. In addition, let-7 and the syn-
thetic miR-cxcr4 repress the translation of their target genes in proliferating cells,
but increase the translation of target genes in serum starved cells, all of which
propose that miRNA’s functions can be shifted depending on cell proliferation
status [42].
5.5 miRNAs in Cancer
It is generally accepted that tumors are initially developed by genetic and epige-
netic alterations of protein-coding oncogenes and tumor suppressors. On account
of structured endeavors to identify additional alterations contributing to human
malignancy, the importance of miRNAs has been realized in the pathogenesis of
cancer over the past several years. Extensive profiling studies have shown that
various miRNAs are differentially expressed in distinct types of human cancer
[43–50]. Furthermore, comprehensive repression of miRNA biogenesis by target-
ing DGCR8, Drosha, or Dicer1 enhances cellular transformation and tumorigen-
esis, which distinctly signifies the role of miRNAs in cancer-related signaling
pathways [51]. Deregulation of miRNAs can result from various causes such as
genomic alterations (i.e., regional gain and loss at chromosomal loci), modification
of transcription factors, epigenetic mechanisms, and abnormal processing of miR-
NAs. Undoubtedly, a number of miRNAs have been identified to have a promising
role in the pathophysiology of many types of tumors. Overexpressed miRNAs in
cancer can serve as oncogenes by targeting protein-coding tumor suppressor genes.
On the contrary, miRNAs have been suggested as tumor suppressors based on their
reduced expression, deletion status, and interaction with protein-coding oncogenes
(Tables 5.1 and 5.2).
Table 5.1 Tumor-suppressive miRNAs
microRNA Expression pattern Identified targets Regulation factors References
miR-15a/16-1 Downregulated in BCL2, MCL1, Deletion [52–55, 57–59]
prostate cancer, CCND1,
pituitary WNT3A,
adenomas, multi- FGF2, FGFR1,
ple myeloma, MAP3KIP3
and CLL
miR-26a-1, miR- Reduced in EZH2, CDK6, Transcribed by [60, 63, 65–68]
26a-2 HCC and CCNE1, C/EBP alpha
nasopharyngeal CCNE2,
carcinoma CCND2,
BDNF, GSK3B
miR-34a, miR- Loss in breast, lung, CCND1, CCNE2, Deletion, hyper- [61, 69, 70]
34b/34c colon, pancreatic CDK4, CDK6, methylation,
cancer, HCC, c-MYC, MET, transactivated
and neuroblas- E2F3, SIRT1, by TP53
toma BCL2, CD44
miR-122 Downregulated in ADAM10, SRF, Controlled by [62, 71–73, 77, 78]
HCC IGF1R, HNF1A,
ADAM17, HNF3A,
CUTL1, HNF3B, and
SMARCD1 HNF4A.
Transcription-
ally activated by
C/EBP alpha
miR-143/145 Downregulated in DNMT3A, Repressed by Ras [79, 80, 82–85, 87,
colon, lung, RREB1, MLL– activation. 88]
cervical, and AF4, ERK5, Epigenetically
prostate cancer OCT4, SOX2, repressed in
KLF4, EGFR, ALL
NUDT1,
c-MYC,
MMP11,
ADAM17,
CCNA2
5.5.1 Tumor-Suppressive miRNAs
Association of miRNAs with human cancer was first suggested in 2002. Both miR-
15a and miR-16-1 are located on chromosome 13q14 and are frequently deleted
or downregulated in chronic lymphocytic leukemia (CLL) [52]. Expression of
both miRNAs is also downregulated in pituitary adenomas, prostate carcinoma,
multiple myeloma, and diffuse B-cell lymphomas [53–55]. The tumor-suppres-
sive function of these two miRNAs is corroborated by their specific target genes.
Anti-apoptotic proteins, B-cell lymphoma 2 (BCL2) and myeloid cell leukemia-1
(MCL1) are critical cellular oncogenes which determine proliferation, differentia-
tion, and tumorigenesis [56]. BCL2 expression is inversely proportional to miR-15a
Table 5.2 Oncogenic miRNAs
microRNA Expression pattern Identified targets Regulation factors References
hsa-miR-21 Upregulated in PDCD4, TPM1, Induced by [44, 90, 91, 100,
most cancer SPRY2, Ras/ERK, 101]
TIMP3, RECK, AP-1, NFκB,
MASPIN, deltaEF1,
PTEN and STAT3.
Suppressed by
FOXO3A and
NFIB
miR-221/222 Overexpressed in CDKN1B, Induced by NFκB, [102, 103,
lung, breast, CDKN1C, c-Jun, and 105–108, 110,
prostate cancer, PTEN, TIMP3, FOSL1. Nega- 111]
and HCC TRPS1, PUMA tively regulated
by ER in breast
cancer. Modu-
lated by EGFR
and MET
miR-17-92 Upregulated in ZBTB4, E2F1, Transactivated by [113–119]
colon, lung, PTEN, E2F, MYCN,
breast cancer, BCL2L11, and c-Myc.
and B-cell CDKN1A, Negatively
lymphomas DKK3, regulated by
TGFBR2, p53 under
SMAD4, TSP1, hypoxic
CTGF condition
miR-106b-25 Increased in CDKN1A, Activated by E2F1 [120–125]
pancreas, BCL2L11, and E2F3
prostate, colon ITGB8, PTEN,
cancer, and E2F1, E2F3,
HCC BCL2L11
miR-130/301 Upregulated in HOXA5, GAX, Transcribed by [126–131]
breast, colon, TP53INP1, NFκB
and pancreatic RUNX3,
cancer CDKN1A,
FOXF2,
BBC3, PTEN,
COL2A1,
NKRF
miR-155 Upregulated in SHIP1, TP53INP1, Epigenetically [132–142]
thyroid, breast, HGAL, repressed
colon, cervical, SOCS1, by BRCA1.
lung cancer, PPP2CA, Transcription-
and PDAC FOXO3A ally activated
by NFκB and
MYB
and miR-16-1 in CLL, and both miRNAs post transcriptionally repress BCL2
[57]. Transcriptome and proteome analysis in MEG-01 cells showed that ectopic
expression of miR-15a and miR-16-1 results in upregulation or downregulation
of numerous genes including MCL1 [58]. In prostate cancer cells, reconstitu-

tion of miR-15a and miR-16-1 expression using lentiviral vectors induces apop-
tosis and growth arrest, whereas knockdown of miR-15a and miR-16-1 leads
to tumorigenesis of untransformed prostate cells [53]. Other than BCL-2,
cyclin D1 (CCND1) and wingless-type MMTV integration site family, member
3A (WNT3A) both of which elevate tumorigenic potentials are directly regulated
by the miR-15 and miR-16-1 cluster [53]. Fibroblast growth factor 2 (FGF2) and
fibroblast growth factor receptor 1 (FGFR1) are also repressed by direct interac-
tions with miR-15a and miR-16-1 in prostate cancer–associated fibroblasts result-
ing in promotion of cell growth and migration [59].
Supporting the prominent role of miRNAs in cancers, miR-26a, the miR-34
family, and miR-122 are identified to be commonly downregulated in hepatocel-
lular carcinoma (HCC) [60–62]. Two distinct host mRNA genes, carboxy-terminal
domain, RNA polymerase II, polypeptide A small phosphatase-like (CTDSPL)
and carboxy-terminal domain, RNA polymerase II, polypeptide A small phos-
phatase 2 (CTDSP2) encode pri-miR-26a-1 and pri-miR-26a-2, respectively,
generating identical mature miR-26a. Transcription of pri-miR-26a-1 is directly
activated by C/EBPα [63]. C/EBPα impedes tumor cell growth through stabiliza-
tion of cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1) and is com-
monly downregulated in HCC [64]. miR-26a has tumor-suppressive biologic
effects by targeting several protein-coding oncogenes. For example, miR-26a
regulates cyclin D2, cyclin E1, and cyclin E2, all of which regulate cell cycle
transition from G1 to S phase [65, 66]. Additional targets of miR-26a include
brain-derived neurotrophic factor (BDNF) and enhancer of zeste homolog 2
(EZH2) [67, 68].
Recent work also suggests the connection between the miR-34 family and
tumor suppressor p53. Primary transcripts of mature miR-34a and miR-34b/c
are activated by p53 and further processed into mature forms. Expression of the
miR-34 family is downregulated in human cancers due to frequent functional p53
deficiency (reviewed in [69]). Deletion of miR-34 family members has also been
observed in several types of cancer [69]. Validated target genes of the miR-34 fam-
ily are associated with apoptosis, senescence, migration, and cell cycle mecha-
nisms [69, 70]. This further supports the p53 network that multiple transcriptional
targets of p53 act in concert with miR-34 family to suppress tumor formation.
Several miRNAs are expressed in a tissue-specific manner, implying their
involvement in tissue specification. miR-122 is a liver-specific miRNA whose
expression is controlled by C/EBPα, hepatocyte nuclear factor 1α, 3α, 3β, 4α,
and REV-ERBα. miR-122 is highly abundant in hepatocytes, but undetectable in
other tissues [71–73]. miR-122 modulates hepatitis C virus accumulation and cho-
lesterol metabolic pathways in liver cells [74, 75]. miR-122 is frequently down-
regulated in HCC patients with poor prognosis, suggesting that miR-122 is an
important regulator of HCC pathogenesis [76]. Indeed, reconstruction of miR-122
expression in HCC cells enhances the effects of sorafenib and doxorubicin and
reduces tumorigenic properties such as angiogenesis and metastasis in orthotopic
transplantation HCC models [77, 78].
Additional miRNAs also target multiple protein-coding oncogenes. Expression

of the miR-143 and miR-145 cluster is broadly downregulated in several types of
cancer including colon, lung, cervical, and prostate cancer [79–82]. Expression of
miR-143 by promoter hypermethylation is also repressed in acute lymphoblastic
leukemia (ALL) [83]. In addition, miR-145 is weakly expressed in human embry-
onic stem cells and regulates the pluripotency factors, OCT4, SOX2, and KLF4
[84]. miR-145 also targets EGFR and nucleoside diphosphate linked moiety
X-type motif 1 (NUDT1) resulting in the inhibition of cell proliferation of human
lung adenocarcinoma cells [85]. Overexpression of miR-143 also induces apopto-
sis and impairs the growth of a xenograft model of human colon carcinoma [86].
DNA methyltranferase 3A (DNMT3A) is targeted by miR-143, and overexpres-
sion of miR-143 in colon cells halts tumor growth and colony formation [87].
KRAS and Ras-responsive element-binding protein 1 (RREB1) are direct targets
of the miR-143/145 cluster. Constitutively, active KRAS induces RREB1 and
restrains the transcription of miR-143/145. This suggests that cellular transforma-
tion can be initiated by KRAS along with activation of other oncogenic signals
related with the miR-143/145 cluster [88]. Depending on the cellular context, the
miR-143/145 cluster acts as oncogenic miRNAs. It was reported that expression
of miR-143 is elevated in hepatitis B virus (HBV)-related HCC [89]. Nuclear fac-
tor kappa B (NFκB) transcribes miR-143 and facilitates invasion and metastasis of
HCC in vivo via targeting fibronectin type III domain containing 3B (FNDC3B).
Knockdown of miR-143 expression in vivo also leads to inhibition of metastasis of
HBV-HCC in transgenic mice [89].
5.5.2 Oncogenic miRNAs
One of the first miRNAs identified as oncogenic in the human genome is miR-21.
miR-21 was first identified in glioblastoma [90] and is upregulated in most
types of human malignancies [44]. A term “oncomiR addiction” markedly
describes the momentous function of miR-21 on all stages of tumor development.
Overexpression of miR-21 in a genetically engineered mouse model facilitates
a pre-B malignant lymphoid-like phenotype [91]. Switching off miR-21 expres-
sion leads to tumor regression and recovery from symptoms of lymphoma, clearly
illustrating the role of miR-21 in tumor initiation as well as maintenance of estab-
lished tumors. Furthermore, a survival advantage followed by the suppression of
miR-21 also demonstrates the clinical impact of targeting one aberrantly expressed
miRNA [91]. Posttranscriptional regulation of miR-21 is mediated by the receptor-
mediated SMADs (R-SMADs). Interactions of R-SMADs with primary transcripts
of miR-21 (pri-miR-21) promote Drosha processing by stabilizing the binding
of pri-miR-21 with Drosha. TGF-β and BMP4 enhance the expression of mature
miR-21 through the complex formation of R-SMADs, pri-miR-21, and DEAD
(Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5, RNA helicase, p68, a component
of microprocessor complex) [92]. Moreover, miR-21 is located at a TMEM49
intronic region, and several pathways modulate the expression of miR-21 at the
transcription level. Akt2 transcriptionally activates miR-21 induction through
binding of NFκB, CREB, and CBP/p300 to the miR-21 promoter regions under
conditions of hypoxia [93]. AP-1 and deltaEF1 increase miR-21 expression by
binding to the miR-21 promoter. Treatment of BMP-6 transcriptionally inhibits
miR-21 expression via suppression of AP-1 and deltaEF1 in breast tumor cells
[94]. In addition, signal transducer and activator of transcription 3 (STAT3) also
directly binds to the miR-21 promoter and regulates miR-21 levels [94]. Interferon
(IFN) increases miR-21 through co-regulation of STAT3 and NFκB. Apoptosis
induced by IFN can be suppressed due to an increased amount of mature miR-21
[95]. Enhanced expression of miR-21 resulted from activation of EGFR signaling
and neurotensin treatment in lung cancer and colon tumors, respectively [96, 97].
In contrast, suppression of miR-21 promoter activity is induced by the binding
of FOXO3A to its promoter region. FOXO3A is a trigger of apoptosis of tumor
cells via upregulation of several pro-apoptotic genes such as Bim and PUMA. Fas
ligand (FasL) is a target of miR-21, and upregulation of FasL by FOXO3A partly
results from miR-21 depletion [98]. Nuclear factor I/B (NFIB) also binds to the
miR-21 promoter and acts as a negative regulator of miR-21 transcription [99].
The potential oncogenic activity of miR-21 has been further supported by numer-
ous validated target genes (reviewed in [100] and summarized in Table 5.2). All of
these are closely related with growth, apoptosis, invasion, and metastasis of tumor
cells. Overexpression of miR-21 therefore leads to facilitation of growth, evasion
of apoptosis, potent metastasis, and resistance toward anticancer agents [100, 101].
Deregulation of other oncogenic miRNAs including miR-221 and miR-222
has also been observed in human malignancies. miR-221 and miR-222 are clus-
tered in an intergenic region on chromosome Xp11.3. They have the same seed
sequence, thus share common predicted target genes. They are upregulated in
lung, breast, prostate cancer, and HCC [102–104]. Contribution of the miR-
221/222 cluster to tumorigenesis of lung cancer and HCC has been suggested.
Expression of miR-221/222 is modulated by EGFR and MET receptors in lung
cancer, and they contribute to gefitinib resistance and tumorigenesis of non-small-
cell lung cancer (NSCLC). Overexpression of miR-221 facilitates the growth of
tumorigenic murine hepatic progenitor cells in vivo, suggesting the important role
of miR-221 in hepatocarcinogenesis [105]. Oncogenic activities of miR-221/222
are also related with their transcription modulators. NFκB and c-Jun contribute to
the oncogenesis of prostate carcinoma and glioblastoma and trigger the expression
of miR-221/222 [106]. A downstream transcription factor of EMT-promoting Ras-
ERK pathway, FOSL1, stimulates the expression of miR-221/222 in breast cancer.
FOSL1 is present in basal-like (BL) sub-type breast cancers; therefore, upregula-
tion of miR-221/222 by FOSL1 in breast cancer contributes to aggressiveness of
BL sub-type partly by targeting tricho-rhino-phalangeal syndrome type 1 (TRPS1)
which represses the GATA family at the transcriptional level and decreases ZEB2
expression [107]. miR-221/222 are negatively modulated by the binding of ER
alpha together with nuclear receptor co-repressor (NCoR) and silencing media-
tor of retinoic acid and thyroid hormone receptor (SMRT) [108]. A number of
evaluated target genes of miR-221/222 such as CDKN1B (p27, Kip1), PTEN,

PUMA, and TIMP3 also substantiate the functional significance of miR-221/222
in tumor proliferation, apoptosis, invasion, and metastasis [102, 109–111]. This
combined knowledge suggests the therapeutic potential of miR-221/222 as a treat-
ment for cancer.
Of all of the miRNA clusters, the miR-17~92 polycistronic cluster is one of
the most studied. This oncogenic cluster is located on chromosome 13q31.3 and
is transcribed from a long non-coding miR-17~92 cluster host gene (MIR17HG).
Six different mature miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b,
and miR-92a) are produced from a single primary transcript. Based on homol-
ogy of their seed sequence, six mature miRNAs can be sorted into four groups,
that is, miR-17/miR-20a (AAAGUG), miR-18a (AAGGUG), miR-19a/miR-19b
(GUGCAA), and miR-92a (AUUGCA). Mature miRNAs from this cluster are
overexpressed in breast, lung, pancreas, colon, prostate, stomach cancer, chronic
myeloid leukemia (CML), and B-cell lymphomas (reviewed in [112]). The miR-
17~92 cluster gene is transcriptionally activated by MYC [113] and MYCN [114]
and suppressed by p53 [115]. Overexpression of the miR-17~92 cluster gene
facilitates the onset and progression of c-Myc-induced lymphoma, indicating an
orchestrated interaction between c-Myc and the miR-17~92 cluster for tumorigen-
esis [113]. In addition, stimulating angiogenesis and tumor growth by overexpres-
sion of miR-17~92 is derived from the weakened TGF beta signals by repressing
related target genes, TGFBR2 and SMAD4 [116]. Functional studies related with
their targets have been also successful, suggesting that they are genuine oncogenic
miRNAs [112, 117, 118]. The effects of individual miRNAs of the miR-17~92
cluster in human cancer still remain to be uncovered, but overexpression of miR-
17~92 generally enhances cell cycle progression and inhibits apoptosis of tumor
cells, for example, through repression of E2F1, PTEN, and BCL2L11 [119].
The intronic miR-106b-25 cluster is a paralog of miR-17~92 polycistronic gene
cluster. It is transcribed from intron 13 of its host gene, minichromosome main-
tenance protein 7 (MCM7), and contains mature miR-106b, miR-93, and miR-25
[120]. Inhibition of a miR-106b~25 cluster effectively inhibits proliferation and
anchorage-independent growth of HCC cells [121]. A miR-106b~25 cluster is a
target of E2F1 and E2F3. In turn, E2F1 is repressed by miR-106b and miR-93.
Also, the pro-apoptotic BCL2L11 gene is also targeted by miR-25, all of which
imply that the miR-106b~25 cluster can blunt the effects of overexpressed E2F
which leads to cell death [122, 123]. Recently, overexpression of a single miR-93
enhances tumor growth and angiogenesis in human astrocytoma U87 cells by tar-
geting integrin, beta 8 (ITGB8) [124]. Expression of the oncogenic miR-106b~25
cluster is upregulated in prostate cancer, and all individual miRNAs of this cluster
directly target and repress PTEN expression, suggesting the association of a miR-
106b~25 cluster with prostate tumorigenesis [125].
The miR-130/301 seed family is also known as an oncogenic miRNA. Four
members of this family are miR-130a, miR-130b, miR-301a, and miR-301b. Their
5’ seed sequences are identical and thus would be expected to regulate the iden-
tical target genes. Consistent with the effects of NFκB on regulating oncogenic
miR-21 and miR-221/222 expression, miR-301a transcription is also promoted by

NFκB. In turn, miR-301a directly suppresses NFκB-repressing factor (NKRF),
which implies a positive feedback mechanism to elevate NFκB activity in tumor
cells [126]. miR-130a regulates the angiogenic phenotype of vascular endothelial
cells by direct modulation of anti-angiogenic factors, GAX and HOXA5 [127].
Experimentally confirmed target genes of miR-130b are tumor protein p53-induc-
ible nuclear protein 1 (TP53INP1), runt-related transcription factor 3 (RUNX3),
and CDKN1A (p21, Cip1), indicating the functional contribution to cell growth
and metastasis [128–130]. Upregulation of miR-301 is considered as a poor prog-
nosis factor of invasive ductal breast cancer. Downregulation of miR-301 expres-
sion in breast tumor cells results in reduced cell proliferation and migration. Nodal
or distant relapses of breast cancer are considered to be related with target genes
of miR-301, namely, FOXF2, BBC3, PTEN, and COL2A1 [131].
Double-stranded fold-back motif of B-cell integration cluster (BIC) RNA is the
precursor of miR-155. BIC/miR-155 is greatly overexpressed in Hodgkin’s lym-
phoma and diffuse large-cell B-cell lymphoma [132]. Explicit evidence of involve-
ment of miR-155 in B-cell malignancy is that ALL is generated in a transgenic
mouse overexpressing miR-155 specifically in B-cell lineage [133]. Deregulation
of miR-155 expression has been shown to be related with several other kinds of
cancer. miR-155 is upregulated in thyroid carcinoma, breast cancer, colon can-
cer, cervical cancer, pancreatic ductal adenocarcinoma (PDAC), and lung cancer
(reviewed in [134]). miR-155 is one of the most promising bona fide oncogenic
miRNAs suppressing apoptosis via efficient repression of several tumor suppres-
sor genes [135–139]. It was also reported that Akt activity is enhanced through
downregulation of phosphatase protein phosphatase 2A catalytic subunit alpha
(PPP2A) mediated by miR-155 [97]. Transcription of miR-155 is activated by
NFκB and MYB transcription factors [140, 141]. Recently, it was demonstrated
that BRCA1 associated with HDAC2 epigenetically restrains the expression of
miR-155 through deacetylation of histones on the miR-155 promoter [142].
In conclusion, since the discovery of Lin-4 in C. elegans, much progress has
been made in characterizing miRNAs, in particular, their role in gene regulation.
Better understanding of miRNA involvement in the regulation of the cancerous
networks may lead to successful miRNA-based cancer therapy in the near future.
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Chapter 6
Targeting DNA Repair Pathways
for Cancer Therapy
Conchita Vens and Robert W. Sobol
Abstract DNA repair pathways maintain the integrity of the genome, reducing the
onset of cancer, disease, and aging. The majority of anticancer therapeutics (radia-
tion and chemotherapy) function as genotoxins, eliciting genomic DNA damage
in an attempt to induce cell death in the tumor. However, cellular DNA repair pro-
teins counteract the effectiveness of these therapeutic genotoxins by repairing and
removing the cell death-inducing DNA lesions, implicating DNA repair proteins
as prime targets for improving response to currently available anticancer regimens.
To trigger a tumor-specific cell death response (with minimal normal cell toxicity),
the level of genomic DNA damage must therefore surpass the DNA repair capac-
ity of the tumor without overwhelming the DNA repair potential of normal tis-
sue. Interestingly, cancer-specific DNA repair defects offer novel approaches for
tumor-selective therapy. This has become highly relevant as it is suggested that
most cancer cells are likely to be defective in some aspect of DNA repair. Herein,
we describe the molecular pathways that participate in the repair of DNA damage
induced by radiation- and chemotherapeutics and discuss strategies that are being
developed to target DNA repair for cancer treatment and highlight key DNA repair
inhibitors that can enhance response. Further, we present novel therapeutic strate-
gies being considered to exploit inherent weaknesses in tumor cells such as defects
in one or more DNA repair pathways or related processes that may provide the
opportunity to selectively increase tumor-specific cell death.
C. Vens
Division of Biological Stress Response, The Netherlands Cancer Institute, NL,
Amsterdam, The Netherlands
R. W. Sobol (*)
Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute,
Hillman Cancer Center; University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
R. W. Sobol
Department of Human Genetics, University of Pittsburgh School of Public Health, Pittsburgh,
PA, USA
138 C. Vens and R. W. Sobol
Fig. 6.1 Schematic representation of cellular DNA damage repair. This figure depicts cellular
repair processes that deal with chemo- and radiotherapy-induced DNA lesions. Prevalent DNA
repair targets for cancer treatment are highlighted in bold
6.1 Role of DNA Repair Pathways for Cancer Treatment
Human cells must repair tens of thousands of DNA lesions per day [1]. If they are
not repaired, these lesions lead to mutations or genome aberrations that threaten cell
survival and genomic integrity. To combat these threats, cells have evolved multiple
DNA repair and DNA damage response (DDR) mechanisms that signal the presence
of lesions and promote their repair or regulate cellular processes in response to the
DNA damage (Fig. 6.1) [2]. Defects in these repair and response pathways can pro-
mote tumorigenesis and, indeed, are common in human cancers [3, 4]. On the other
hand, current therapy options for cancer patients exploit the DNA-damaging proper-
ties of certain drugs and agents. The success of radiation exposure during radiother-
apy and the success of most chemotherapy agents rely on the destructive nature that
these agents have on cellular DNA, ultimately resulting in death and hopefully eradi-
cation of the tumor cells. Hence, DNA damage and repair mechanisms play a crucial
role in determining treatment outcome. On a cellular level, resistance to treatment is
profoundly determined by the capacity of the cancer cell to respond to and repair the
individual DNA lesions that are induced by the chemotherapeutic agents or radiation.
Our increasing knowledge of these processes has led to the development of new
concepts that target and exploit the cancer cell DDR [5]. Counteracting resistance to
chemotherapy by targeting the appropriate DNA repair pathway is a promising strat-
egy in cancer treatment. Another is to exploit the DNA repair and response defects
that are present in cancer cells thereby specifically targeting tumor cells while spar-
ing healthy cells from a high load of unrepaired DNA damage [6, 7]. Together,
these strategies might provide promising avenues in the conquest against cancer.
6 Targeting DNA Repair Pathways for Cancer Therapy 139
Here, we will briefly describe essential cellular DNA repair and response mecha-
nisms and illustrate novel concepts and promising strategies to exploit and target
DNA repair for cancer treatment.
6.1.1 DNA Damage Response
The initial DDR of a cell involves the recognition of the DNA damage followed by
the propagation of a series of signals ranging from alterations in RNA or protein
expression and modification of protein function or stability through p ost-translation
modification, among other signals. The cell’s defense to genotoxic lesions is trig-
gered and accomplished by a series of events that mediate and regulate prolifera-
tion, cell death, or DNA repair crucial to its survival [2, 4]. The initial steps for an
appropriate response require detection of the lesion, signaling of its presence and
promotion of repair. Cells act upon DNA damage not only by promoting and exe-
cuting repair but also respond by halting the cell cycle or by promoting cell death
mechanisms in order to prevent propagation of the damage. The DDR therefore has
an impact on transcription, cellular metabolism, cell cycle regulators, as well as
cell death, via apoptosis and senescence.
One of the most prominent members of the DNA damage signaling pathway
that links DNA damage with cell cycle checkpoints is the protein ataxia telangi-
ectasia-mutated (ATM) [8], a protein kinase that is recruited to DNA double-
strand breaks (DSB) such as those induced by ionizing radiation. The formation
of DSBs triggers the activation of ATM and activated ATM then phosphorylates a
wide range of downstream substrate proteins thereby signaling the presence of the
damage throughout the cell to facilitate repair [9]. Initial activation of ATM is pro-
moted by its autophosphorylation that initiates a signaling cascade of further phos-
phorylation events that constitute the DDR [10]. With excessive unrepaired DNA
damage present, this cellular response can culminate in an apoptotic response in
which p53 is central but not necessarily always required.
Another key DDR signaling component is the ataxia telangiectasia-RAD3-
related (ATR) kinase that gets activated after replication stress-induced DNA
damage. Replication stress, caused, for example, by exposure to hydroxyurea (HU),
results in the formation of large stretches of single-stranded DNA coated with
replication protein A (RPA) that triggers activation of ATR. Similar to processes
in ATM-mediated signaling, ATR signaling is promoted by regulatory proteins.
ATRIP and TopBP1, together with RAD17-mediated 9-1-1 (Rad9-Rad1-Hus1)
clamp loading, “sense” the damage and trigger the activation of ATR.
Further downstream of these initial events, the cell cycle checkpoint-regulating
protein kinases CHK1 and CHK2 are among the most important targets (substrates)
of ATM and ATR. Supported by the activation of p53 and mediated via multiple
paths, this signaling cascade ultimately results in the reduction of cyclin-dependent
kinase activity that drives cell cycle progression. The halt in cell cycle progression is
thought to allow time for repair and, most importantly, if not successfully repaired,
to prevent propagation of the DNA damage. Cell cycle checkpoints are in place at
the border from G1 to S, within S and at the G2/M border. The prevalent blocks
and their extent depend on the damaging agent and the number and type of lesion.
Another important downstream target of ATM and ATR is p53, an essential player
in the induction of apoptosis upon DNA damage. Thus, DDR mechanisms have a
crucial role in the protection against genome instability and chemo/radiotherapy
response.
ATM/ATR signaling also enhances repair by recruiting repair factors to the
site of the lesion and activating DNA repair proteins through phosphoryla-
tion or indirectly, by modulating acetylation, ubiquitylation, SUMOylation, or
DNA repair gene transcription. These kinases also influence chromatin structure
through phosphorylation of the histone H2A variant (γH2AX). Thereby, they
facilitate recruitment of DDR factors and expedite DNA repair while amplifying
DSB signaling that is crucial to cellular survival following exposure to DNA-
damaging agents.
The role of the DDR is broad with respect to the type of cancer therapeutic. DDR
activity is involved upon exposure of a whole range of chemotherapeutic agents and
upon radiation. Indeed, DDR and cell cycle blocks are induced by radiation, topoi-
somerase I and II poisons, anthracyclines, alkylating drugs including platinum ana-
logues and antitumor antibiotics. Interference in DDR by the use of inhibitors will
likely affect the response and cellular survival in most cancer treatment options.
6.1.2 Direct-Reversal Repair and Mismatch Repair
One of the first DNA repair proteins to be considered as a viable target for improving
chemotherapy was O6-alkylguanine-DNA alkyltransferase (MGMT or AGT), a pro-
tein encoded by the O6-methylguanine-DNA methyltransferase gene (MGMT) [11].
In depth, discussion on the function of MGMT and its role in cancer and chemother-
apy can be found in many excellent reviews [12–14]. MGMT falls within the cate-
gory of direct-reversal (DR) DNA repair proteins that also include the AlkB family of
proteins [13, 15]. Unlike most other DNA repair pathways that correct lesions by
removing the base containing the lesion [base excision repair (BER), see below],
removing a short oligonucleotide containing the lesion [nucleotide excision repair
(NER), see below] or removing long tracts of DNA (mismatch repair, MMR) fol-
lowed by a DNA synthesis step (repair-directed DNA synthesis), DR proteins such
as MGMT or the AlkB proteins reverse the damage to the DNA base directly, and
the mechanism of repair does not involve a DNA synthesis step. This section will
focus on the role of MGMT in DNA lesion repair and the subsequent role that the
MMR proteins play in the cellular response when MGMT is unable to repair the
O6-alkylguanine lesion. Further discussion on the mechanism of action of AlkB pro-
teins can be found elsewhere [13].
Like many DNA repair proteins, MGMT repairs lesions from both carcinogenic
compounds and from chemotherapeutic agents. As such, MGMT acts to suppress
cancer formation by removing lesions induced by carcinogens such as methylnitro-

sourea (MNU), the tobacco smoke lung carcinogen 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (NKK), and the colon carcinogen azoxymethane. Conversely,
many chemotherapeutic agents, including Temozolomide (Temodar, TMZ), dacar-
bazine, streptozotocin, procarbazine, BCNU (camustine), CCNU (lomustine), and
gliadel trigger cell death by inducing the formation of an alkyl lesion (methyl- or
chloroethyl-) on the O6 position of guanine bases in DNA [12–14, 16]. Upon MGMT
binding to the DNA containing the alkyl lesion, the O6-alkyl group is transferred
from the guanine base onto a Cysteine (Cys) residue (amino acid residue Cys145 in
humans) in the MGMT protein [12, 14]. Upon transfer of the alkyl group to MGMT,
the protein undergoes a conformational change that both releases the protein from the
repaired DNA and promotes the ubiquitylation and subsequent proteasome-mediated
degradation. This suicide mechanism of MGMT has been taken advantage of clini-
cally, as will be described below regarding the development and evaluation of MGMT
inhibitors (see Sect. 6.3.2).
The chemotherapeutic agents mentioned above induce the formation of
methyl or chloroethyl adducts on the O6 position of guanine bases in DNA. If not
repaired, the majority of the chloroethyl lesions are converted to G-C interstrand
DNA cross-links. Such lesions are primarily repaired by a concerted effort of the
NER, HR, and fanconi anemia (FA) pathways (see below). In general, interstrand
DNA cross-links are highly genotoxic, inducing cell death. Conversely, if the
methyl lesion (O6-MeG) is not removed by MGMT, during cellular replication, the
mispairing of O6-MeG with thymine leads to the formation of a O6-MeG:T mis-
pair, a substrate for MMR. Repair mediated by the MMR pathway facilitates the
removal of the DNA strand containing the newly synthesized “T” base. However,
re-synthesis of the DNA in the process of MMR will regenerate the O6-MeG:T
mispair, perpetuating the O6-MeG lesion and the presence of the mispair. As such,
in the absence of MGMT-mediated repair, O6-MeG is suggested to initiate a futile
cycle of MMR or alternately to trigger ATR protein kinase activation through the
action of several MMR proteins [17], leading to apoptosis and cell death [18–20].
Details on the MMR pathway can be found elsewhere [21]. However, for the pur-
pose of this discussion, we should consider the MMR pathway as an essential sen-
sor to trigger cell death from chemotherapeutic agents that induce the O6-MeG
lesion. Briefly, recognition of the O6-MeG:T mispair by the MMR protein MSH2
induces recruitment and activation of ATR and subsequently CHK1 and CHK2 to
activate an apoptotic response [22, 23]. In fact, much of the resistance to agents
such as TMZ observed clinically is due to high expression of MGMT (and sub-
sequent repair of the lesion) or loss of MMR (therefore preventing the initiation
of apoptotic signaling) [24–26]. Currently, TMZ along with radiation and surgery
are the standard of care for glioblastoma multiforme (GBM), the most common
and aggressive primary brain tumor [27]. Median survival is less than two years
[28–30], and unfortunately, almost all patients eventually recur with the disease
and the large majority of recurrent tumors are resistant to chemotherapy [31, 32].
Inhibition of MGMT-mediated repair has been taken advantage of experimentally
and in many clinical trials [33] since MGMT can be inhibited with the O6-MeG
analogue O6-benzylguanine [34] (Sect. 6.3.2). Improved prognosis has also been
reported in tumors with loss of MGMT expression due to promoter methylation
[35] whereas poor prognosis is observed when MGMT expression levels are high
or MMR capacity is compromised. Hence, elevated expression of MGMT and/or a
non-functional MMR pathway contribute much of the observed resistance to TMZ
in many tumor cell lines and in clinical trials.
6.1.3 Base Excision Repair
As suggested in its namesake, the BER pathway is the primary mechanism to

remove and repair base lesions. A special sub-pathway of BER, single-strand
break repair (SSBR), is also essential for the repair of single-strand DNA breaks
[13, 36, 37]. The types of base lesions repaired by the proteins of the BER path-
way range from base deamination products (e.g., conversion of C to U or 5 meC
to T) to oxidative modification of bases (8-oxo-7,8-dihydro-2′- deoxyguanosine;
8-oxodG), alkylation products such as N7-meG and N3-meA that are induced by
chemotherapeutic agents such as TMZ [38] and many others [36, 37]. These and
many other lesions are induced in genomic (and mitochondrial) DNA by a multi-
tude of anticancer treatments including radiation, monofunctional alkylators such
as TMZ, cisplatin, and 5FU, among others. There are over 20 proteins brought to
bear to facilitate the complete process of BER [36]. Repair is initiated following
recognition of the base lesion by one of the eleven DNA glycosylases in humans.
This group of proteins is further subdivided into two classes: Bifunctional and
monofunctional DNA glycosylases. A β-bifunctional glycosylase such as OGG1
excises the modified base and hydrolyzes the DNA backbone (via a β-elimination
step) 3′ to the incised base, leaving a 3′ unsaturated aldehyde (after β-elimination)
and a 5′ phosphate at the termini of the repair gap. Alternatively, a β,δ-bifunctional
glycosylase such as NEIL1 hydrolyzes the glycosidic bond to release the lesion
and then cleave the DNA backbone 3′ to the resulting apurinic/apyrimidinic
(AP or abasic) site via β-elimination and 5′ to the abasic site via δ-elimination.
More detail on the mechanism of these bifunctional DNA glycosylases and the
specific BER proteins involved in processing the resulting repair gaps can be
found elsewhere [36, 37].
For the purpose of describing the complete BER pathway, we will focus on
the initiation of BER by monofunctional DNA glycosylases, with an emphasis
on the methylpurine DNA glycosylase (MPG) (also called AAG or ANPG).
MPG is the primary glycosylase for the repair of the chemotherapy-induced
DNA lesions such as N7-meG and N3-meA. These lesions are removed by
hydrolysis of the glycosidic bond, producing an abasic site, a substrate for
AP-Endonuclease 1 (APE1). Given the highly toxic nature of the intermedi-
ates in BER [13], it has been suggested that the product of each BER reac-
tion “hands off” the toxic BER intermediate to the next enzyme in the
pathway likening the complete reaction to the hand-off of a baton in a relay
race [39]. Such a process or hand-off mechanism has the advantage of elimi-
nating or avoiding the accumulation of free BER intermediates that are prone
to induce cell death [13]. Once formed by the glycosylase, the resulting aba-
sic site is then handed off to APE1 to be hydrolyzed on the 5′ end. The resulting
single-nucleotide repair gap contains a 3′OH and a 5′deoxyribose-phosphate
(5′dRP) moiety at the margins. It has been suggested that this BER intermediate
(a single-strand break with a 5′dRP moiety) recruits poly(ADP)ribose polymerase
(PARP)1 to the lesion site. Recruitment then triggers activation of PARP1. Activated
PARP1 polymerizes NAD+ to yield the polymer poly (ADP) ribose (PAR), an
essential posttranslational modification. The first protein to be modified by PAR is
PARP1 itself (auto-modification). Subsequently, it has been observed that XRCC1
and many other proteins are modified [40]. Once modified, activated PARP1 then
facilitates chromatin relaxation (likely to provide access to the lesions for repair)
[41, 42] and recruitment of the remaining BER proteins required to complete repair,
including XRCC1, DNA Ligase III, and DNA polymerase β (Polβ). Whereas
XRCC1 is a scaffold protein, Polβ carries out two essential enzymatic functions in
BER. First, the repair gap is tailored by the 5′dRP lyase activity of Polβ. Next, Polβ
fills the single-nucleotide gap, preparing the strand for ligation by either DNA ligase
I (LigI) or a complex of DNA ligase III (LigIII), and XRCC1 [36].
Although some BER substrates (base lesions) induced by chemotherapeutic
agents are cytotoxic [43], most are found to be mutagenic. However, essentially,
every intermediate throughout the BER pathway (abasic sites, 5′dRP lesions, and
single-strand DNA breaks) is toxic [13] and as such, there has been considerable
interest in developing BER inhibitors to enhance the accumulation of the cytotoxic
repair intermediates following chemotherapy or radiation treatment. This is dis-
cussed further in the sections below.
6.1.4 Nucleotide Excision Repair
Another multi-protein, highly complex DNA repair pathway is the NER pathway.
NER plays an important role in the repair of DNA lesions induced by many geno-
toxins and chemotherapeutics including DNA cross-linking agents such as chloro-
ethylating agents (see Sect. 6.1.2), cisplatin, carboplatin, and lesions induced by
photodynamic therapy (PTD). Put simply, NER facilitates the removal of bulky
DNA adducts that grossly distort the DNA double helix and those that cause a
block to transcription. Molecular details on the proteins involved in NER can be
found in several excellent reviews [44–47]. Overall, the pathway consists of two
complementary sub-pathways that have some overlap. The two sub-pathways are
distinct regarding the lesion recognition step but converge and utilize the same
proteins to remove the oligonucleotide containing the lesion and for the steps
involving new DNA synthesis.
The global genomic NER (GG-NER) pathway surveys the entire genome
for DNA helix distorting lesions whereas the transcription-coupled repair NER
(TC-NER) pathway is recruited to facilitate removal of DNA lesions that block

the elongating RNA polymerase and stall transcription. The GG-NER pathway
utilizes the DDB1/DDB2 heterodimer, part of the DDB1-Cul4A-DDB2 E3 ubiq-
uitin ligase, to facilitate lesion recognition and repair [48]. As such, targeting the
proteasome (Sect. 6.4.2) or deubiquitinating enzymes (DUBs) (Sect. 6.5.4) would
therefore indirectly impact NER function. The TC-NER pathway partners with the
TFIIH transcription complex to recognize and repair lesions that halt transcription.
Upon lesion recognition, XPG mediates cleavage of the DNA strand containing
the lesion on the 3′ side of the lesion, and subsequently, the ERCC1/XPF heterodi-
mer hydrolyzes the DNA strand containing the lesion on the 5’ side. Replication
factors then facilitate DNA synthesis and ligation. Of all the proteins in this path-
way, ERCC1 has emerged as a valuable biomarker of response to chemotherapeu-
tic agents that induce DNA damage repaired by NER (e.g., cisplatin) and is under
consideration as a drug target [49, 50]. Currently, biomarker measurements have
included both mRNA and protein analysis. However, it is not yet clear whether
protein levels of ERCC1 are a valid biomarker [51, 52].
6.1.5 Non-Homologous-End-Joining
One of the most cytotoxic lesions is a DNA double-strand break (DSB). If not
repaired, DSBs lead to chromosome breaks, loss of genetic material, and gross
genomic rearrangements. Whereas tolerance to the presence of DSBs might vary
in different cell types and cellular states, only a few DSBs will cause cell death or
prevent clonogenicity in most cells including cancer cells [53]. These lesions are
induced by multiple agents such as ionizing radiation, bleomycin, and topoisomer-
ase II inhibitors, but can also be induced indirectly at replication forks when con-
verting DNA single-strand breaks (SSBs) induced by topoisomerase I inhibitors
(camptothecin).
Two major cellular pathways deal with the repair of DNA DSBs. The use of
homologous DNA for repair distinguishes those repair pathways. As indicated
by the name, the non-homologous-end-joining (NHEJ) repair pathway does not
require any homologous sequences. Proteins of the NHEJ pathway can repair the
two ends in a DSB by simple end joining while the homologous recombination
(HR) repair pathway (see Sect. 6.1.6) requires homologous DNA stretches as tem-
plates for DNA synthesis and repair.
After the initial recognition of the DSB that is held in place and stabilized
by the binding of the MRN complex (MRE11, RAD50, NBS) and promoted by
ATM (see above), DSB repair is executed by the DNA-dependent protein kinase
(DNA-PK). DNA-PK is comprised of the catalytic subunit DNA-PKcs and the rel-
atively small Ku proteins (Ku70/80). They promote the simple ligation of the two
broken DNA ends. Damage-induced DSBs, in particular after ionizing radiation,
are rarely re-ligateable, and some end processing might be required that is accom-
plished by other enzymes such as Artemis. The DNA ligase IV-XRCC4 complex
finally re-ligates the two ends. DNA PK–independent DSB end-joining activity
has been observed in cells that have impaired NHEJ activity, the so-called alterna-
tive or B-NHEJ pathway. PARP and Ligase III activity appears to be implicated in
this cellular DSB repair option [54, 55].
The role of ATM seems to be of particular importance in the repair of a certain pro-
portion of DSBs, namely those in heterochromatic regions of the genome [56, 57].
These, judging from the repair kinetics, require more time to repair but influence sur-
vival substantially as indicated by the hypersensitivity to radiation of cells with impaired
ATM function.
Based on the cytotoxic nature of DSBs, cells impaired in any step of the NHEJ
process are highly sensitive to ionizing radiation. Genetic defects in or inhibition
of NHEJ also profoundly affects survival of cells by other DNA-damaging agents
that cause DSBs (directly or indirectly) such as DNA cross-linkers, bleomycin,
and topoisomerase inhibitors.
6.1.6 Homologous Recombination DSB Repair

and the Fanconi Pathway
The HR repair pathway, in contrast to NHEJ (see above), requires homologous

DNA stretches as templates for DNA synthesis and repair [44, 58]. To assure
accurate repair, HR tends to use the sister chromatid as a template, restrict-
ing this pathway to the S and G2 phase of the cell cycle. Together with the FA
pathway, it has a crucial role in the surveillance of replication fork progression.
As anticipated by its requirement for a homologous template, HR mainly deter-
mines survival of S- and G2-phase cells. HR’s involvement upon radiation is not
only required at directly induced DSBs but also at secondarily induced DSBs that
result from replication attempts on nicked DNA [59]. In agreement with such a
function, HR has been shown to determine radiosensitivity in a cell cycle phase-
dependent manner. HR and the FA pathway are also crucial in resolving blocked
replication forks [60]. Such blocks are, for example, caused by interstrand
cross-links (ICLs) that tether the two DNA strands together and prevent separa-
tion during replication. Survival upon other cancer therapeutic agents that induce
replication-blocking lesions such as alkylating agents and topoisomerase inhibi-
tors is strongly determined by the functionality of HR. These blocks must be
repaired or bypassed to allow cells to survive.
A complex multi-member process assures HR-driven repair. In brief, an
ordered assembly of nucleoprotein filaments of RAD52, RAD51, and RAD54
upon RPA coating of the resected DNA promotes and catalyzes homologous DNA
pairing. The extent of the resection at the break site is mediated by the MRN com-
plex and appears to partly define the use of HR instead of NHEJ. Strand exchange
is assisted by the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2, and
XRCC3. In concert, these proteins direct and provide the recombinase activity,
that is, crucial to resolve the complex-branched structures that arise in this pro-
cess. Notably, the products of the breast cancer susceptibility genes BRCA1 and
BRCA2 are involved in the FA and HR repair pathways, assisting DSB and cross-
link repair.
To allow the resolution of blocked replication fork structures, in particular fol-
lowing exposure to DNA cross-linking agents, another replication-associated
repair process is required, the FA pathway. Its members were discovered while
analyzing FA patients, victims of a human genetic disease that is characterized,
among other features [61], by extreme cellular sensitivity to drugs that pro-
duce ICLs. Subsequently, their role and actions in cellular cross-link repair was
revealed.
The products of at least 15 genes have been currently implicated in this path-
way [61–63]. This pathway constitutes a major signaling cascade upon replication
fork stalling: the FA “core complex” consists of at least 8 FA elements (FANCA,
FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) and acts by
realizing the mono-ubiquitylation of the FA ID complex (FANCD2 and FANCI).
This activation of the ID complex allows chromatin binding and is thought to
facilitate DNA repair, in particular HR. The FA-mediated recruitment of the
RAD51 recombinase and the BRCA1-FANCJ helicase activity allows re-establish-
ment of the replication fork. Resolution of stalled replication forks appears to be
also supported by the translocase activity of FANCM that can remodel branched
DNA structures. The FA core complex is regulated by ATR and cell cycle check-
point elements (CHK1) allowing the activation of the pathway [64]. Importantly,
any mutation upstream of the FA pathway that will disrupt the mono-ubiquityla-
tion of FANCD2 will result in the cellular ICL hypersensitivity phenotype.
As illustrated above, upon exposure to DNA-damaging agents, it is the multi-
tude of cellular repair capacities that ultimately determines survival. HR and FA
have been shown to determine the cellular sensitivity to a wide range of cancer
therapeutics. HR-defective cells are hypersensitive to cross-linkers, IR, topoi-
somerase inhibitors, and alkylators as they induce DSBs and replication stalling.
6.2 Strategies Targeting DNA Repair for Cancer Treatment
The requirement for DNA repair and genome maintenance in response to radiation
and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets
for improving response to currently available anticancer regimens. In addition, fre-
quent cancer-specific DNA repair and DDR defects offer tumor-selective therapy
options. Thus, strategies targeting DNA repair pathways represent promising new
avenues to improve outcome in cancer treatment.
Targeting DNA repair pathways in cancer treatment has been proposed in
several settings (Fig. 6.2). Most evidently, inhibition of cellular DNA repair will
cause increased sensitivity to chemotherapeutic agents or radiotherapy [65].
As illustrated above, cellular death upon exposure to most chemotherapeutic
Fig. 6.2 Strategies to improve cancer treatment by targeting DNA damage response and repair.
The overall goal is to increase tumor cell kill (y-axis) while sparing normal tissue by increasing
tumor specificity (x-axis) of the cancer treatment. While some strategies will achieve increased
cell death and thereby an increased probability to control the tumor (for example by applying
DNA-PK inhibitors in combination with radiotherapy or by re-sensitizing chemotherapy-resistant
tumors), others (such as those based on the exploitation of tumor-specific defects) will increase
tumor specificity of cancer therapies. However, in the manner in which current chemo- and radio-
therapy regimens are largely applied, each of the targeted approaches will be only beneficial in a
small fraction of patients with tumors that harbor the respective DNA repair defects. Neverthe-
less, considering the wide range of combination possibilities and the high number of targets and
exploitation opportunities, together they represent a promising avenue in cancer treatment
agents and ionizing radiation partly depends on the repair capacity for the
respective lesions. HR, NHEJ, BER, and DR processes are responsible for the
resistance to these agents. Hence, inhibitors to DNA repair elements might be
useful as dose intensifiers, augmenting the cell-killing properties of many if
not all therapeutic agents. This can be particularly useful in a setting in which
cancer cells obtained resistance to certain agents due to an improved repair capac-
ity. Thus, counteracting marked cancer cell resistance is one strategy in which DNA
repair inhibitors are proposed to act as dose intensifiers. By lowering the tolerance
and inhibiting alternative repair routes while augmenting cell kill, the application
of “intelligent” dose intensification by DNA repair inhibition can also prevent the
development of chemotherapy resistance.
Dose intensification will not be, in general, however, well tolerated, since
chemotherapeutic drugs and radiotherapy doses are often administered at
maximum-tolerated levels. Non-cancerous cells, with few exceptions, are as
much exposed to the chemotherapeutic agents as are the cancer cells. Indeed, it
is the normal tissue response that defines the dose level and use of dose intensi-
fiers. Some tumor properties could, however, provide a tumor-specific effect when
targeting DNA repair. Further, in line with the rationale of most classical cancer
therapeutics, the proliferative nature of tumors is exploitable. For example, target-
ing replication-associated repair pathways such as HR with novel targeted agents
could be beneficial in radiotherapy regimens in which the healthy cells in the irra-
diated area are nonproliferative. A gain could also be expected if chemotherapy
dose limits are not defined by the proliferative cells, allowing dose intensification
by targeting replication-associated DNA repair. Other tumor-specific properties
such as exposure to hypoxic conditions or the altered metabolic status in cancer
cells can offer opportunities to achieve tumor-specific dose intensification and will
be discussed in the following paragraphs.
Another implication of DNA repair-targeting strategies has, however, become
highly relevant. It is suggested that most cancer cells are likely to be defective in
some aspect of DNA repair. Considering the multitude of repair options of healthy
cells, DNA repair defects in cancer cells can be exploited by targeting the remain-
ing repair processes. The combined lethal effect of two genetic variations that are
otherwise non-lethal is termed “synthetic lethality” [66]. In compliance with the
synthetic lethality concept, cancer cells defective in the primary repair pathway are
viable but rely heavily on secondary backup repair for survival. As this is not only
restricted to repair of endogenously produced lesions, this concept also applies
to cells exposed to exogenous damage by exacerbating the effects of chemo- and
radiotherapy in the defective cancer cells only (also may be called synthetic sick-
ness). Despite mutations and genetic defects, the differential expression of DNA
repair proteins or the altered engagement of DNA repair sub-pathways can be a
base of tumor-specific activities. Hence, these tumor-specific DDR and repair
defects offer promising novel approaches to tumor-selective therapy.
Investigation of the functionality of the individual DNA repair pathways in can-
cer cells and the knowledge on which pathways are implicated upon the inhibition
of DNA repair drug targets are necessary to combine these cancer therapy options
in an intelligent manner while focusing on a differential effect in the cancer versus
normal cells.
6.3 DNA Repair Targets
The recognition that DNA repair processes are prime targets for chemo-
and radiosensitization has driven the development of specific inhibitors to
elements of DDR, NHEJ, HR, DR, and BER. The more recent discovery of
tumor-specific targeting opportunities by the inhibition of DNA repair processes
fueled such attempts and has yielded a multitude of DNA repair inhibitors [5].
Some of these novel agents are currently being evaluated in the clinic while oth-
ers are being tested preclinically. Novel DNA repair targets have been identified,
Table 6.1 Targets in DNA damage response and repair
DNA Repair DNA Repair Compounds (DNA
protein target pathway involved repair inhibitors) Context Strategy Development stage Reference
PARP1/2 BER Olaparib Monotherapy BRCA1 or BRCA2 Clinical validation [248]
deficiency
PARP1/2 BER Olaparib Chemosensitizer Combined with cisplatin Clinical validation [249]
and gemcitabine
PARP1/2 BER Olaparib Radiation sensitizer Combined with radiation Preclinical [250]
PARP1/2 BER Veliparib Monotherapy BRCA1 or BRCA2 Preclinical [251]

deficiency
PARP1/2 BER Veliparib Chemosensitizer Combined with Clinical validation [134, 251–
cyclophosphamide, and 255]
carboplatin, preclinical
temozolomide
or topotecan
PARP1/2 BER Veliparib Radiation sensitizer Combined with radiation Preclinical [254, 256–
6 Targeting DNA Repair Pathways for Cancer Therapy
258]
MGMT DR O6-benzylguanine Chemosensitizer Combined with carmustine Clinical validation [259]
MGMT DR O6-benzylguanine Chemosensitizer Combined with Clinical validation [260]

temozolimide
MGMT DR Lomeguatrib Chemosensitizer Combined with Clinical validation [261, 262]
temozolimide
CHK1 DDR AZD7762 Radiation sensitizer Combined with radiation Preclinical [263]
CHK1 DDR AZD7762 Chemosensitizer Combined with irinotecan Preclinical [115]
CHK1 DDR SCH900776 Chemosensitizer Combined with SN38 Preclinical [264]
(continued)
149
Table 6.1 (continued)
150

CHK1 DDR PF-0477736 Monotherapy Myc-driven cancers Preclinical [265]
None (Abasic BER TRC102 Chemosensitizer Combined with pemetrexed Preclinical [142]
site in DNA) (methoxyamine)
None (Abasic BER TRC102 Chemosensitizer Combined with temozolimide Preclinical [133–135]
site in DNA) (methoxyamine)
None (Abasic BER TRC102 Radio- and Combined with Preclinical [136]
site in DNA) (methoxyamine) chemosensitizer iododeoxyuridine +
radiation
None (Abasic site BER TRC102 Chemosensitizer Combined with BCNU Preclinical [137]
in DNA) (methoxyamine)
None (Abasic site BER TRC102 (methoxyamine) Chemosensitizer Combined with manumycin A Preclinical [138]
in DNA)
None (Abasic site BER TRC102 (methoxyamine) Chemosensitizer Combined with fludarabine Preclinical [139]
in DNA)
None (Abasic site BER TRC102 (methoxyamine) Radiation sensitizer Combined with radiation Preclinical [140, 141]
in DNA)
ATR DDR NU6027 Chemosensitizer Combined with PARP inhibitors Preclinical [151]
ATR DDR NVP-BEZ235 Monotherapy Cyclin-E overexpressing cells Compound screen [152]
ATM DDR KU55933 Radiation sensitizer Combined with radiation Compound screen [266]
and preclinical
ATM DDR KU60019 Radiation sensitizer Combined with radiation Preclinical [167]
ATM DDR CP466722 Radiation sensitizer Combined with radiation Preclinical [153]
(continued)
C. Vens and R. W. Sobol
Table 6.1 (continued)
DNA-PK DDR NU7026 Radiation sensitizer Combined with radiation in Preclinical [164]
EGFRvIII-expressing tumors
DNA-PK DDR NU7441 Radio- and Chemosen- Combined with doxorubicin or Compound screen [267]
sitizer radiation and preclinical
DNA-PK DDR DT01 (dbait, DRIIM) Radiation sensitizer Metastatic melanoma with Compound screen [158]
relapsed cutaneous tumors and clinical
validation
DNA-PK DDR DT01 (dbait, DRIIM) Chemosensitizer Combined with 5-fluorouracil or Preclinical [268]
irinotecan
DNA-PK DDR HNI-38 Radiation sensitizer Combined with radiation Preclinical [269]
DNA-PK DDR KU-0060648; A dual Chemosensitizer Combined with etoposide Preclinical [159]
inhibitor of DNA-PK
and PI-3 K
NAMPT NAD+ biosyn- FK866 (Apo866) Chemosensitizer Combined with various Preclinical [190–192]
thesis chemotherapeutics
6 Targeting DNA Repair Pathways for Cancer Therapy
NAMPT NAD+ biosyn- GMX1778 Chemosensitizer Combined with various Preclinical [183]
NAMPT NAD+ biosyn- CB30865 Chemosensitizer Combined with various Preclinical [184]
NAMPT NAD+ biosyn- CHS-828 Chemosensitizer Combined with various Preclinical [185]
151
and compounds that specifically inhibit their activity are sought in order to apply
tumor-specific anticancer strategies.
We will list currently explored DNA repair targets and some of the most
advanced compounds according to their developmental stage (Table 6.1).
Rationales and applied strategies will be discussed while pointing to opportunities
on combinations and other DNA repair targets.
6.3.1 Poly(ADP)Ribose Polymerase
One of the most advanced and applied DNA repair target inhibitors to date are the
PARP inhibitors [67–69]. Since the discovery that cells with defects in the BRCA
genes are selectively killed by the inhibition of PARP, PARP inhibitors have rap-
idly made their way into the clinic [70, 71]. As tumors from carriers of mutations
in the breast cancer susceptibility genes BRCA1 and BRCA2 are almost exclu-
sively composed of such BRCA-defected cells while normal cells of these carri-
ers still carry a functional allele, hence are HR proficient, these PARP inhibitors
achieve tumor-specific kill with little normal cell toxicity. The proposed mecha-
nism that causes such selectivity points to the dependence of BER-inhibited cells
on HR due to secondarily induced cytotoxic DSBs (Fig. 6.3a) [60]. Indeed, early
synthetic lethality screens in yeast indicated such an opportunity revealing a cru-
cial link between BER and HR for cellular survival [72]. Other hypotheses assume
a direct role of PARP inhibitors in replication fork stalling [73]. Most compounds
with PARP inhibitory activity target PAR generation by blocking the catalytic
activity of the enzyme. In principle, these compounds compete with NAD+ for the
PARP catalytic site and are therefore not necessarily specific to PARP1 and could
impact the activity of the other PARP isoforms [74]. Several PARP inhibitors are
in clinical development. To date, the leading compounds Olaparib (AZD2281,
AstraZeneca; originally developed by KuDos) and Veliparib (ABT-888, Abbott)
are probably the two most extensively studied in the clinic whereas at least one
compound, namely Iniparib (BSI-201; Sanofi-Aventis), has been reported to lack
effective PARP inhibitory activity [75, 76].
Although registration of these drugs is still awaiting approval, several studies
have shown their beneficial application [77, 78]. One obstacle could be that these
PARP inhibitors were expected to act in a fraction of tumors, those exhibiting HR
defects due to BRCA1 & BRCA2 mutations only. It should be noted that those early
clinical trials revealed that not all BRCA mutation carriers benefit, indicating that a
certain degree and type of HR defect is required to be exploitable with PARP inhi-
bition. The impact of individual BRCA mutations with respect to HR functionality
and/or PARP inhibitor sensitivity could be variable [79]. The status and propensity
to use the remaining DSB repair mechanism NHEJ, for example via 53BP1 chan-
neling, also influences the extent of PARP inhibitor toxicity [60, 80]. In addition,
other general drug resistance mechanisms such as increased compound rejection by
(a)
(b)
Fig. 6.3 Cellular functions of PARP and opportunities for cancer treatment. a The mode of
action of PARP inhibitors is based primarily on inhibition of the poly(ADP)ribosylation activ-
ity of the enzyme PARP1. As a result, BER or SSBR cannot be executed. In addition, the lack of
poly(ADP)ribosylation is likely to negatively impact chromatin and lesion accessibility. Auto-
poly(ADP)ribosylation of PARP1 is thought to promote its repulsion from DNA. A failure to
recruit downstream BER elements or an increase in lesion shielding by trapping PARP1 on the
DNA further inhibits BER. BER intermediates accumulated upon chemo- and radiotherapy, how-
ever, will then cause replication problems that in turn will induce DSBs. Those will ultimately
lead to cell death via apoptosis and/or mitotic catastrophe in particular if not repaired by HR.
b PARP activity has several cellular roles that can be taken advantage of in cancer treatment.
PARP promotes DNA repair and its inhibition, when combined with chemotherapy or with
tumor-specific defects, enhances tumor cell kill. NAD+ depletion, as a consequence of chemo-
or radiotherapy-induced PARP activity, also induces cell kill. Agents depleting cellular NAD+
levels could indirectly inhibit PARP. Conversely, PARP inhibitors can have the effect of lowering
chemotherapy-induced NAD+ depletion, thereby altering the mode of cell death. Lastly PARP
also has regulatory functions regarding DNA damage–induced gene expression that is often con-
nected to an inflammatory or fibrotic response. PARP inhibitors have been reported to exhibit
anti-inflammatory properties
drug transporters, decreased tumor perfusion, cellular metabolism, or simply phar-

macogenetics of a certain compound might also be responsible for a lack of benefit.
However, new studies indicate that other defects that supposedly result in
impaired HR or DSB repair, such as when ATM is mutated, result in PARP
inhibitor-mediated selective kill [81, 82]. Mantle cell lymphoma harbors ATM
defects and preclinical studies demonstrate sensitivity of these tumors to PARP
inhibition [83, 84]. Other genetic analyses indicated frequent ATM mutations in
human cancer. These studies therefore warrant the application of PARP inhibi-
tors for cancer treatment in a much larger patient population. In agreement with
a synthetic lethal interaction with HR-driven processes, cells with an impaired FA
pathway are hypersensitive to PARP inhibition, thus further enlarging the potential
patient population that might benefit from such therapies [81]. Some studies indi-
cated an impact of PTEN deletion on HR via a reduction of Rad51 or Rad51 para-
logs, a condition that could be exploited by PARP inhibition [85, 86]. However,
this could only be partly confirmed in a separate study [87]. HR impairment was
observed when cells experience hypoxia [88, 89] which sensitized them to PARP
inhibition. PARP inhibitor sensitivity in hypoxic cells or those with defects in
FA, ATM, or PTEN is, however, generally less pronounced than when BRCA-
defective. Yet, a benefit, in particular when combined with radio- or chemotherapy,
can be anticipated since PARP inhibitors appear to enlarge the therapeutic window
and could spare healthy cells from chemo- to radiosensitization.
Historically, prior to the discovery of specific killing of tumors with defective
HR, PARP inhibitors have been actively studied in preclinical and clinical investi-
gations to potentiate the cytotoxic effects of chemo- and radiotherapy (Fig. 6.3a).
PARP inhibitors, due to their DNA repair-inhibiting properties, are radiation sen-
sitizers and are highly effective in sensitizing cells to chemotherapeutic agents
such as DNA alkylators (e.g., TMZ) and topoisomerase I inhibitors (irinotecan
and topotecan). Combining PARP inhibitors with chemo-/radiotherapy could be
beneficial in a large patient population with the added advantage of single-agent
activity in a fraction of patients that happen to have tumors with exploitable DNA
repair defects. As noted above, in such dose-intensification strategies, the benefit
of tumor-specific killing needs to be evaluated against normal tissue effects that
are connected to the use of DNA repair inhibitors. To exemplify, since the radio-
sensitizing effect of PARP inhibitors is most effective in proliferating cells, PARP
inhibitors have been proposed in clinical radiotherapy settings in which normal tis-
sue toxicity within the radiation field is not defined by a highly proliferating (stem)
cell fraction such as in the treatment of lung cancer and glioblastoma [90, 91].
Radiation-induced lung toxicity is strongly determined by inflammatory and fibrotic
processes. Therefore, PARP inhibition might impact lung toxicity in a beneficial
way by altering the inflammatory DDR [92, 93].
Despite crude chemosensitization and synthetic lethal activity, other strate-
gies exploit the BER inhibitory properties of PARP inhibitors. These strategies
aim to prevent the quick development of resistance to alkylators such as temo-
zolomide (TMZ). Resistance to TMZ is associated with MGMT expression and
MMR defects (see above). PARP inhibitors, however, are able to (re-) sensitize
MMR-defective cells to the anti-tumor effect of TMZ [94–98] thereby counteract-

ing the resistance to TMZ.
Other PARP inhibitor combinations have been pursued. Assuming that BRCA
defects do limit HR functions but do not fully impair HR, BER inhibition and con-
sequently increased engagement of HR might be the basis for the observed syner-
gistic cytotoxicity of Olaparib and Cisplatin [99, 100]. Depending on the cytotoxic
agents and genetic background, BRCA-defected cells die by apoptosis or mitotic
catastrophe upon PARP inhibition.
DNA damage–induced and PARP-activation-mediated consumption of NAD+
has been implicated in the increase in genotoxin-induced cell death [74, 101].
The PARP1 and PARP2 proteins [102] act as sensors of DNA damage such as
DNA single-strand breaks and become hyperactivated, consuming NAD+ as a sub-
strate to synthesize PAR [103]. Consumption of NAD+ after DNA damage leads
to ATP depletion, likely due to continued re-synthesis of NAD+ as well as ongoing
cellular utilization of NAD+ and ATP for metabolic functions [74, 101]. Following
up on the observation that cell death due to BER inhibition and the accumulation
of BER intermediates results in PARP hyperactivation [103], it was shown that
the combination of BER and NAD+ biosynthesis inhibition significantly sensi-
tizes glioma cells to TMZ. Dual targeting of these two interacting pathways (DNA
repair and NAD+ biosynthesis) may prove to be an effective treatment combina-
tion for patients with resistant and recurrent GBM. Thus, in summary, several
distinct roles of PARP including the promotion of DNA repair and its impact on
damage-induced gene expression as well as the relationship to NAD+ levels can
be exploited for cancer therapy (Fig. 6.3b).
6.3.2 O6-Methylguanine-DNA Methyltransferase
As described in Sect. 6.1 above, MGMT is the sole protein responsible for the
repair of O6-alkylguanine lesions (formed by chemotherapeutic agents such as
TMZ). Tumors with elevated MGMT expression are resistant to TMZ and related
chemotherapeutic agents, and so, an active area of investigation has been the devel-
opment of MGMT inhibitors. If MGMT is inhibited (or MGMT is not expressed),
the tumor becomes highly sensitive to the agent (provided the tumor cell is
proficient in MMR—see Sect. 6.1.2). There have been multiple methodologies
proposed to inhibit or overcome resistance mediated by MGMT expression in the
tumor as well as to prevent sensitivity of normal tissue (primarily hematopoietic
cells) [12]. As we alluded in Sect. 6.1.2, the mechanism of action of MGMT in the
repair or de-alkylation of guanine suggested O6-benzylguanine as an ideal inhib-
itor [104]. This inhibitor, also called BG, is an analogue of the O6-alkylguanine
base and contains a benzyl ring instead of an alkyl group. The BG compound read-
ily reacts with MGMT, and the benzyl moiety is transferred to Cys145 as shown
(Fig. 6.4), releasing free guanine and rendering MGMT inactive. In some cases,
this has been shown to trigger ubiquitylation and proteasome-mediated destruction
Fig. 6.4 Schematic
representation depicting
the mechanism of action
of the MGMT inhibitor
O6-benzylguanine (BG).
The benzyl moiety of BG
is transferred to the Cys145
residue in MGMT, releasing
free guanine, rendering
MGMT inactive. In some
cases, this has been shown
to trigger ubiquitylation
and proteasome-mediated
destruction of MGMT
of MGMT [105]. Further details on BG and related analogues to inhibit or regulate

MGMT can be found elsewhere [106]. A second inhibitor with greater inhibitory
activity that has been widely tested is the alkylguanine analogue 6-[4-bromo-2-
thienyl]methoxypurin-2-amine [107], also called lomeguatrib [108, 109].
A significant challenge with all or most chemosensitizers is the observed
increase in normal cell/tissue sensitivity or cell death. This has been addressed
with regard to MGMT by the use of an ex vivo gene therapy approach to express
a mutant of MGMT in the bone marrow or hematopoietic stem cells (cells are
modified ex vivo and re-delivered to the patient), as described [12]. This is feasi-
ble since the G156A or P140K mutants of MGMT are 60-fold and 500-fold more
resistant to BG than the wild-type protein, respectively [110].
6.3.3 Cell Cycle Checkpoints
DNA damage activates checkpoints to arrest proliferation. Normal cells have

intact G1, S, and G2 checkpoints that are mediated by the ATM/CHK2 and
ATR/CHK1 pathways. Owing to mutations in the p53 or pRB tumor suppressor
genes, cancer cells, however, lack a G1 checkpoint and as a result, rely on G2
checkpoints to prevent cell division and propagation of the damage. Thus, the
S/G2 checkpoint is an attractive target for cancer-specific sensitization to DNA-
damaging agents [111]. In order to exploit the cancer-specific defects, inhibitors
have been developed that abrogate the G2 checkpoint. CHK1 has been a prime
target for such attempts, as activated CHK1 mediates the arrest by phosphorylat-
ing Cdc25A and Cdc25C leading to their degradation and inactivation that oth-
erwise promote S-phase progression and entry into mitosis. Loss of the G2 cell
cycle checkpoint, despite the presence of unrepaired damage, is thought to pro-
voke mitotic catastrophe and ultimately cell kill. Consistent with this idea, loss of
intra-S or G2/M checkpoints increases the cytotoxicity of DNA-damaging agents
such as ionizing radiation and cisplatin. CHK1 knockdown sensitizes cells to
5-fluorouracil, doxorubicin, and etoposide. Despite the proposed mechanism that
CHK1 inhibition causes sensitization in p53-defective cancer cells due to the G2
block abrogation in a G1 block-deficient background, the data to support this are
contradictory [112–114]. Xeno-transplant studies on human triple-negative breast
cancer demonstrated the benefit of combining irinotecan with CHK1 inhibitors,
inducing checkpoint bypass and apoptosis. A role of p53 was supported by the
gain of CHK1 sensitization after p53 knockdown in the resistant tumors [115].
A more complicated rationale, however, argues that a series of DDR defects in
tumors should be considered and could be exploited by the inhibition of CHK1.
These are based on the secondary effects of CHK1 inactivation on replication and
are discussed below.
A large battery of CHK1 inhibitors is available to support cancer treatment in
combination with radio- and chemotherapy [116]. Older CHK1 inhibitors such as
UCN01 were not very selective but did demonstrate potent chemosensitization to
cisplatin and camptothecin. More potent and specific CHK1 inhibitors were devel-
oped; however, concomitant CHK2 inhibition to some degree is common to most
CHK1 inhibitors. In general, cells appear to depend on a functional G2 checkpoint
when exposed to agents that cause replication stress. Hence, potentiation is greatest
to cross-linkers, topoisomerase I poisons and nucleoside analogues such as gemcit-
abine. Consistent with replication stress hypersensitivity when G2 arrest is abrogated,
PARP inhibitors also cause problems. PARP inhibition can cause a G2 checkpoint
dependence and the combination of PARP inhibitors with CHK1 inhibitors is syn-
thetic lethal [117]. These cellular sensitivity features are the basis for the clini-
cal trials testing the combination of older (UCN01) or later generation compounds
(for example AZD7762, AstraZeneca; PF477736, Pfizer; LY2606368, Eli Lilly and
SCH900776, Schering Plough) with cisplatin, topotecan, and gemcitabine. For a
more detailed review, see [116]. Unfortunately, safety requirements as assessed in
these initial studies were not met in at least one compound (AZD7762) [118]. Similar
to chemotherapy regimens, CHK1 inhibitors prevent ionizing radiation (IR) induced
S and G2 arrest and demonstrated some potential to radiosensitize in a p53-depend-
ent manner. CHK1 is upregulated in Myc-overexpressing lymphomas, and single-
agent activity is expected since CHK1 inhibition is cytotoxic to these cells.
Interestingly, recent data indicate that CHK1 activity might prevent
replication-induced DNA damage or is implicated in DNA repair [119, 120]. A
conversion of halted replication forks (induced by the chemotherapeutics) into

persistent and cytotoxic DSBs has been postulated. The CDK-driven unsched-
uled initiation of replication origins can be accounted for with regard to this
DSB induction and explains chemosensitization beyond G2/M checkpoint abro-
gation [120]. The notion that CHK1 inhibition promotes replication stress
merits their evaluation in synthetic lethal strategies exploiting tumor-specific DNA
repair defects [121].
6.3.4 AP-Endonuclease 1
Targeted knockout (KO) of AP(apurinic/apyrimidinic)-endonuclease 1 (Ape1

or Ref1) in mice is lethal [122], and deletion of the Ape1 gene in mouse cells
induces apoptosis within 24 h [123]. Interestingly, granzyme A(GzmA)-mediated
cell death is enhanced by GzmA-mediated cleavage of Ape1. It is sug-
gested that the proteolysis of APE1 enhances GzmA-mediated cell death
by promoting apoptosis [124]. In human cells, RNA interference–mediated
depletion of APE1 suppresses cell and tumor growth [125]. These and many
other studies therefore support the development of APE1 inhibitors to enhance
radiation and chemotherapy response [126, 127].
The first such compound to be tested clinically that impacts APE1 function
in BER is methoxyamine (TRC102; Tracon Pharmaceuticals). Methoxyamine
hydrochloride (MX) was first suggested to be a mutator, inducing the formation of
5,6-dihydro-6-methoxyaminecytosine residues in DNA [128]. It was subsequently
determined that MX traps aldehyde groups, forming a stable intermediate [129].
The AP site in DNA is not a chemically unique species but exists as an equilib-
rium mixture of the ring-closed cyclic hemiacetals and open-chain aldehyde, and
hydrate forms [130]. The transient open-chain aldehyde form is reactive with
aldehyde-specific reagents such as methoxyamine, allowing the trapping or quan-
titative measurement of AP sites in DNA [131]. It was subsequently determined
that the reaction between MX and the open-chain aldehyde form of an AP site
blocks repair of DNA base lesions by BER. The trapped AP site is a highly cyto-
toxic intermediate and sensitizes cells to the cytotoxic effects of alkylating agents
[132–134] and other DNA-damaging agents that may give rise to spontaneous
or enzyme-mediated AP sites, such as TMZ [135], iododeoxyuridine + radiation
[136], BCNU [137], manumycin A [138], fludarabine [139], radiation [140, 141],
and pemetrexed [142]. Interestingly, elevated expression of the downstream BER
protein DNA polymerase β (Polβ) reverses MX sensitization of alkylating agents,
suggesting that the cleaved open-chain aldehyde may be the preferred MX sub-
strate [134]. MX is in the clinic under the brand name TRC102 and is undergoing
clinical evaluation in Phase II trials for solid tumors as a sensitizer to Temodar®
(TMZ) or Alimta® (Pemetrexed) and in hematologic malignancies with Fludara®
(Fludarabine).
In parallel, there are a number of groups developing direct APE1 active site
inhibitors [143–146], and an overview of the development of APE1 inhibitors has
just been reported [147]. As might be expected, many of these APE1 inhibitors
are themselves cytotoxic and enhance the cytotoxicity of DNA-damaging agents
such as TMZ [144, 145] or other alkylating agents [143]. Although Ape1 KO or
GzmA-mediated Ape1 cleavage in mouse cells induces apoptosis, the cell death
mechanism(s) induced by these recently developed APE1 inhibitors has yet to be
resolved. Interestingly, the cell death mechanism triggered by some of these APE1
inhibitors may involve the accumulation of DNA DSBs since it was observed that
the compounds are more cytotoxic in cells deficient in the HR proteins BRCA1 or
BRCA2 [148].
6.3.5 Ataxia Telangiectasia-RAD3 Related
Similar strategies and rationales that apply to the CHK1 target also apply to
ATR. A direct link connects ATR with CHK1 [121]. After initial Rad17 binding,
Claspin-loaded single-strand DNA mediates the activation of CHK1 by ATR upon
replication stress. ATR is essential in the surveillance of replication stress, espe-
cially when associated with exposed single-stranded DNA mostly connected to
replication problems. Stalled replication forks can collapse which results in the for-
mation of DSBs, a signal that mainly, but not exclusively, triggers ATM whereas
the single-stranded DNA-induced damage response appears to be ATM independ-
ent. Endogenous and exogenous damage induces replication stress that requires
a proficient ATR/CHK1 response for survival. One prevalent rationale to inhibit
ATR for cancer therapy is to exacerbate the levels of replication stress that might
be augmented in cancer cells due to inherited defects in the DDR and DNA repair
pathways. Cancer cells are exposed to a higher load of replication stress com-
pared to normal cells and will suffer most from targeting ATR. In addition, since
targeting replication-associated processes, any selective killing property will be
augmented in highly proliferative cells. Such a strategy is supported by two obser-
vations: (1) the activated DDR found in early stages of tumorigenesis [149, 150]
indicates an increased load of “endogenous” DNA damage and (2) the discovery
that a wide variety of oncogenes generate such damage.
Based on this proposed endogenous damage (replication stress)-induced kill-
ing mechanism, such ATR inhibitors have been proposed to act as single agents.
However, combination strategies similar to those for the CHK1 inhibitor are envi-
sioned. As noted before, the response of normal cells should be carefully taken
into consideration. A few compounds have been pursued by industry. The ATR
inhibitor NU6027 appears to impair HR and enhances the cytotoxicity of PARP
inhibitors [151], a theme that is consistent to other DDR inhibitors. One com-
pound (NVP-BEZ235) has been recently discovered and found to trigger pref-
erential cell kill in cyclin-E overexpressing cells [152] and is awaiting entry into
clinical trials for cancer therapy.
6.3.6 Ataxia Telangiectasia-Mutated
ATM inhibitors are less advanced in their clinical development than PARP or
CHK1 inhibitors. Since involved in DSB repair, NHEJ, and HR, ATM inhibition
results in reduced cellular DSB repair activity and cell cycle checkpoint defects.
As a result, ATM inhibitors are highly potent radiosensitizers while exhibiting
little toxicity on their own. Therefore, they have been proposed to be applied in
this context [153]. Several ATM-inhibiting compounds have been identified and
pursued in preclinical studies: KU55933 and KU60019 (KuDos/AstraZeneca) and
CP466722 (Pfizer).
Combination strategies for ATM inhibitors have been proposed to enhance the
cytotoxicity of PARP inhibitors (see above). Interestingly, p53 disruption in normal
cells sensitized those cells to the combination of PARP and ATM inhibitors [83].
Due to its crucial role in DSB repair, the inhibition of ATM will radio- and chemo-
sensitize most cells, including normal cells, with no evident DNA repair defects.
However, synergistic cytotoxicity can be observed under certain conditions that
could be exploited for tumor-targeted strategies. Cells with BER defects for exam-
ple rely on secondary DSB repair pathways such as HR for survival upon dam-
aging agents. Similar to the PARP inhibitor/HR-defect synthetic lethal interaction,
the conversion of unrepaired SSBs to DSBs could be a mechanism that underlies
such dependence. BER defects of different kinds have been reported in tumors, and
it is suggested that inhibition of DSB repair processes will be beneficial in such a
setting [154].
6.3.7 DNA-Dependent Protein Kinase
DNA-PK is crucial to DSB repair as well as cellular survival following radiation

and topoisomerase II poisons, and so, DNA-PK has been a long sought target for
drug development [155]. Kinase activities are relatively easy to target, and drugs
inhibiting DNA-PK activity have been available for some time. One should note
that a considerable fraction of first generation tyrosine kinase inhibitors do also
inhibit DNA-PK to some extent. The radiosensitization phenotype by some of these
agents should therefore be re-evaluated with respect to its origin (see below). Some
examples of currently pursued inhibitors are NU7026 and NU7441 [156, 157].
Other targeting strategies apply short modified DNA molecules that are supposed
to interfere in DNA-PK signaling (DT01, DNA therapeutics) or short peptides that
resemble Ku80 (HNI-38) and disrupt the interaction with DNA-PK [158]. The dual
DNA-PK and PI3-K inhibitor KU-0060648 takes advantage of the inhibition of two
cellular processes central to promoting survival upon chemo- and radiotherapeu-
tic insults. Surprisingly, this inhibitor enhanced etoposide-induced xenograft tumor
growth delay substantially without exacerbating etoposide toxicity in mice indicat-
ing that the combination provided some tumor specificity [159].
Most DNA-PK-inhibiting agents are very potent radiosensitizers, enhancing

clonogenic cell kill markedly [160]. Radiosensitization by one compound, the
DNA-PK inhibitor BEZ235, has been shown to be associated with an accelerated
p53-dependent senescence [161]. Although proposed as radiation dose intensifiers,
DNA-PK inhibitors, similar to ATM inhibitors (though often to a greater extent),
will sensitize normal tissue to radiation at least as much as it does sensitize the
tumors. However, some DNA repair defects inherent to tumors might provide
some therapeutic gain when applying low inhibitor doses that could spare normal
tissue. Opportunities exist for example in the treatment of ATM-deficient tumors
since DNA-PK inhibition induces tumor cell kill in ATM-deficient cells [162].
Considering that disruption of the catalytic activity of DNA-PK confers severe
immunodeficiency, DNA-PK inhibitors will have to be evaluated carefully for tox-
icities in preclinical studies before entering clinical trial [163]. Tumor-targeted
delivery strategies could, however, make use of these highly potent radiation dose
intensifiers. Other strategies exploiting the differential expression or use of DNA-
PK and Ku-dependent DNA repair in some tissues with respect to the tumors
could apply these inhibitors successfully. An example for this is the sensitization
of EGFRvIII-overexpressing cells with DNA-PK inhibitors [164].
6.4 Indirect DNA Repair Modulators
6.4.1 Signaling Pathways, EGFR and the PI3K/AKT Pathway
Targeting the cellular signaling pathways via EGFR, PI3K/AKT, or MAPK can
modulate the DNA repair status of cells. The effects are multiple and involve dif-
ferent pathways. Cellular signaling influences DNA repair in multiple ways includ-
ing changes in the expression levels of crucial DNA repair proteins such as RAD51
or the regulation of the activation or translocation of enzymes and kinases such as
DNA-PK. For example, the inhibition of the MAPK pathway can lead to reduced
DSB repair by both homologous and non-homologous pathways [165, 166]. Links
between the AKT, MAPK, and EGFR pathways and DNA repair have been found,
particularly with DSB repair by NHEJ or HR [166–171]. Hence, targeting these
pathways can result in the concomitant inhibition of DNA repair.
Based on knowledge that the PI3K pathway promotes DNA repair and survival,
inhibitors of AKT have been evaluated as chemosensitizers for alkylating agents
[172, 173]. AKT inhibitors such as LY294002 and wortmannin radiosensitized and
caused enhanced sensitivity to alkylating agents such as TMZ, or the cross-linkers
cisplatin in various human tumor cell lines.
The suppression of ATR/CHK1 checkpoints has been observed upon treatment
with wortmannin (a fairly non-specific PI3K inhibitor). For more details, see [174]
and references within. Note that an influence on Rad51 expression or on NHEJ via
DNA-PK has also been observed.
Co-targeting PARP and the PI3K pathway has been shown to synergistically
decrease growth and further induce apoptosis [175]. The molecular mechanism of
this combination effect is not completely clear but could originate from secondary
effects on DNA repair pathways and the DDR from the inhibition of cellular sign-
aling in human cancer cells. Further detail on AKT inhibitors is discussed in more
detail in Chap. 13.
6.4.2 Proteasome Inhibitors
Proteasome-mediated destruction and removal of key DNA repair and DDR pro-
teins are an essential aspect of the cellular response to genotoxins [4, 176]. Failure
to remove critical DNA repair and DDR proteins in a timely fashion effectively
halts the process, triggering a cascade of events leading to cell death [177].
As the endpoint in ubiquitin-mediated protein turnover, targeting the proteasome
is a high-profile target to enhance DNA damage–induced cell death [178, 179].
Further detail on the proteasome pathway and proteasome inhibitors is discussed
in more detail in Chap. 12.
6.4.3 NAD+ Biosynthesis Inhibitors
The metabolite NAD+ is an essential substrate for all PARPs [74]. Since PARP1,
as well as PARP2 and PARP3, is a critical protein involved in DNA repair
and the DDR [36, 103], we have included NAD+ biosynthesis inhibitors as an
indirect DNA repair modulator. All of the NAD+ biosynthesis inhibitors developed
to date target NAMPT, a pivotal and rate-limiting enzyme in the salvage pathway of
NAD+ biosynthesis [180]. NAMPT catalyzes the synthesis of nicotinamide mononu-
cleotide (NMN) from nicotinamide to 5-phosphoribosyl-1-pyrophosphate [181]. The
resulting NMN is then converted to NAD+ by one of the three isoforms of NMNAT
(1, 2 or 3), located in the nucleus, cytosol, or mitochondria, respectively [182]. The
most studied inhibitor of NAMPT is FK866 (also referred to as Apo866) although
several other NAMPT inhibitors have been reported including GMX1778 [183],
CB30865 [184], and CHS-828 [185], and one group is actively screening for NAMPT
inhibitors [186]. FK866 binds at the interface of the NAMPT dimer [187] and effec-
tively inhibits NAMPT and depletes cellular NAD+ levels within 24 h, inducing apop-
tosis [188] although the overall level of cell death may be offset by FK866-mediated
induction of autophagy [189]. Further detail on the shift of cell death triggered by
NAD+ biosynthesis inhibitors is discussed in Chap. 2.
As an NAMPT inhibitor, FK866 does have a chemo-potentiating effect
[190–192] that is more pronounced when combined with a second DNA repair inhibi-
tor [133] or when combined with TRAIL [193]. Interestingly, it has been suggested
that some tumors are deficient in the NAPRT1-mediated NAD+ biosynthesis pathway
that is responsible for generating NAD+ from nicotinic acid (NA) [183, 194]. In sum,
these studies suggest that NAMPT is a potentially valuable target to impact both
metabolism and DNA repair and may provide a type of synthetic lethality based on
DNA repair and NAD+ biosynthesis status [133, 195].
6.5 Promising Future Targets and Opportunities
6.5.1 ERCC1
An emerging DNA repair target protein is ERCC1, encoded by the excision

repair cross-complementing rodent repair deficiency, complementation group 1
gene (ERCC1) [49, 50]. As an essential NER protein, elevated ERCC1 expres-
sion enhances the repair of cisplatin-induced genomic DNA damage [15] and
decreased expression is suggested to improve response to platinum-based
therapies [196]. As described above (Sect. 6.1.4), ERCC1 exists as an obligate
heterodimer with the protein XPF to yield an essential NER structure-specific
nuclease ERCC1/XPF [15]. The ERCC1/XPF heterodimer is recruited to func-
tion in NER by interaction with the NER protein XPA [197] although multiple
interaction sites are found to facilitate recruitment of ERCC1/XPF to the NER
complex [198]. In this regard, it has been suggested that the function of ERCC1
in NER can be disrupted by interfering with the interaction between ERCC1
and XPA [199]. Alternatively, since heterodimer formation (ERCC1/XPF) is
needed to maintain the stability of ERCC1 [200], it might be feasible to inter-
rupt the heterodimer complex and force ERCC1 proteolysis [201]. Finally, one
might also consider deregulating expression of ERCC1 through modulation of the
RAS kinase or ERK1/2 pathways [202]. Using the Raf kinase and VEGF receptor
inhibitor sorafenib reduces ERCC1 levels and effectively enhances the response
to radiation and chemotherapy [203]. Of course this treatment would have multi-
ple effects on gene regulation.
6.5.2 DNA Polymerases
There are 15 DNA polymerases in the human cell [204] and several have been
suggested to be viable drug targets either because they facilitate repair of DNA
damage or may be overexpressed in cancer [205]. DNA polymerase β (Polβ) is a
member of the X family of DNA polymerases [204, 206] and is an essential BER
protein, as detailed above. Polβ has two active sites and several critical functional
domains that may be considered as targets to inhibit Polβ and BER. Further, the
central and pivotal role of Polβ in BER implicates this protein as a prime target
to enhance the response to chemotherapeutic agents or radiation. Although many
inhibitors of Polβ have been developed and characterized very few, if any, show
specificity and all are cytotoxic even in Polβ KO cells. Three recent reviews on
Polβ inhibitors should be referred to for more detail [195, 207, 208].
Other polymerases have been also identified as potential targets. A siRNA screen
and large-scale tumor sample analysis identified DNA polymerase theta (POLQ) as a
target that determines radiosensitivity and is overexpressed in tumors. POLQ depletion
radiosensitized these cells arguing for the development of POLQ inhibitors [209–211].
6.5.3 DNA Glycosylases
DNA glycosylases are the initiating enzymes in BER (see above). In general,
DNA glycosylases probe the DNA helix for base lesions by a base-flipping
mechanism, interrogating each base as it is flipped out of the major groove as
the enzyme moves along the DNA helix. Identification of the lesion then pro-
motes hydrolysis [212]. As discussed above, radiation and chemotherapeu-
tic agents induce the formation of BER substrates and many of these base
lesions block replication and are therefore cytotoxic. For example, the methyl-
ated base 3-methyladenine, induced by the chemotherapeutic agent TMZ and
repaired by MPG, triggers lesion-induced apoptosis as well as sister chroma-
tid exchange, chromatid and chromosome gaps and breaks, and S-phase arrest
[213]. As such, it has been hypothesized that inhibition of MPG would enhance
response to TMZ and other alkylators. Similarly, UNG, the primary glycosy-
lase responsible for the removal of deoxyuracil, is reported to govern the effi-
cacy of pemetrexed [142], and therefore, UNG might be considered a potential
target to enhance the response to this chemotherapeutic agent. Finally, it was
recently demonstrated that the DNA glycosylase OGG1 is in a complex with
the BER protein PARP1. The OGG1–PARP1 complex promotes PARP1 activ-
ity whereas loss of OGG1 suppressed PARP1 activity [214]. From this study, it
might be suggested that an inhibitor designed to disrupt the OGG1–PARP1 inter-
action may function as a pseudo- or indirect PARP1 inhibitor. However, there are
multiple challenges to developing effective DNA glycosylase inhibitors such as
the large overlap in enzyme substrate specificity [36, 37] and the requirement for
some glycosylases in normal cell survival [215, 216] and immunoglobulin class-
switch recombination [217]. To date, we know of no DNA glycosylase inhibitors
with the exception of Ugi, a virally encoded UNG inhibitor [218].
6.5.4 DUBs
As with most posttranslational modifications, both the synthesis and removal of

the ubiquitin modification is a highly regulated process [219, 220]. Recently, sev-
eral studies have revealed the potential for targeting DUBs to enhance radiation
and chemotherapeutic response [221, 222]. Toward that end, several groups have
reported the development of DUB inhibitors that are effective in preclinical studies
[223–226].
6.5.5 HR/FA
HR and FA strongly determine cellular survival upon cross-linkers, topoisomerase

poisons, alkylators, and radiation. These pathways are mostly engaged in a prolif-
eration-dependent manner with less influence on damage repair and response in
quiescent cells. Thus, targeting HR and FA can be an effective means to potentiate
radio- and chemotherapy [227].
Novel agents targeting RAD51, albeit indirectly, are now emerging in clinical tri-
als. MP470 (Supergen) is a tyrosine kinase inhibitor with RAD51 protein–suppressing
properties. More specific inhibitors are sought in high-throughput screening efforts of
which some provided some compounds with convincing specificity and potency [228].
The value of HR inhibition can be deduced from experiments in which ele-
ments of the HR or FA pathway such as RAD51 or BRCA2 have been downreg-
ulated, supporting the potential of HR/FA-targeting strategies. Downregulation
of Rad51 or BRCA2 sensitized glioma cells to the cytotoxic effects of TMZ or
nimustine in cells with low MGMT levels [229]. The marked resistance of glio-
mas to chemo- or radiotherapy regimens that might be partly defined by HR or
increased Rad51 levels can be defined by targeting RAD51, indicating a similar
strategy option to counteract resistance by HR inhibition [230].
FA- and HR-defected cells have been found to lack the increased radio-resist-
ance that is observed in hypoxic areas of the tumors [231]. Inhibitors targeted to
HR or FA will not only preferentially sensitize proliferating cancer cells but also
hypoxic tumors to radiation. Owing to less-effective DSB induction under low
oxygen concentration, radiotherapy often fails in patients with hypoxic tumors.
Tumor specificity of HR-targeting drugs will be increased when applied to
BER-defected cancer cells as illustrated above. BRCA2-knockdown radiosensitizes
cells that express a dominant-negative Polβ mutant to a greater extent than isogenic
controls [232]. Similarly, NHEJ-defected cancer cells are expected to rely on HR
for survival upon genotoxic threats. Together, these interactions and data support
strategies applying HR- or FA-inhibiting drugs for tumor-specific sensitization.
6.5.6 Poly(ADP)ribose Glycohydrolase
Poly(ADP)ribose glycohydrolase (PARG) [233] is an enzyme involved in BER

and is the major factor responsible for removing the PAR molecules synthesized
by PARP1 and PARP2, among other PARPs [234]. As seen with PARP1, PARG
is also a substrate for caspase-3 [235], supporting the significance of PAR metab-
olism in cell death processes. Mouse KO studies or RNA interference studies to
knockdown PARG in human cells have suggested that blocking PAR degrada-
tion is an effective means to modulate chemotherapy and radiation response
[134, 236, 237]. Pre-clinical evaluations using early phase small-molecule inhibi-
tors such as gallotannin, tannic acid, and related small molecules have implicated
PARG in genotoxin or chemotherapy sensitivity [238–240]. The inhibitor GPI-
16552 (N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide) has shown early suc-
cess in cell and mouse models as a chemotherapy sensitizer [97] but has mostly
been effective to reduce inflammation [241–245]. Recent drug discovery efforts
seem promising and have yielded PARG inhibitors with increased specificity and
cell permeability [246, 247].
6.6 Summary and Concluding Remarks
Inducing tumor-specific or selective cell death is a significant challenge in the

treatment of cancer. Although radiation and most chemotherapeutic treatments
are designed to induce significant genotoxic damage to the tumor cells, a multi-
tude of DNA repair and DDR gene products respond to the presence of genotoxic
stress (DNA damage) by orchestrating a massively complex cellular response to
survive and repair the genome insult. Most DNA repair pathways focus on specific
damage types (e.g., DSBs), but there is considerable overlap and backup capac-
ity when evaluating the overall involvement of DR, BER, MMR, NER, HR, and
NHEJ proteins in response to these agents (radiation and chemotherapy). It has
been observed that some cancer cells can survive the loss of key DNA repair pro-
teins (e.g., BRCA1/2). In fact, we have come to realize that many cancer cell types
are defective in one or more DNA repair or DDR genes/proteins, precipitating the
need to identify the key stress nodes in these defective cells in response to radia-
tion and classical chemotherapy treatments. With this in mind, we have described
the molecular pathways that participate in the repair of DNA damage induced by
radiation and chemotherapeutics. Further, we present novel therapeutic strategies
being considered to exploit inherent weaknesses in tumor cells such as defects in
one or more DNA repair pathways that may provide the opportunity to selectively
increase tumor-specific cell death.
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Chapter 7
Molecular Chaperones and How Addiction
Matters in Cancer Therapy
Monica L. Guzman, Maeve A. Lowery, Tony Taldone, John Koren III,

Erica DaGama Gomes and Gabriela Chiosis
Abstract Constitutive expression of HSP90 in normal cells is required for its

evolutionarily conserved housekeeping function of folding and translocating cel-
lular proteins to their proper cellular compartment. Under the stress of malignant
transformation, cellular proteins and networks become perturbed requiring spe-
cific maintenance by a stress-modified HSP90. We here detail the many functions
this oncogenic HSP90 takes on in cancer cells and discuss how the addiction of
cancer-altered networks on HSP90 can be harvested therapeutically by small mol-
ecule HSP90 inhibitors.
7.1 HSPs as Buffers of Cellular Instability
To allow for normal functioning, cells use intricate molecular machineries com-
prised of thousands of proteins programmed to execute well-defined functions.
Dysregulation in these pathways may lead to altered functions that confer a patho-
logic phenotype. In cancer, not one but multiple pathways and molecules lose their
inherited state leading to cells characterized by many defects such as aberrant pro-
liferation, cell cycle, invasive potential, and evasion of apoptosis [1]. While at the
M. L. Guzman
Division of Hematology and Oncology, Weill Cornell Medical College, NY, USA
M. A. Lowery
Department of Medicine, Gastrointestinal Service,
Memorial Sloan-Kettering Cancer Center, NY, USA
T. Taldone· J. Koren III· E. D. Gomes· G. Chiosis (*)
Department of Medicine, Breast Cancer Service,
Memorial Sloan-Kettering Cancer Center, NY, USA
T. Taldone· J. Koren III· E. D. Gomes· G. Chiosis
Department of Molecular Pharmacology and Chemistry, Sloan-Kettering Institute, NY, USA
182 M. L. Guzman et al.
cellular level such dysregulation may be advantageous (i.e., increased survival), at

the molecular level these changes occur at a cost of protein robustness that further
translates into local energetic instability. To regain a pseudo-stable state, cells must
co-opt chaperones, such as the heat shock proteins (HSPs), to bind such aberrant
proteins and maintain them in a functional conformation. By these mechanisms,
HSPs buffer the molecular instability and help maintain a functional cellular state
under the transforming pressure [2]. As such, cancer cells often express high lev-
els of several HSPs, which augment the aggressiveness of tumors and also allow
cells to survive lethal conditions, including killing by therapies. In addition to con-
ferring resistance to treatment, elevated HSP expression also facilitates cancer by
inhibiting programmed cell death and by promoting autonomous growth [3–5].
It is not surprising that the cell uses the HSPs to buffer proteome instability.
After all, the HSP chaperones are the evolutionarily evolved cellular machinery
that maintains protein folding and functional conformation [6]. They are also one
of the most abundant proteins with a long half-life and with a quick production
mechanism. Indeed, HSPs were first identified as stress proteins that confer resist-
ance to physical stresses, such as elevated temperatures and chemical insults in
all cellular organisms. These stresses may lead to acute protein misfolding from
whose potential cytotoxicity the cells become protected by the quick induction of
HSPs [7].
While also expressed under normal conditions, HSPs are rapidly elevated after
stress and confer a temperature-resistant phenotype. In cancer cells, cells that are
under numerous acute and chronic stresses caused by internal factors such as pro-
teome and genome instability and alterations, as well as environmental factors such
as hypoxic conditions and nutrient deprivation, HSPs become vital for survival [8].
As we will discuss below, in cancer cells, HSPs are used to re-wire molecular path-
ways so that the cells can gain survival advantage under the transforming pressure.
7.2 HSP90 as an Important Cancer HSP
The major cancer HSPs are the HSP90 and HSP70 family of chaperones. In
spite of several recent efforts to target HSP70, HSP90 remains to date the only
one with a timely promise of delivering a new anticancer therapy [9, 10]. It is
the most studied and perhaps understood cancer HSP family with four main
known mammalian paralogs, the cytoplasmic heat shock protein 90 alpha and
beta (HSP90α and β) [11], the endoplasmic reticulum (ER) glucose-regulated
protein94 (GRP94) [12], and the mitochondrial tumor necrosis factor receptor–
associated protein-1 (TRAP-1) [13]. Each is known to be highly expressed in
cancer, but while HSP90α/β have been widely investigated, and important roles
assigned to them in maintaining the functional conformation of a large number
of aberrant malignancy-driving proteins, little is known on the roles of GRP94
and TRAP-1 [14, 15]. Because most small molecules used to investigate the
7 Molecular Chaperones and How Addiction Matters in Cancer Therapy 183
role of HSP90 are pan-HSP90 inhibitors and few studies differentiate the role of
HSP90α from that of HSP90β, we will here refer to HSP90 without delineating
the role of individual paralogs.
HSP90 is a member of the GHKL family, proteins characterized by an atypi-
cal fold in their ATP-binding regulatory pocket, referred to as the Bergerat fold
[16]. Its structure consists of three domains: mainly an amino-terminal region
(N-domain) that contains the ATP pocket and co-chaperone-interacting motifs,
a middle domain that provides docking sites for client proteins and co-chaper-
ones and that participates in forming the active ATPase, and finally a carboxy-
terminal domain that contains a dimerization motif, a second ligand-binding
region and interaction sites for other co-chaperones [17, 18]. Dimerization of
two HSP90 molecules through their C-domains is believed to be necessary for
chaperone function.
HSP90 function is regulated by a plethora of co-chaperones, of which
approximately 20 have been identified to date. A full list of these co-chaperones
has recently been reviewed by Johnson [6]. Many of these co-chaperones com-
pete for the same binding site on HSP90 or bind alternate HSP90 conforma-
tions, and thus, distinct HSP90-co-chaperone complexes likely exist at any time.
Several of these co-chaperones have tetratricopeptide (TPR) domains and bind
to the MEEVD acceptor site on HSP90. Others, such as SGT1 and GCUNC-
45, bind to a sequence in the N-terminal domain of HSP90. Most have no effect
on the ATPase activity of HSP90, while others stimulate (i.e., AHA1) or inhibit
(i.e., p23, Cdc37) it. Co-chaperones also regulate the conformation of HSP90,
such as p23 that is believed to stabilize the closed conformation, and control the
chaperoning of client proteins. Cdc37, for example, pre-binds clients that are
kinases and delivers them to HSP90, whereas HSP-organizing protein (HOP)
presents HSP70-bound clients to HSP90.
HSP90 interacts with and regulates its client proteins in a cycle that is driven
by the binding and hydrolysis of ATP. Through this catalytic cycle, HSP90
undergoes considerable structural changes, and this dynamic nature of HSP90
is key to its ability to function as a chaperone [19]. This conformational flux is
constantly altered by the binding of regulatory nucleotides (i.e., ATP/ADP) and
co-chaperones (i.e., HOP, Cdc37, p23, AHA1, and immunophilins) and regu-
lated in part by a series of tyrosine phosphorylation events.
Because HSP90 activity is intrinsically linked to its conformation, which
is in turn dependent on the binding and release of ATP/ADP, co-chaperones,
and client proteins, regulating the HSP90 cycle offers several ways of interfer-
ing with HSP90 chaperoning function. Specifically, small molecules that bind
to the regulatory ATP/ADP pocket, interfere with binding of its co-chaperones
or with the conformational flexibility of HSP90, have all been shown to lead
to HSP90-mediated activities in cancer cells [10]. To date, most advanced in
development are those inhibitors that bind to the N-terminal regulatory pocket,
with several in clinical evaluation in cancers [20, 21]. Section 7.5 will detail
these efforts.
Fig. 7.1 Pivotal steps in the validation and translation of HSP90 as a target in cancer
7.3 Differences in Tumor and Normal Cell

HSP90: A Rationale for Targeting HSP90 in Cancers
In fact, much of our understanding on the biology of HSP90 in cancer comes

from studies with such small molecules [22]. Key milestones in these studies,
leading to validation of HSP90 as a cancer chaperone, are outlined in Fig. 7.1.
While the HSP90 protein was first discovered in the 1980s, it has drawn lit-
tle interest as a potential target in cancer. After all, it is abundantly (~1–3 % of
total cellular protein) and ubiquitously expressed in most if not all human cells
and not particularly variable in expression between normal and cancer cells.
Knockdown of even 50 % of HSP90 in cancer cells has little effect on their
viability, while knockout of at least the HSP90β paralog is embryonically lethal
[23]. Such findings match poorly with the belief that a good therapeutic target
has to be crucial to the malignant phenotype and be of low expression in vital
organs and tissues [24].
Fig. 7.2 a Within normal cells, constitutive expression of HSP90 is required for its evolutionar-
ily conserved housekeeping function of folding and translocating cellular proteins to their proper
cellular compartment (“housekeeping complex”). Upon malignant transformation, cellular pro-
teins are perturbed through mutations, hyperactivity, and retention in incorrect cellular compart-
ments or other means. The presence of these functionally altered proteins is required to initiate
and maintain the malignant phenotype, and it is these oncogenic proteins that are specifically
maintained by a subset of stress-modified HSP90 (“oncogenic complex”). Certain HSP90 inhibi-
tors specifically bind to the “oncogenic complex”. b By acting specifically on the “oncogenic
HSP90”, pharmacologic HSP90 inhibition is more efficient than genetic means at lowering
HSP90 function below the threshold necessary for cancer cell survival
Interest on the target potential of HSP90 grew, however, after the serendipi-
tous discovery of a natural product, geldanamycin (GM) [25]. Found in a screen
searching for compounds able to revert the phenotype of cells transfected with the
v-src oncogene, it was later demonstrated to do so by specifically binding to the
N-terminal regulatory pocket of HSP90 and, by this, to inhibit its function. While
surprising in light of the available genetic data, low concentrations of this natural
product were active on many cancer cells and induced differentiation, reduced cell
proliferation, and/or induced death, while being of no significant toxicity at such

concentrations to normal cells [25]. Subsequent crystal structures of HSP90 in
complex with GM and the regulatory nucleotides have uncovered the unique char-
acteristics of this pocket and its high potential for druggability, further spurring the
interest in HSP90 as a drug target [10, 11, 17].
The low toxicity seen with HSP90 inhibitors on normal cells remained unexplained
for more than a decade. Then, Kamal et al. [26] proposed that, while no specific muta-
tions differentiated HSP90 from normal and cancer cells, in cancer cells the chaper-
one was found entirely in complexes of high affinity and sensitivity to small molecule
inhibitors. In normal cells, on the other hand, a dynamic complex of HSP90, of low
affinity and decreased sensitivity to pharmacologic inhibition, was present. This mech-
anism provided a satisfactory explanation for the distinct sensitivity of normal and
cancer cells to GM and other HSP90 inhibitors. It, however, fell short on explaining
other observations, such as the little effect 50 % reduction in HSP90 levels had on
cancer cells. An explanation came 8 years later when Moulick et al. [27] demonstrated
that HSP90 in cancer cells was not in its entirety in the high-affinity form, but rather it
was composed of a “housekeeping HSP90” species, of low affinity to small molecule
inhibitors, such as the HSP90 found in normal cells, and also of a distinct HSP90,
defined as the “oncogenic HSP90” species. The “oncogenic HSP90,” of high affin-
ity to certain small molecule inhibitors, comprised a functionally distinct HSP90 pool,
enriched or expanded in cancer cells (Fig. 7.2a). The authors showed it to be a highly
co-chaperone-dependent HSP90 that certain cancer cells use to maintain the altered
proteins and protein networks that are needed to drive the malignant phenotype. Thus,
while the tumor becomes addicted to survival on a network of HSP90-oncoproteins,
these proteins become dependent on “oncogenic HSP90” for functioning and stability.
In this view, small molecules by their ability to interact specifically with the “onco-
genic HSP90” will primarily and selectively affect these complexes and will act on
the “housekeeping HSP90” only at higher or at saturating concentrations (Fig. 7.2b;
HSP90 inhibitor). On the other hand, genetic means will equally reduce the expres-
sion of both “oncogenic” and “housekeeping” HSP90 pools, and thus, it is conceiv-
able that more than 50 % reduction in HSP90 levels would be necessary to lower
HSP90 to the threshold level required for cell survival (Fig. 7.2b; HSP90 siRNA).
With these concepts in mind, it is therefore important to understand that the
pharmacologically targeted tumor HSP90 is not identical with the tumor HSP90,
but rather encompasses a small portion of it (Fig. 7.2a; HSP90 inhibitor).
7.4 HSP90 as a Regulator of Important Cancer-Mediating

Proteins
Now that we understand that HSP90 chaperones a significant portion of the altered
cancer proteome, we will detail below the nature of such proteome. Significant
research over the past 30 years has identified that HSP90 regulates many cancer pro-
teins and protein networks involved in signaling, cell cycle and proliferation, DNA
Fig. 7.3 Mechanisms by which HSP90 chaperones the cancer proteome. Cancer cells require
HSP90 to regulate the folding and stability of several cancer-driving proteins (a) and to facilitate
the complex formation of molecules involved in aberrantly activated complexes (b, c). In these
cases, HSP90 acts a scaffolding molecule that maintains protein complexes in an active configu-
ration (b) or as a facilitator of protein complex assembly (c)
damage and repair, transcriptional and translational activity, and epigenetic regulation.
These, while potentially partly dependent on HSP90 in normal cells, may become
addicted to HSP90 for their function in the course of cell progression from a normal
to a cancerous state. In such cases, the cancer cells require HSP90 to regulate the fold-
ing and stability of overexpressed (i.e., HER2), mutated (i.e., mutant B-Raf , mutant
FLT3), activated (i.e., AKT), or chimeric proteins (i.e., BCR-ABL ) [28] (Fig. 7.3a).
Recent studies have started to unveil additional roles for HSP90, such as to facilitate
complex formation of molecules involved in aberrantly activated complexes. In these
cases, HSP90 acts as a scaffolding molecule that maintains protein complexes in an
active configuration (i.e., STAT5) [27] (Fig. 7.3b) or as a facilitator of protein com-
plex assembly (i.e., RNA polymerase II, RISC) [29] (Fig. 7.3c).
7.4.1 HSP90 as a Regulator of Onco-Protein Client

Stability and Folding
Traditionally, HSP90 was known as a regulator of protein stability (Fig. 7.3a). It

binds to near native proteins that have metastable characteristics and regulates
their stability and helps to keep them in a folded, functional state [30, 31]. Among
the proteins that require HSP90 for this activity are several kinases (Sect. 7.4.2),
transcription factors (Sect. 7.4.3), epigenetic regulators (Sect. 7.4.4), and several
proteins involved in DNA repair (Sect. 7.4.5). In general, inhibition of HSP90
leads to a reduction in the steady-state levels of these proteins. The major path-
way by which the cell degrades these HSP90 client proteins is the proteasome.
Specifically, upon HSP90 inhibition, an E3 ligase is recruited to the complex, and
upon ubiquitination, the protein is directed to the proteasome for degradation,
thus short-circuiting further cycles of HSP90-mediated refolding [22, 28, 32].
HSP90, therefore, sits at the crossroad of folding/stabilization and degradation
pathways, with small molecule inhibitors pushing the equilibrium strongly toward
the degradative fate.
7.4.2 HSP90 and Protein Kinases
Activation of kinases is necessary to maintain the increased signaling state charac-

teristic of cancer cells, and thus, these proteins have emerged early on as impor-
tant cancer targets. Concordantly, the nature of the HSP90-addicted kinases and the
mechanism of such interrelationship were widely studied. In effect, kinases were
the first known HSP90 onco-clients since HSP90 was identified in 1980s as part of
pp60v-src-HSP90-p50 complexes immunoprecipitated from Rous sarcoma virus-
transformed cells [33]. Since then, a number of mutated, activated, overexpressed,
or chimeric kinases that are key mediators of disease have been shown to be reg-
ulated by HSP90 in a variety of cancers [32]. Among these are the HER-tyrosine
kinases, AKT, Raf kinases, including c-Raf and B-Raf, JAK kinases, c-KIT, MET,
FLT3, and a variety of chimeric kinases, including BCR-ABL and NPM-ALK,
to list a few. Several key cell cycle regulator proteins such as cyclin-dependent
kinases are also HSP90 regulated. In all of these cases, HSP90 is required to main-
tain the functional conformation of the kinase, and inhibition of HSP90 results in
kinase inactivation and degradation. An updated list of potential HSP90-interacting
kinases as well as other proteins can be found at the Picard laboratory website
(www.picard.ch/downloads/Hsp90interactors.pdf). While HSP90 may help fold pol-
ymorphic variants of the same protein, the folding requirement for each variant may
vary. This was observed with v-SRC, an unstable protein kinase, which has a greater
requirement for HSP90 than its cellular counterpart c-SRC [34]. Similarly, mutant
forms of p53 [35] and B-Raf protein kinase [36] and the chimeric BCR-ABL [27]
have a greater requirement for chaperones than do their wild-type counterparts.
7.4.3 HSP90 and Transcription Factors
Transcription factors are the downstream effectors of many cancer-

promoting
pathways and thus play an important role in cancer therapy. Many of them,
including the steroid receptors, the STAT family, and p53, appear to require
HSP90 to facilitate increased signaling and transcriptional robustness.
7.4.3.1 Steroid Hormone Receptors
Most steroid hormone receptors such as glucocorticoid receptor (GR), mineralo-

corticoid receptor (MR), and androgen receptor (AR), validated anticancer targets
in several cancers including breast and prostate cancer, appear to require HSP90
for activity [37, 38]. HSP90 has a complex role in integrating the subcellular dis-
tribution as well as the transcriptional activity of these receptors. One of the best
studied is the GR. In the absence of a ligand, HSP90 binds to GR and maintains
the receptor in a hormone-binding competent conformation. The dynein/dynactin
motor complex also associates with the HSP90-FKBP52 complex bound to GR
and MR, suggesting that this mega-complex may power the active retrograde
movement of steroid receptors [39, 40]. As seen with HSP90 clients, treatment
of cells with HSP90 inhibitors resulted in disruption of hormone-binding activity
and increased proteasomal degradation of the receptors [41].
7.4.3.2 Tumor Suppressor p53
The tumor suppressor p53 is another transcription factor that requires HSP90.
Somatic mutations in p53 are found in many types of human tumors and often
result in the inactivation of its tumor suppressor function. These mutations result in
conformation changes and prolonged protein half-life that require HSP90 for their
stabilization [35]. Furthermore, binding of HSP90 to mutant p53 inhibits the ability
of MDM2 to promote p53 ubiquitination and degradation, leading to the stabiliza-
tion of both mutant p53 and MDM2 [42, 43]. Interestingly, while HSP90 associates
with both the wild-type and the mutated p53, inhibition of HSP90 upregulates the
wild-type protein but downregulates the mutant one, suggesting a distinct role for
HSP90 in the regulation of the normal (wild-type) and aberrant (mutant) p53 [44].
7.4.3.3 HIF-1α
Hypoxia inducible factor-1α (HIF-1α) is a nuclear transcription factor involved

in the transactivation of numerous target genes, many of which are implicated in
the promotion of angiogenesis and adaptation to hypoxia. In normoxic conditions,
HIF-1α is expressed at low levels being targeted for proteasome-dependent deg-
radation by VHL, but hypoxia normally impairs VHL function, allowing HIF to
accumulate [45]. In these conditions, HIF-1α interacts with HSP90. HIF protein
from HSP90-inhibited cells is unable to bind DNA, suggesting that HSP90 is nec-
essary for mediating the proper conformation of HIF and/or recruiting additional
cofactors [46].
7.4.3.4 BCL6 Transcriptional Repressor
In diffuse large B-cell lymphoma (DLBCL), HSP90 was found to be an impor-

tant regulator of the transcription repressor BCL6 [47]. The oncogenic effects
of BCL6 are related to its ability to directly repress key genes such as ATR
(encoding ataxia telangiectasia and Rad3-related) and TP53 (encoding tumor
suppressor protein p53). In these conditions, HSP90 stabilized BCL6 mRNA and
protein and formed a complex with BCL6 at its target promoters. HSP90 inhibi-
tion derepressed BCL6 target genes, inducing reactivation of key BCL6 target
genes and apoptosis.
7.4.4 HSP90 and the Epigenetic Machinery
Increasing evidence indicates that epigenetic alterations contribute to malignant

states. These alterations result in gene silencing and activation as a consequence
of chromatin remodeling driven by modifications including histone methylation,
acetylation, and DNA methylation. Aberrant epigenetic changes can lead to tumor
formation as a result of dysregulated gene expression. Thus, histone-modifying
enzymes and DNA methyltransferases are important therapeutic targets. Of note,
molecules involved in epigenetic regulation have been identified to be directly or
indirectly affected by HSP90 inhibitory therapies.
7.4.4.1 DNA Methyl Transferase 1
Gene silencing by CpG island methylation is a common mechanism of tumor sup-

pressor gene repression in cancers. The DNA methyltransferase DNMT1 plays a
critical role in the maintenance of methylation. It has been shown that DNMT1
can cooperate with histone deacetylases (HDAC) to initiate and maintain epige-
netic gene silencing [48]. Studies that blocked HSP90 activity revealed that it
resulted in the ubiquitin–proteasome degradation of DNMT1 in breast cancer and
in other tumor cells [49, 50]. By altering the stability of DNMT1, HSP90 inhibi-
tion resulted in decreased DNMT1 levels followed by the derepression of JunB,
indicating DNMT1 as another cancer client of HSP90.
7.4.4.2 Histone Methyltransferases
HSP90 has also been shown to interact with proteins involved in chromatin regu-
lation by modifying histone methylation, including EZH2, PRMT5, CARM1,
SMYD3, and SMYD2. EZH2 is the catalytic core in the polycomb repressor com-
plex 2 (PRC2) that is involved in the trimethylation of histone-3 lysine-27 (H3K27),
resulting in chromatin condensation and gene silencing [51]. EZH2 is aberrantly
regulated in a wide range of cancer types, and it is involved in stem cell mainte-
nance and tumorigenesis [52, 53]. Thus, disruption of EZH2 is of interest as a thera-
peutic strategy for cancer. HSP90 was recently shown to regulate EZH2 in leukemia
cells, with its inhibition leading to decreased EZH2 protein expression, suggesting
that HSP90 is required to maintain the stability of EZH2 [54]. PRMT5 is a protein
arginine methyltransferases (PRMTs) involved in arginine methylation of histones
and other proteins that play a role in the regulation of major signaling pathways
that control survival and malignant transformation [55]. PRMT5 is a client protein
of HSP90 in ovarian cancer cells, and PRMT5 protein levels decreased upon treat-
ment with an HSP90 inhibitor [56]. PRMT4/CARM1 (co-activator-associated argi-
nine methyltransferase 1), an enzyme that catalyzes the transfer of a methyl groups
to arginine residues resulting in the activation of signal transduction cascades and
transcriptional activation [57], has been found to be deregulated in tumor cells [58].
CARM1 was recently reported to be a client protein of HSP90 in leukemia cells,
and this interaction shown to be critical for their survival [27]. The SMYD (SET and
MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in
various cellular processes, including gene expression regulation and DNA damage
response. Initially identified as genuine histone methyltransferases, specific mem-
bers of this family have recently been shown to methylate non-histone proteins such
as p53 , VEGFR, and the retinoblastoma tumor suppressor (pRb). Both SMYD3 and
SMYD2 (SET and MYND domain–containing protein 3) are histone methyltrans-
ferases that interact with HSP90 [59, 60]. HSP90 inhibition decreased the expression
of SMYD3 and inhibited migration and proliferation of breast cancer cells. [60]
7.4.5 HSP90 and DNA Damage and Repair System
The DNA damage response refers to a series of tightly regulated, complex cellular
mechanisms that repair DNA breaks occurring through normal cellular metabolism or
environmental exposures. These pathways include base excision repair (BER), nucleo-
tide excision repair (NER), mismatch repair (MMR), non-homologous end-joining
(NHEJ), and homologous recombination (HR). The choice of repair pathway used is
determined by the type of lesion and by the expression of repair proteins at specific
cell cycle phases to ensure that the most accurate repair is performed [61]. DNA dou-
ble-strand breaks (DSBs), which pose the most significant threat to genomic integrity,
are repaired by two major DNA repair pathways, NHEJ and HR. Deficiency in the
HR pathway results in repair of all DSBs by the error-prone NHEJ pathway leading to
the accumulation of chromosomal aberrations. All steps of the DNA damage response
require the recruitment and modification of multiple key proteins; several of which
have been proposed as potential client proteins of the molecular chaperone HSP90
[62]. Several proteins crucial to the HR and NHEJ pathways for repair of DSBs
including BRCA2, FANCA, ATR, CHK1, and DNA-PK are also HSP90-chaperoned
[63–65]. Interestingly, HSP90 can be modified during the DNA damage response.
HSP90 is phosphorylated by DNA-PK, and both phosphorylated HSP90 and DNA-PK
have been found to co-localize to the H2AX apoptotic ring that contains activated
ATM, CHK2, and H2AX [65, 66]. HSP90 inhibition may therefore promote the deg-
radation of proteins involved in DNA repair and thereby potentiate DNA damage sus-
tained by ionizing radiation (IR) or chemotherapy. These observations are supported
by the enhancement of radiosensitivity of multiple human cancer cell lines including
glioblastoma, breast, colon, sarcoma, and pancreatic cancer cell lines by treatment
with inhibitors of HSP90 [67, 68].
7.4.6 HSP90 as a Scaffolding Molecule to Maintain

Signaling Complexes in an Active Configuration
In addition to regulating protein stability and folding, HSP90 has been recently shown
to act as a scaffolding molecule that increases the competency of protein complexes
involved in signaling and in transcription (Fig. 7.3b). Specifically, in chronic myeloid
leukemia (CML), HSP90 was found to facilitate increased STAT5 signaling by binding
to and influencing the conformation of STAT5 and by maintaining STAT5 in an active
conformation directly within STAT5-containing transcriptional complexes [27]. In the
cytosol, HSP90 binding to STAT5 modulated the conformation of the protein to alter
STAT5 phosphorylation and dephosphorylation kinetics. In addition, HSP90 maintained
STAT5 in an active conformation in STAT5-containing transcriptional complexes.
Unlike the classic HSP90 clients that require HSP90 for stability (Sect. 7.4.1), HSP90
inhibition led to inhibition but not degradation of STAT5.
7.4.7 HSP90 as a Protein Complex Assembly Facilitator
Recently, HSP90 has also been identified as a critical protein in promoting and
maintaining the assembly of several cancer-related multi-protein complexes
(Fig. 7.3c). The involvement of HSP90 in these complexes varies, and in some, it
relates to stabilizing an unstable protein subunit and facilitating its incorporation into
the mega-complex. In other cases, HSP90 promotes a change in the composition of
a given complex. In any case, HSP90 is no longer present in the final assembled
complex. Such effects of the chaperone on the assembly of the following seven com-
plexes: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase–related protein
kinase (PIKK), telomere complex, kinetochore, RNA-induced silencing complexes
(RISC), and 26S proteasome was recently detailed in a review article [29].
7.4.8 Global HSP90 Proteome Investigations in Cancer
While the above studies led to the identification of important HSP90 client
proteins, it is clear that due to the complexity of the altered proteome in any given
cancer cell, large proteomic and genomic analyses combined with bioinformatic
analyses are needed to understand the HSP90-addicted proteome. Because malig-

nant evolution is characterized by alterations in a multitude of proteins and path-
ways, and perhaps no two tumors present an identical spectrum of defects, it is
likely that addiction of cancer cells to HSP90 occurs in a tumor-specific manner,
and thus, no two tumors will have an identical set of HSP90-sheltered proteins and
protein networks. Indeed, several recent investigations have used such methods to
demonstrate the complexity of HSP90-sheltered networks. A thorough review on
this topic was recently published by the Matts group [69].
The yeast has been a useful system to study HSP90 functions, several of these
findings being eventually validated in cancer cells. As such, several systematic prot-
eomic and genomic methods were used to map HSP90 interactions in yeast [69]. In
one such study, physical interactions were identified using genome-wide two-hybrid
screens combined with large-scale affinity purification of HSP90-containing protein
complexes [70]. Genetic interactions were uncovered using synthetic genetic array
technology and by a microarray-based chemical-genetic screen of a set of about 4,700
viable yeast gene deletion mutants for hypersensitivity to the HSP90 inhibitor GM.
These analyses proposed that HSP90 may interact with 10 % of the yeast proteome
and that its interacting proteins were involved in a wide range of cellular functions,
including cell cycle and DNA processing, cellular communication, fate/organization,
and transport, as well as energy production, metabolism, transcription, translation, and
transport facilitation. This study identified two novel HSP90 interactors, namely Pih1
(Protein interacting with HSP90) and Tah1 (TPR-containing protein associated with
HSP90), which interact physically and functionally with the conserved AAA(+)-type
DNA helicases Rvb1/Rvb2, forming the R2TP complex. The complex is required for
chromatin remodeling, box C/D small nucleolar ribonucleoprotein (snoRNP) biogen-
esis, apoptosis, phosphatidylinositol-3 kinase–related protein kinase (PIKK) signaling,
and RNA polymerase II assembly, implicating HSP90 in these processes.
In a yet another classic approach, changes in the proteome of HeLa cer-
vical cancer cells were analyzed upon treatment with the HSP90 inhibitor
17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) using
SILAC and high resolution, quantitative mass spectrometry [71]. This study found
that the proteome most sensitive to HSP90 inhibition and thus most significantly
downregulated by 17-DMAG was that fitting the functional categories of “protein
kinase activity” and “DNA metabolic processes”. The kinome regulated by HSP90
in HeLa was associated with a multitude of signaling and signaling-associated
pathways including the PI3K -AKT-, IL-6-, HER-, FAK-, Ephrin receptor-, ERK
-MAPK-, NFκB-, inositol phosphate metabolism, and G-protein-coupled recep-
tor–mediated networks. In the “DNA metabolic processes” group, enriched were
GO annotated processes such as “cellular response to DNA damage,” “DNA modi-
fication,” “response to DNA damage stimulus,” and “regulation of transcription”.
In a novel approach combining chemical proteomics with bioinformatic analy-
ses, Moulick et al. analyzed the HSP90-sheltered proteome in the K562 CML cells
[27]. In this approach, HSP90 in complex with its client proteins and co-chaperones
was isolated by chemical purification using a solid-support-attached HSP90 inhibi-
tor, namely the purine-scaffold PU-H71. PU-H71/HSP90 protein cargo was then
analyzed by mass spectrometry. The method took advantage of the fact that PU-H71
bound preferentially to the fraction of tumor HSP90 associated with altered client
proteins (i.e., “oncogenic HSP90”; Fig. 7.2a) and moreover, upon binding, locked
HSP90 in an onco-client bound configuration. Together these features greatly facili-
tated the chemical affinity purification of tumor-associated protein clients by mass
spectrometry, providing tumor-by-tumor global insights into the HSP90-sheltered
proteome, including in primary patient specimens. Top scoring networks identified
by this method to be HSP90-sheltered in CML were those used by BCR-ABL to
propagate aberrant signaling: PI3K /AKT/mTOR -, Raf -MAPK-, NFκB-, STAT5-,
and FAK-mediated signaling pathways. Other important transforming pathways in
CML, driven by MYC and TGF-β as well as others involved in disease progres-
sion and aberrant cell cycle and proliferation of CML, were identified. In addition
to signaling proteins, proteins that regulate carbohydrate and lipid metabolism, pro-
tein synthesis, epigenetics, gene expression, and cellular assembly and organization
were identified, in accord with the postulated broad roles of HSP90 as an impor-
tant mediator of cell transformation. The method also identified the histone-arginine
methyltransferase CARM1 as a novel HSP90 interactor, implicating the HSP90-
facilitated CARM1 activity in CML leukemogenesis.
7.5 Targeting HSP90 in Cancer
As delineated in Sect. 7.4, HSP90 shelters a complex proteome vital for cancer
progression and maintenance. The HSP90-chaperoned networks and pathways
differ from tumor-to-tumor, depending on the molecular wiring of the alterations
that characterize a specific tumor. These are appealing features for a cancer target,
because by inhibiting one protein, HSP90, one could have beneficial effects in a
variety of tumors. Such effects were not appreciated early on when HSP90 was
discovered, because as indicated above, HSP90 is an abundant protein expressed
in most human cells. However, its persuasive roles in regulating the malignant pro-
teome, the observation that it can be easily pharmacologically modulated by small
molecule ligands, and findings demonstrating the differences between tumor and
normal cell HSP90 have together given it credibility as a cancer target. Indeed, at
the moment, more than 20 inhibitors have been or still are in clinical testing, with
many more coming in the pipeline (Fig. 7.4) [20, 21].
As indicated, the first compounds identified as HSP90 inhibitors were those
that modulate HSP90 chaperone activity by inhibiting the N-terminal domain ATP-
binding site [25, 72]. Targeted approaches such as structure-based drug design,
biochemical and cell-based screening, virtual screening, fragment-based drug
design, and medicinal chemistry have since led to the identification and devel-
opment of several novel inhibitors that act by similar or alternate mechanisms to
inhibit HSP90 function [10]. The molecules currently advanced to clinical trials
are those that bind to the regulatory pocket. These constitute a diverse array of
structures, which at a close inspection classify according to their similarity to GM
(Sect. 7.5.1), to the purine-scaffold (Sect. 7.5.2), or to radicicol (Sect. 7.5.3).
Ansamycins Purine-scaffold
benzoquinone NH2 Br O
O Cl- N
N
NH2 I
R H H OH O S O
O N N N N
C17 O N
S O
N
H N N N
O H
OH
OH H3 CO
H3CO OH H3 CO N
H3CO HN O
OCONH2
MPC-3100
OCONH2
PU-H71 HO
Geldanamycin R= -OMe IPI-504
17-AAG R= -NHCH2 CHCH2 NH2 N Cl
17-DMAG R= -NHCH2 CH 2 N(CH 3) 2 N O
N N
IPI-493 R= -NH2 N
S
N O N
H2N N
Resorcinols O
N N
O NH CH3
Cl
HO
H 3C OCH3
O HO
O O
Debio 0932 BIIB021
OH O N
H
OH O N
Radicicol Other
NVP-AUY922
H2N O
N
H
N
O
N
NH2
HO O O
N
N O N
CH3 O
OH O O N
F3C
N NH
HO O O SNX-5422
AT13387 N
O H2N N
O
O H3CO N
N F
OH O
N
KW-2478
HO H2N
N O NVP-HSP990
N
O
NH
OH N NH
XL888
HN
STA-9090 O
Fig. 7.4 Chemical structures of HSP90 inhibitors in clinical evaluation
7.5.1 Geldanamycin Derivatives
Initial efforts in the discovery of clinically translatable HSP90 inhibitors

have focused on GM and several of its derivatives (Fig. 7.4, ansamycins).
GM is a benzoquinone ansamycin first isolated from a fermentation broth of
Streptomyces hygroscopicus in 1970 [25]. Inhibition of HSP90 was subse-
quently shown to be the mechanism of its anticancer activity [34] and more
specifically was later shown to inhibit its ATPase activity by competing with
ATP for binding to the N-terminal nucleotide-binding pocket [25]. GM was

never evaluated in clinical trials because of its poor pharmaceutical proper-
ties, including poor solubility, limited in vivo stability, and hepatotoxicity in
animals [10, 25]. Despite the complexity of the molecule, attempts have been
made to structurally modify geldanamycin so as to improve these proper-
ties and make it more “drug-like”. Early attempts focused on modifying the
17-position of the quinone ring. Synthetically, the C17 methoxy group can
readily be substituted with amines, an approach that resulted in two clinically
viable candidates, 17-AAG and 17-DMAG. 17-AAG, the first HSP90 inhibitor
to enter clinical studies in cancers, has served to establish proof-of-principle
for HSP90 inhibition in humans [19–21]. 17-DMAG is a related analog with
an N,N-dimethylethylamino group in place of the C17-methoxy. This ion-
izable amine group led to improved solubility and better oral bioavailability
for 17-DMAG when compared with 17-AAG. 17-DMAG showed activity in
refractory metastatic breast cancer and in refractory acute myeloid leukemia
patients, but its development was halted as a result of unacceptable side effects
[19–21]. IPI-504, the reduced form of 17-AAG, was designed to resolve a
number of issues concerning 17-AAG [10]. As a hydrochloride salt, IPI-504
was highly water soluble, and because it lacked the benzoquinone characteris-
tic of 17-AAG, it showed an improved toxicity profile. In vivo, however, IPI-
504 and 17-AAG interconvert through the action of oxidoreductases. IPI-504
showed clinical benefit in a study in patients with non-small-cell lung cancer
(NSCLC) in tumors harboring ALK rearrangements [19–21].
7.5.2 The Purine and Purine-Like Scaffold HSP90 Inhibitors
The availability of crystal structures of ADP, GM, and radicicol bound to the
N-terminal nucleotide-binding domain of HSP90 and the realization that HSP90
possesses a unique ATP-binding pocket made the rational design of synthetic
inhibitors possible [10]. As mentioned, HSP90 is part of a small protein family
called the GHKL (Gyrase, HSP90, histidine kinases, and MutL) family, which
contains a unique fold in their regulatory pocket region, called the Bergerat fold
[16], a feature that portends selectivity for its ligands over other ATP-binding
pockets. Taking advantage of the distinct shape of the ATP pocket, Chiosis et al.
[72] designed PU3 as the first reported synthetic inhibitor. PU3, a purine-scaf-
fold compound, maintained the purine core of the endogenous ligands. This was
linked at the 8-position to an aryl moiety via a methylene linker (Purine-CH2-
Aryl). This essential motif was maintained, with some variations, for essentially
all the purine-scaffold HSP90 inhibitors (Fig. 7.4 purine-scaffold). From this
interesting lead, a large effort from multiple groups resulted in four molecules
to enter clinical evaluation in cancers: PU-H71, BIIB021, MPC-3100, and Debio
0932 (formerly CUDC-305) [20, 21].
7.5.3 The Resorcinol Class
The compounds within this class are distinguished by the presence of a resorcinol
moiety, and for this, they are related to radicicol (Fig. 7.4 resorcinols), a natural
product inhibitor of HSP90 isolated from Monosporium bonorden. Although a
potent HSP90 inhibitor, radicicol is unstable in vivo, and no attempts to directly
modify its structure have resulted in a clinical candidate. However, a number of
agents currently investigated maintain the resorcinol moiety. One of these, NVP-
AUY922, was developed from a lead molecule discovered during a high-through-
put screen designed to measure the ability of a compound to inhibit yeast HSP90
ATPase activity [10, 21, 22]. AT13387, on the other hand, was developed using
a fragment-based approach using NMR screening and X-ray crystallography.
A number of other novel resorcinol compounds are currently being evaluated in
clinical trials, including STA-9090 (ganetespib), KW-2478, and DS2248 [20, 21].
7.5.4 Other HSP90 Inhibitor Classes
Serenex used a chemo-proteomics approach in the discovery of a novel chemo-

type HSP90 inhibitor that ultimately resulted in the clinical candidate SNX-5422
[10, 20]. To accomplish this, purine-binding proteins from porcine lung or liver
were loaded onto an ATP-affinity column and subsequently were challenged with
a focused library of 8,000 compounds in a parallel search for both a suitable target
protein and hit compound. The clinical development of this agent has been halted
due to severe ocular toxicity [20–22]. Other HSP90 inhibitors advancing to clini-
cal studies include XL888 and NVP-HSP990 (Fig. 7.4 others).
7.6 Clinical Experience with HSP90 Inhibitors
Given their potential to inactivate a number of different onco-client proteins and

affect multiple cancer networks, HSP90 inhibitors have been hypothesized to be
active, and consequently tested, in a wide variety of cancers [20, 21, 73]. To date,
the greatest clinical activity as evidenced by objective tumor regressions has been
observed with the GM analog tanespimycin (17-AAG) when given in combina-
tion with trastuzumab to patients with HER2+ metastatic breast cancer in whom
tumor progression was seen with trastuzumab alone (i.e., 59 % overall clinical
benefit rate in trastuzumab-refractory disease). Preclinically, HER2 is among the
most sensitive client proteins to HSP90 inhibition, and this may account for the
activity observed in this setting. Recently, objective tumor responses were also
reported with ganetespib (STA-9090) in NSCLC tumors that harbored the ALK
rearrangement. ALK rearrangements, particularly the EML4-ALK fusion protein,

found in a subset of NSCLC patients, are also a sensitive HSP90 client protein
[20, 21, 73].
While these results validate HSP90 as a relevant anticancer target, the full
potential of HSP90 inhibitors to be active in a broader spectrum of tumor types
has yet to be achieved. This differential response could be because we are unable
to select patients who might best benefit from therapy and perhaps have not suc-
cessfully optimized the dosing and scheduling of this class of agents. As a class,
HSP90 inhibitors demonstrate rapid clearance from normal tissues and the blood
compartment with prolonged retention in tumors; hence, traditional serum phar-
macokinetics is insufficient to guide dosing and scheduling [10]. Additionally, the
current drug development paradigm based on identifying maximum tolerated dose
may not be applicable in the case of these inhibitors where target modulation is
critical to producing their anti-tumor effects. The limited value of plasma phar-
macokinetics and the importance of tumor concentration in predicting therapeu-
tic response suggest the need for the clinical development of an assay of tumor
pharmacokinetics for HSP90 inhibitors. In addition, there is presently no clear
consensus on how to identify those patients most likely to benefit from HSP90
therapy. Unlike for other targets, where either protein expression (i.e., HER2 lev-
els for trastuzumab selection in treatment of breast cancer patients) or the presence
of a mutation (i.e., EGFR mutation for Tarceva selection in treatment of NSCLC
patients) drives patient selection, there is no such knowledge for HSP90. No
mutation characterizes HSP90 in tumors nor is its expression considerably vari-
able between responsive and un-responsive tumors. The lack of a biomarker for
proper patient selection is especially problematic since successful development
of targeted agents requires identification of the patient sub-population that should
receive the drug (i.e., tumors with EGFR mutations for Tarceva). Such selection
may reduce the number of patients receiving ineffective treatment, reduce costs
to health care and facilitate the clinical development and route to approval for the
HSP90 inhibitors [10, 20, 21, 73].
7.7 Conclusion
HSPs and HSP90, in particular, have emerged as important targets in cancer due to
their ability to buffer wide proteome alterations such as are characteristic in malig-
nant transformation. While not easily accepted initially as potential cancer targets,
several studies now spanning almost three decades have revealed important dif-
ferences in the biochemical and functional roles of HSP90 in cancer cells that are
different from those in normal cells. These findings, together with the now almost
explosive interest in the discovery and development of small molecule HSP90
inhibitors, and with the encouraging clinical studies with these agents, have
cemented HSP90 as a cancer target. The journey is, however, yet to be complete.
Several issues, including the lack of a biomarker for patient selection and the ina-
bility to measure target inhibition in clinic, remain to be addressed.
Acknowledgments G Chiosis is funded in part by Mr. William H. and Mrs. Alice Goodwin
and the Commonwealth Foundation for Cancer Research and “The Experimental Therapeutics
Center of Memorial Sloan-Kettering Cancer Center”, the Geoffrey Beene Cancer Research
Center of MSKCC, Leukemia and Lymphoma Society, Breast Cancer Research Fund,
the SPORE Pilot Award and Research and Therapeutics Program in Prostate Cancer, the
Hirshberg Foundation for Pancreatic Cancer, the Byrne Fund, National Institutes of Health
(1U01 AG032969, 1R01CA155226, 1R21AI090501, 1R21CA158609, 3P30CA008748,
P50CA086438), MSKCC Society, Department of Defense (R03-BC085588), Susan G Komen
for the Cure and the Institute for the Study of Aging and The Association for Frontotemporal
Dementias (Grant #281207 AFTD). T Taldone discloses a grant support from the Department of
Defense (PDF-BC093421). M Guzman is funded by the US National Institutes of Health (NIH)
through the NIH Director’s New Innovator Award Program, 1 DP2 OD007399-01, National
Cancer Institute (R21 CA158728-01A1), Leukemia and Lymphoma Foundation (LLS 6330-11),
and she is a V Foundation Scholar.
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Chapter 8
Sphingolipid Metabolism and Signaling
as a Target for Cancer Treatment
Vinodh Rajagopalan and Yusuf A. Hannun
Abstract Sphingolipids play key roles in the regulation of several biological

processes that are integral to cancer pathogenesis. Among the sphingolipid metab-
olites, ceramide and sphingosine-1-phosphate (S1P) have been shown to modulate
cancer development and progression. The biological roles of other metabolites,
such as sphingosine, and ceramide 1-phosphate, are also beginning to emerge. In
general, ceramide plays a role as a tumor-suppressing lipid-inducing anti-prolif-
erative response such as cell cycle arrest, induction of apoptosis, and senescence
whereas S1P plays a role as a tumor-promoting lipid-inducing transformation, cel-
lular proliferation, and inflammation in various cell models. Glycosphingolipids,
another emerging class of bioactive sphingolipids, are believed to play anti-apop-
totic roles and offer drug resistance to currently used chemotherapeutic drugs.
These emerging biological roles of sphingolipids and its potential usefulness in
treating cancer in the form of anticancer therapeutics are discussed in this chapter.
8.1 Sphingolipid Metabolism
Sphingolipids are a class of lipids with a sphingosine back bone that are formed
from non-sphingolipid precursors in the ER and get metabolized further within
different sub-cellular compartments thereby giving rise to a plethora of metabo-
lites. Of all these metabolites, ceramide is one of the most widely studied bioactive
molecules. It is formed through three distinct pathways (Fig 8.1) (1) de novo
synthesis—synthesis from non-sphingolipid precursors; (2) turnover p athways—
break down products from complex sphingolipids; and (3) recycling and sal-
vage pathways—The de novo pathway starts with the condensation of serine
V. Rajagopalan
Department of Biochemistry and Molecular Biology, Medical University of South Carolina,
Charleston, SC, USA
Y. A. Hannun (*)
Stony Brook University, Stony Brook, NY, USA
206 V. Rajagopalan and Y. A. Hannun
Serine + palmitoyl coA Ethanolamine-1-phosphate

+ Hexadecenal
SPT S1P lyase

3-keto-dihydrosphingosine Sphingosine-1-Phosphate
KDS S1PPase SK
Dihydrosphingosine Sphingosine
CerS + Fatty acyl coA CDase CDase CerS + Fatty acyl coA
DES C1PP
Dihydroceramide Ceramide
Ceramide-1-phosphate
CK
SMase GCS
CGT GCase
SMS
Sphingomyelin Glucosylceramide
Galactosylceramide
Glycosphingolipids
Fig. 8.1 Ceramide can be formed by de novo pathway from serine and palmitoyl-coA or from
hydrolysis of sphingomyelin or cerebrosides (glucosyl or galactosyl ceramide). Ceramide, thus
formed, can be phosphorylated by CK to yield ceramide-1-phosphate or serves as a substrate for
the synthesis of sphingomyelin or glycosphingolipids. Ceramide can be deacylated by cerami-
dases to form sphingosine which can be phosphorylated by SKs to generate S1P which can be
acted upon by phosphatases to generate sphingosine or by lyase to form ethanolamine-1-phos-
phate and hexadecanal, an aldehyde. Abbreviations: SPT serine palmitoyl transferase, KDS
3-keto-dihydrosphingosine reductase, DES dihydroceramide desaturase, SPPase Sph-1- phosphate
phosphatase, CK cer kinase, C1PP C1P phosphatase, SMS SM synthase, GCS glucosylceramide
synthase, GCase glucosyl CDase, CGT UDP-galactose ceramide-galactosyltransferase
and palmitoyl-CoA catalyzed by serine palmitoyl transferase (SPT) to generate

3-keto-dihydrosphingosine which is subsequently reduced to form dihydrosphin-
gosine (sphinganine). Ceramide synthases (CerS) then act on dihydrosphingosine
(or sphingosine) to form dihydroceramide (or ceramide) [1]. Dihydroceramide is
subsequently desaturated by dihydroceramide desaturase (DES) which introduces
a 4, 5-trans-double bond, thereby generating ceramide that occurs at the cytosolic
face of the endoplasmic reticulum (ER) [2]. Ceramide, thus generated, can be
used in biosynthetic reactions for the synthesis of sphingomyelin (SM), glucosyl-
ceramide (GluCer), galactosylceramide (GalCer) or ceramide 1-phosphate (C1P)
by the attachment of head groups comprised of either phosphocholine, glucose,
galactose or phosphate by sphingomyelin synthase (SMS), glucosyl ceramide syn-
thase (GCS), UDP-galactose: ceramide-galactosyltransferase (CGT), or ceramide
kinase, respectively (CK) [3, 4, 5]. In the turnover pathways of ceramide genera-
tion, sphingomyelinases act by cleaving sphingomyelin as a substrate [6] whereas
8 Sphingolipid Metabolism and Signaling 207
Fig. 8.2 Ceramide is generated in the ER by de novo pathway. It is transported to the Golgi

membranes for SM synthesis in a CERT-dependent way or glucosylceramide (GlcCer) synthesis
in FAPP2-dependent way. SM and complex GSLs are transported to the plasma membrane via
vesicular trafficking where sphingomyelin gets metabolized by aSMase or neutral SMase to gen-
erate ceramide, or they are shuttled to lysosomes where aSMases and glucosidases metabolize SM
and glucosylceramide, respectively, into ceramide. It is hydrolyzed by acid ceramidase to form
sphingosine. Sphingosine may then traverse the lysosomal membrane to the cytosolic side where
it might have two cellular fates. It can either be recycled into sphingolipid pathway in the ER
or can be phosphorylated by SK1 or SK2. In mitochondria, ceramide is generated by the activa-
tion of n-SMase. Abbreviations: 3KdhSph 3-keto-dihydrosphingosine, dhSph dihydrosphingosine,
CERT ceramide transfer protein
hydrolases such as β-glucosidases and galactosidases act on glycosphingolipids

such as glucosylceramide (GlcCer) and galactosylceramide as substrates to gen-
erate ceramide, respectively [7]. In the recycling and salvage pathways, ceramide
generated in the lysosomes from the hydrolysis of complex sphingolipids is fur-
ther broken down to sphingosine by the action of ceramidases [8] which is then
re-acylated outside the lysosome by the action of ceramide synthases (CerS) to
form ceramide. Since sphingosine derived from ceramide is salvaged to regener-
ate ceramide, it is referred to as the recycling and salvage pathway. Alternatively,
sphingosine can be acted upon by sphingosine kinases (SK1 or SK2) [9] to form
sphingosine-1 phosphate (S1P). S1P phosphatases can dephosphorylate S1P to
regenerate sphingosine [10]. On the other hand, S1P lyase metabolizes S1P irre-
versibly to release ethanolamine phosphate and hexadecenal [11].
The sphingolipid enzymes discussed above are distributed in different intra-
cellular locations. De novo ceramide synthesis takes place on the cytosolic
surface of the ER and its associated membranes. Ceramide formed in the ER is

transported through ceramide transfer protein (CERT) to the trans-Golgi wherein
it serves as a substrate for sphingomyelin synthase for formation of SM [12], or
through vesicular transport to the Golgi wherein it serves as substrate for GCS for
the formation of glucosylceramide. The transport protein, four-phosphate-adaptor
protein 2 (FAPP2) delivers glucosylceramide to appropriate sites in the Golgi for
synthesis of more complex glycosphingolipids (GSL) [13]. Subsequently, SM and
complex GSLs are transported to the plasma membrane via vesicular trafficking.
In the plasma membrane, SM can be hydrolyzed to ceramide and metabolized
further by acid sphingomyelinase (aSMase) possibly acting on the outer leaf-
let, or by neutral sphingomelinase (nSMase) residing on the inner leaflet of the
plasma membrane [14, 15].
From the plasma membrane, sphingolipids are recycled through the endo-
somal pathway. In lysosomes, aSMases and glucosidases metabolize complex
sphingolipids (SM and glucosylceramide, respectively) into ceramide which is
hydrolyzed by acid ceramidase to form sphingosine. Sphingosine may then trav-
erse the lysosomal membrane to the cytosolic side where it has two cellular fates.
Cytosolic sphingosine is either recycled into the sphingolipid pathway in the ER
or phosphorylated by SK1 or SK2 [9] (refer to Fig 8.2 [16]).
8.2 Biological Targets of Ceramide
Ceramide regulates many biological processes such as cancer cell growth, dif-
ferentiation, apoptosis, and senescence [17, 18]. Many signals such as cytokines,
anticancer drugs, and stress-inducers upregulate ceramide through the de novo
or salvage pathways [19, 20]. Ceramide triggers signaling cascades by regulat-
ing phosphatases, cathepsin D, or kinase suppressor of RAS (KSR) as described
below.
Phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2A
(PP2A) are activated by ceramide in vitro. Inhibition of these phosphatases inhib-
its the ability of ceramide to dephosphorylate (inactivate) several pro-proliferative
proteins such as PKCα, Akt/PKB, c-Jun, Bcl-2, Rb, and SR [21, 22].
Many studies have documented the translocation of cathepsin D from lys-
osomes in response to oxidative stress followed by activation of caspase 3 and
cell death [23]. Interestingly, cathepsin D was found to be a ceramide-binding and
ceramide-activated protein [24]. Besides, acid sphingomyelinase-derived ceramide
has been shown to favor autocatalytic proteolysis of inactive cathepsin D to enzy-
matically active cathepsin D isoform [25].
Similarly, KSR has been found to be ceramide responsive [26–30]. Mammalian
KSR activates Raf, and activation of this pathway results in apoptosis [31, 32].
Also, ceramide has been shown to activate the zeta isoform of protein kinase C
(PKCz) by phosphorylation [33]. Ceramide-activation of PKC-zeta has been
CDase
SK
Ceramide Sphingosine-1-phosphate
CAPPs, Cathepsin D,KSR

RAF, MEKK ERK, NF- B, Cox-2
Telomerase, c-MYC, Caspases,

CDKs Inhibition of apoptosis
Malignant transformation
Angiogenesis
Inflammation
Growth arrest
Apoptosis
Senescence
Fig. 8.3 Ceramide regulates many protein signaling molecules such as cathepsin D and
ceramide-activated protein phosphatases (CAPPs), KSR, RAF, MEKK. Proteins that are modulated
by these pathways include RB,SR, AKT, PKC-α, c-JUN, Bcl-2,telomerase, c-MYC, caspases, and
cyclin-dependent kinases (CDKs) which in turn brings about cellular responses such as growth
arrest, apoptosis, and/or senescence. Ceramide that gets metabolised to S1P by ceramidase and SK,
regulates proteins such as ERK, NF-κB, Cox-2 which in turn brings about cellular responses such
as inhibition of apoptosis, malignant transformation, angiogenesis, and inflammation
shown to be necessary for inactivation of Akt-dependent mitogenesis in vascular

smooth muscle cells [34] (refer to Fig. 8.3).
In contrast to ceramide, S1P has emerged as a potential regulator of biologi-
cal processes such as proliferation, inflammation, vasculogenesis, and tumor pro-
motion [35]. S1P is a soluble molecule, secreted outside the cell which acts in
autocrine and paracrine manner to bring about receptor-mediated cellular func-
tions like cell motility and proliferation [35]. S1P acts directly on members of the
S1P1-5 receptor family, which are G protein-coupled receptors. Non-receptor-
mediated functions of S1P such as activation of TNF receptor–associated factor
2 (TRAF2) in the TNF pathway leading to NF-kB activation have been reported
[36]. It has also been shown that interaction of TRAF2 with SK and activation of
SK is critical for prevention of TNFα-mediated apoptosis [36] (refer to Fig. 8.3).
Thus, these findings underscore the significance of uncovering bioactive sphin-
golipid-mediated cellular pathways that would help to conceive novel therapeutic
strategies for many pathological conditions, importantly cancer.
8.3 Ceramide as a Tumor Suppressor
The involvement of sphingolipids in cancer pathogenesis is brought to light from

the observation that the levels of sphingolipid metabolizing enzymes are altered in
many tumors. For example, in a study, SK expression has been found to be upregu-
lated in many human tumors originating from tissues such as breast, colon, lung,
stomach, uterus, etc., [37]. In another study done by Riboni et al., an inverse cor-
relation between the levels of ceramide and tumor malignancy in glial tumors
(astrocytomas) was observed [38]. In general, exogenous ceramide-induced anti-
proliferative responses like apoptosis, differentiation and growth inhibition, senes-
cence, and autophagy. Therefore, it is considered as a tumor suppressor lipid. These
anti-proliferative roles of ceramide are discussed in detail in the following section.
8.3.1 Ceramide and Apoptosis
Ceramide has been implicated in many cell death paradigms. Birbes et al. [39]
showed that selective targeting of bacterial sphingomyelinase (bSMase) to mito-
chondria and not to any other compartments such as plasma membrane, ER, or
Golgi resulted in apoptosis that was associated with generation of ceramide and
release of cytochrome c in MCF-7 cells. Overexpression of Bcl-2 prevented the
mitochondria-targeted bSMase effects on apoptosis. Dai et al. [40] showed that
UV-induced apoptosis was marked by increase in SM in all sub-cellular loca-
tions, particularly mitochondria, in HeLa cells. Ceramide levels were found to
be elevated in mitochondria at 2–6 h, consistent with the cell death time course.
D609, an inhibitor of sphingomyelin synthase to a marked extent and fumonisin
B1 (FB1), a ceramide synthase inhibitor to a lesser extent, rescued the cells from
increases in SM and ceramide, and consequently cell death. On the other hand,
the SPT inhibitor myriocin did not rescue the UV effects on cell death, suggest-
ing the involvement of the turnover pathway-generated ceramide in bringing about
UV-triggered cell death.
In another study in Caenorhabditis elegans, upon inactivation of ceramide syn-
thase, somatic apoptosis was unaffected, but ionizing radiation-induced apoptosis
of germ cells was obliterated, and this phenotype was reversed by microinjec-
tion of long-chain natural ceramide. Radiation-induced ceramide accumulation in
mitochondria consequently activated CED-3 caspase and apoptosis [41].
In Ramos B cells, surface B-cell receptor (BcR)-triggered cell death was marked
by an early increase in C16 ceramide. Pulse labeling with sphinoglipid precursor,
palmitate, in the presence of ceramide synthase inhibitor, FB1, demonstrated that the
de novo ceramide-generating pathway was activated following BcR activation. The
apoptotic cell death induced by cross-linking of BcR was mediated through mito-
chondrial cell death pathways followed by caspase activation [42]. In LNCaP pros-
tate cancer cells, androgen ablation, which is considered as one of the therapeutic
modalities, was found to increase C16 ceramide level followed by G0/G1 cell cycle
arrest and apoptosis. 5alpha-dihydrotestosterone (DHT) or fumonisin B1 treatment
rescued LNCaP cells from apoptosis [43].
In another study, ceramide acting via PP1, dephosphorylated SR proteins that
regulated the alternate splicing of Bcl-x(L) and caspase 9. In A549 lung adeno-
carcinoma cell lines, cell-permeable D-e-C(6) ceramide downregulated the mRNA
levels of anti-apoptotic Bcl-x(L) and caspase 9b with concomitant increase in the
mRNA of pro-apoptotic Bcl-x(s) and caspase 9. The chemotherapeutic agent,
gemcitabine, induced de novo generation of ceramide and brought about afore-
mentioned alternate splicing of Bcl-x(L) and caspase 9b and consequent loss of
cell viability as measured by MTT assay [44].
In several studies, aSMase was shown to be necessary for radiation-induced
apoptosis in endothelial cells and mice lacking aSMase were protected from gas-
trointestinal and CNS apoptosis [45–47]. In another study, the endolysosomal
aspartate protease cathepsin D (CTSD) was identified as a target of ceramide gen-
erated by acid sphingomyelinase in response to TNFα. CTSD cleaved pro-apop-
totic Bid and activated it in vitro. The lack of Bid activation in cathepsin-deficient
fibroblasts suggested Bid is downstream of cathepsin D in bringing about apopto-
sis as a result of TNFα treatment [48].
In addition to aSMase, neutral sphingomyelinase has been implicated in stress
response pathways initiated by TNFα in MCF-7 cells [49]; amyloid-β peptide in
neuronal cells [50]; ethanol in HepG2 hepatoma cells [51]; and staurosporine in
several neuronal cell lines [52].
In addition to the sphingomyelinases, ceramidases (CDases) were also found to
regulate apoptosis. In one study, nitric oxide induced the degradation of nCDase,
thereby, enabling ceramide accumulation and cell death [53]. In another study, the
degeneration of photoreceptor cells was marked by an increase in ceramide which
was rescued by overexpression of CDase that cleared the ceramide and prevented
its apoptotic effect [54]. These studies clearly implicate ceramide in apoptosis and
in mediating the cellular response to various stress causing stimuli.
8.3.2 Ceramide in Senescence
Ceramide has been implicated in cellular senescence. Their relationship stems

from the observation that in WI-38 human diploid fibroblasts (HDF), there was
increased neutral sphingomyelinase activity with generation of ceramide when
cells entered senescence. These changes were not seen when cells entered qui-
escence achieved with serum withdrawal or contact inhibition [55]. Exogenous
administration of ceramide (15uM) onto young WI-38 cells induced retino-
blastoma protein dephosphorylation and inhibited serum-induced AP-1 activa-
tion, DNA synthesis, and mitosis, thereby inducing a senescence phenotype
[55]. Involvement of ceramide in replicative senescence has been shown in
human umbilical vein endothelial cells (HUVEC) as well [56]. In another study,
gemcitabine induced senescence in pancreatic cancer cells, and sphingomyelin

treatment enhanced chemosensitivity to the drug by reducing the induction of
senescence and redirected the cells to enter apoptosis. The authors concluded that
ceramide inhibited cell cycle progression at low levels, induced senescence at
moderate levels, and apoptosis at high levels [57]. Besides these studies, the yeast
aging genes lac l and lag1 were subsequently identified as ceramide synthases,
thereby providing a genetic link between ceramide to senescence and aging [58].
Ceramide also regulates senescence by inhibiting telomerase which is the
enzyme that prevents the shortening of the telomeres, the long tandem repeats of
G-rich sequences (5′-TTAGGG-3′) found at the ends of chromosomes. Telomerase
is found to be frequently activated in many immortal cells in culture represent-
ing different tissues and malignant tumors, suggesting its role in cellular immor-
talization and tumorigenesis [59, 60]. In the A549 lung carcinoma cell line,
daunorubicin treatment or sphingomyelinase overexpression increased ceramide
generation followed by inhibition of telomerase activity. Clearance of ceramide
by overexpression of GCS prevented the telomerase inhibition [61]. These studies
suggest ceramide is an upstream regulator of senescence and aging.
8.3.3 Ceramide in Cell Differentiation and Growth Inhibition
Historically, the role of ceramide in cellular differentiation was discovered with

the observation that vitamin D3-induced monocytic, but not neutrophilic-type cell
differentiation in HL-60, and U037 leukemia cells was accompanied by increase
in nSMase activity and a concomitant spike in ceramide levels [62]. In turn, exog-
enous ceramide was found to induce monocytic differentiation of these cells. In
neuronal cell lines, ceramide-induced differentiation in T9 glioma cells, purkinje
and hippocampal neurons [63].
In another study, incubation of exponentially growing Saccharomyces cerevisiae
with short-chain ceramide inhibited cell growth with the involvement of an okadaic
acid-sensitive protein phosphatase [64]. Another study uncovered the mechanism
of ceramide-induced growth suppression in that serum withdrawal in MOLT-4 cells
resulted in significant dephosphorylation of Rb, correlating with the induction of
G0/G1 cell cycle arrest [65]. Taken together, these studies implicate a role for cera-
mide in cell differentiation and cell cycle progression.
8.3.4 Ceramide and Autophagy
In mammalian cells, ceramide and/or dihydroceramide have been shown to

induce autophagy. For example, in glioma cells, ceramide has been shown to acti-
vate the transcription of death-inducing mitochondrial protein, BNIP3, and sub-
sequent autophagy [66]. In the human colon cancer HT-29 cells, C2 ceramide
inhibited activation of protein kinase B, which is a negative regulator of interleukin

13-dependent macroautophic inhibition. In MCF-7 breast cancer cells, ceramide
stimulated the expression of Beclin-1 which is an autophagy gene product. This
study also showed that tamoxifen-induced autophagy was blocked using the SPT
inhibitor myriocin (ISP1) [67]. In another study, ceramide was shown to induce
autophagy by regulating calpain in MEFs [68]. In DU145 cells, fenretinide (4HPR)
treatment favored autophagic induction possibly due to the increase in endogenous
dihydroceramide [69].
In a study by Signorelli et al. [70], resveratrol induced autophagy in HGC-
27 cells with an increase in dihydroceramides possibly by inhibition of dihy-
droceramide desaturase. Inhibitors of dihydroceramide desaturase mimicked the
autophagic induction induced by resveratrol.
Mechanistically, Beclin-1 has been shown to be physiologically associated with
the mammalian class III phosphatidylinositol 3-kinase (PI 3-kinase) Vps34, and
the knockdown of Beclin-1 blunted the autophagic response of the cells to nutri-
ent deprivation or C2-ceramide treatment [71]. Class I PI3K and AKT pathway
are known to suppress autophagy and ceramide has been shown to inhibit AKT by
activation of PP2A, thereby establishing a possible mechanistic link between cera-
mide and autophagy induction [67, 72, 73].
Based on the above studies showing ceramide effects on mammalian
autophagic regulation, combined with the observation that yeast subjected to
heat stress exhibits growth suppression accompanied by upregulation of cera-
mide synthesis and downregulation of nutrient transporters on their cell surface
[74, 75], Edinger and colleagues hypothesized that ceramide-induced mamma-
lian autophagy might be mediated through a yeast-like response to heat stress by
downregulation of nutrient transporters. They showed that C2 ceramide produced
a profound downregulation of nutrient transporter proteins in mammalian cells.
Inhibition of autophagy or acute limitation of extracellular nutrients increased the
sensitivity of cells to ceramide. Supplementation of cells with the cell-permeable
nutrient methyl pyruvate protected the cells from ceramide-induced cell death and
delayed autophagic induction. So the authors concluded that ceramide killed cells
(apoptosis) by provoking nutrient limitation via downregulation of nutrient trans-
porters and subsequent autophagy [76]. Taken together, these studies implicate
ceramide in the regulation of autophagic response.
8.4 S1P/S1PR as Tumor Promoters
S1P is considered a pro-survival lipid. For example, S1P stimulated the invasive-
ness of glioblastoma tumor cells [77], promoted estrogen-dependent tumorigen-
esis of breast cancer cells [78] and conferred resistance to the cytotoxic actions of
TNF-α and daunorubicin [10]. A number of studies documented the role of S1P/
S1PR in proliferation, inhibition of apoptosis, vasculogenesis/angiogenesis, and
inflammation. These topics will be discussed in the following sections.
8.4.1 S1P in Proliferation and Inhibition of Apoptosis
Sphingosine kinase (SK) phosphorylates sphingosine to form S1P. Overexpression

of the SK1 isoform induced oncogeneic transformation in NOD/SCID mice. Using
inhibitors of SK, investigators implicated SK in the involvement of oncogenic
H-Ras-mediated transformation [79]. In another study, addition of exogenous S1P
reversed the cell death induced by ceramide [80]. Mechanistically, S1P counter-
acted ceramide-induced activation of stress-activated protein kinase (SAPK/JNK)
and activated the extracellular signal-regulated kinase (ERK) pathway in govern-
ing the fate of the cell [80].
Neutralizing antibody to S1P substantially reduced tumor progression in
murine xenograft and allograft models. The antibody arrested tumor-associated
angiogenesis, neutralized S1P-induced proliferation, attenuated release of pro-
angiogenic cytokines, and blocked the ability of S1P to protect tumor cells from
apoptosis [81].
In yet another study, S1P-mediated inhibition of apoptosis in C3H10T 1/2
fibroblasts depended on ERK activation and MKP-1, which downregulated
SAPK/JNK to bring about inhibition of apoptosis [82]. In male germ cells, S1P
inhibited stress-induced cell death, possibly by inhibiting nuclear factor kappa B
(NF-kappa B) and AKT phosphorylation [83].
8.4.2 S1P and Vasculogenesis/Angiogenesis
S1P promotes vasculogenesis and angiogenesis. S1P, the natural ligand for S1P3
receptor or KRX-725, a synthetic peptide that mimics S1P action on this recep-
tor, favored angiogenesis, as demonstrated by assessment of vascular sprouting
using aortic rings as an ex vivo model of angiogenesis. When S1P or KRX-725
were combined with other growth factors such as basic fibroblast growth factor
(b-FGF), stem cell factor, or vascular endothelial growth factor (VEGF), the inves-
tigators observed synergistic induction of angiogenesis [84]. In a cultured mouse
allantois explant model of blood vessel formation, Argraves et al. [85] showed that
S1P, synthesized via the action of SK2, promoted vasculogenesis by promoting
migratory activities of angioblasts and early endothelial cells to expand the vascu-
lar network.
VEGF has been shown to stimulate SK1 activity with an increase in the pro-
duction of S1P and activation of H and N Ras oncogenes in T24 bladder tumor
cell lines [86]. Endothelial cells undergo morphogenesis into capillary networks
in response to S1P involving G protein receptors [87]. S1P has been shown to
induce endothelial cell invasion and morphogenesis in physiologically relevant
collagen and fibrin matrices [88]. Based on studies employing inhibitors and
functional antagonists of S1P receptors, it has been hypothesized that the angi-
ogenic function of S1P is mediated by S1P1 and S1P3 signaling [87, 89, 90].
Knockout of the S1P1 receptor resulted in vascular deficiencies in mice [91]. In

a recent study, SphK1–SphK2 double-knockout mice manifested defective neu-
ral and vascular systems and exhibited embryonic lethality. The authors inferred
that S1P was required for “functionally intertwined pathways of angiogenesis and
neurogenesis” [92].
8.4.3 S1P in Inflammation
The SK1/S1P pathway has been implicated in inflammation. For instance, TNF-
alpha resulted in activation of SK1/S1P pathway specifically leading to extracel-
lular signal-regulated kinases and NF-kappa B activation [36] and consequently
expression of vascular cell adhesion molecule (VCAM) and intercellular adhe-
sion molecule (ICAM) [93]. S1P also induced cyclooxygenase 2 (COX2) and
prostaglandin E2 (PGE2) production in L929 fibrosarcoma and A549 lung ade-
nocarcinoma cells and genetic knockdown using siRNA blocked their produc-
tion. Additionally, preventing S1P clearance using siRNAs against S1P lyase/
phosphatase resulted in increased production of COX2 and PGE2, implicating a
key role of S1P in this pathway [94]. Microglial activation has been implicated
in neuroinflammation. LPS treatment increased SK1 mRNA and protein levels
and consequently upregulated expression of proinflammatory cytokines such as
TNF-alpha, IL-1beta, and iNOS in microglia. Chronic production of inflamma-
tory cytokines by microglia has been implicated in neuroinflammation [95, 96].
Further, the SK/S1P pathway has been implicated in many other inflammatory
disease paradigms such as asthma, rheumatoid arthritis, and inflammatory bowel
diseases [97].
In summary, these data collectively demonstrate that S1P regulates cancer cell
viability, angiogenesis, and inflammation which favor cancer pathogenesis.
8.5 Role of Other Metabolites of Sphingolipid

Metabolism in Tumorigenesis
In addition to ceramide and S1P, ceramide -1 phosphate has been implicated in

cell survival pathways. Chemotherapeutic agents changed the alternative splicing
of caspase 9 and Bcl-x pre-mRNA into pro-apoptotic forms which was mediated
by ceramide-dependent activation of PP1 [98]. C1P has been found to inhibit PP1,
and therefore, it might antagonize ceramide-mediated apoptosis, functioning as a
pro-survival lipid. In another study, C1P blocked apoptosis in part by activating
PI3-K/PKB/NF-κB pathways and production of anti-apoptotic Bcl-XL [99]. CERK
might play an important role in regulating the balance between ceramide and C1P
and therefore cell death and cell survival similar to S1P.
Sphingosine which is the breakdown product of ceramide is also implicated

in apoptotic responses. In one study, gamma irradiation along with TNF-α
induced sphingosine and S1P levels. The elevation of sphingosine by exog-
enous administration of sphingosine or by treatment with SK inhibitor induced
apoptosis in LNCaP prostate cancer cell lines [100]. In another study, phorbol
myristate acetate (PMA) and tumor necrosis factor (TNF) separately induced
apoptosis marked by elevation of sphingosine in HL-60 cells and neutrophils,
respectively. Exogenous administration of sphingosine or its methylated deriva-
tive N, N,-dimethylsphingosine (DMS) also induced apoptosis in cells of both
hematopoietic and carcinoma origin [101]. In an attempt to find the mechanism
of sphingosine-induced apoptosis, Domae and colleagues found that sphingosine
induced c-Jun expression and apoptosis in HL-60 cells and inhibition of pro-
tein kinase A (PKA) potentiated this effect [102]. In another study by Houghton
and co-workers, rhabdomyosarcoma cell lines were found to be more sensitive
to the induction of apoptosis with an increase in the cellular levels of sphingo-
sine. Mechanistically, sphingosine-mediated cell death involved mitochondrial
events such as Bax activation and translocation to the mitochondria, release of
cytochrome c and Smac/Diablo, but not apoptosis-inducing factor (AIF), endonu-
clease G, and HtrA2/Omi, from mitochondria and finally activation of caspase-3
and caspase-9 [103].
Gangliosphingolipids have been implicated in the epithelial-to-mesenchy-
mal cell transition (EMT) which is believed to play a role in cancer progression.
Pharmacological inhibition of GlcCer synthase has been shown to result in down-
regulation of E-cadherin, a major epithelial marker, and upregulation of vimentin
and N-cadherin, major mesenchymal cell markers, with marked changes in gan-
gliosphingolipids (Gg4 or GM2) and increased motility, implying these specific
glycosphingoplipids (Gg4 or GM2) might play a role in inhibition of EMT [104].
Some glycosphingolipids have been recognized as tumor antigens and thus could
participate in tumor cell regulation and as immune targets. In many studies, it was
shown that GM3 modulated receptor tyrosine kinase activity in cells [105–107].
In a recent study, ganglioside GM3 inhibited autophosphorylation of the EGFR
kinase domain, thereby inactivating it in response to ligand binding, and removal
of neuraminic acid of the GM3 headgroup or expression of the K642G mutant
released this inhibitory effect [108].
8.6 Sphingolipids in Cancer Therapy
Among the bioactive sphingolipids, ceramide and S1P act as pro-apoptotic and
anti-apoptotic lipids, respectively, and therefore, modulation of these lipids may be
effective as a treatment strategy for cancer (refer to Fig. 8.4 [109]). Such strategies
to increase the accumulation of ceramide and attenuation of S1P are discussed in
detail in the following section.
FTY 720
Tumor proliferation
S1P Angiogenesis
Inflammation
SK1
Chemoresistance
S1P
Sphingosine
GSL
P-gp
Golgi Apoptosis
CDase
Glucer
B13
GCS PP1, PP2A

OGT2378
Pyridinium
SMase Ceramide Ceramide
SMS
SM
nSMase
D609 Ceramide
analogues
Mitochondria
Chemotherapy
Fig. 8.4 Chemotherapeutic agents increases ceramide levels and induces apoptosis through the
de novo pathway or through the neutral sphingomyelinase (N-SMase) pathway. Induction of SK1
in colon cancer leads to accumulation of S1P, possibly leading to tumor proliferation, angiogene-
sis, and inflammation. Clearance of ceramide to Glucosylceramide by GCS in breast cancer cells
leads to the development of drug resistance. P-glycoprotein (P-gp) expression might potentiate
the chemo-resistant phenotype. Marked in red arrows are modulators of SPL metabolism. B13 is
an inhibitor of acid CDase, OGT2378 is GCS inhibitor, D609 is an inhibitor of SMS, FTY720 is
a sphingosine analogue, and ceramide analogues mimic endogenous ceramides, and Pyridinium
ceramide targets mitochondria and promotes mitochondria mediated apoptosis. Abbreviations:
CDase ceramidase, ER endoplasmic reticulum, PP1 protein phosphatase 1, PP2A protein phos-
phatase 2A, GSL glycosphingolipid, GCS glucosylceramide synthase
8.6.1 Targeting Ceramide Generation
Cytotoxic chemotherapeutic agents such as daunorubicin, etoposide, camptothecin,

fludarabine, and gemcitabine were shown to induce de novo ceramide generation, and
inhibition of this pathway reduced the cytotoxic responses to these drugs meaning
Table 8.1 Sphingolipid analogues and inhibitors of ceramide metabolism

Compounds Mode of action Cancer types
B13 Acid ceramidase inhibitor Prostate and colon
D609 Sphingomyelin synthase Monocytic leukemia
inhibitor
C16 Serinol, 4,6-diene-cera- Ceramide analogue Neuroblastoma and breast
mide, 5R-OH-3E-C8-cera-
mide, adamantyl-ceramide,
and benzene-C4-ceramide
Pyridinium ceramide Ceramide analogue Head and neck squamous cell
carcinoma, breast and colon
Pegylated liposomes with Improved delivery Breast
ceramide
Vincristine in SM-liposomes Improved delivery Acute lymphoid leukemia
FTY720 Sphingosine analogue Lymphoma, bladder, glioma,
prostate
OGT2378 GCS inhibitor Melanoma
that these drugs manifest their cytotoxic effects partly through ceramide production
[110–112]. Besides the de novo pathway, certain drugs, including daunorubicin,
also induce ceramide generation through the activation of nSMase which hydrolyzes
sphingomyelin to generate ceramide. For instance, in leukemia cells, cytosine arabi-
noside (Ara-C) induced activation of nSMase [113]. Mechanistically, this induction of
nSMase was brought about by generation of reactive oxygen species followed by Jun
N-terminal kinase phosphorylation and apoptosis [114]. In another study, actinomycin
D and etoposide induced nSMase activity in a p53- and ROS-dependent manner [115].
Alternatively, studies on reagents that inhibit the enzymes that favor the cera-
mide clearance pathway, leading to accumulation of ceramide and, thereby, poten-
tiating the cytotoxic effects were tested. For instance, compounds such as B13 that
inhibited acid CDase or tricyclodecan-9-yl-xanthogenate (D609) that inhibited
SMS, induced apoptosis in colon cancer and in U937 human monocytic leukemia
cells, respectively [116, 117] (refer to Fig. 8.4 and Table 8.1).
Interestingly, SM was found to potentiate the chemotherapeutic response of gemi-
citabine in prostate cancer cell lines [118]. In a study involving combination of SM
with chemotherapeutic agents, doxorubicin, epirubicin, or topotecan, it was found
that the combination therapy increased the cytotoxic effect of the drugs by increas-
ing their bioavailability, possibly by modulation of plasma membrane lipophilicity,
facilitating entry of these agents into the various cancer cell lines studied [119].
8.6.2 Mimicking Ceramide Action (Analogues of Ceramide)
Ceramide analogues (such as the soluble short-chain C2- and C6-ceramides) have
been shown to bring about cell death in many types of cancer cell lines tested [120].
C16-serinol, 4, 6-diene-ceramide, 5R-OH-3E-C8-ceramide, adamantyl-ceramide,
and benzene-C4-ceramide (Table 8.1) are some of the ceramide analogues that
induced cell death in cell lines such as neuroblastoma and breast cancer [121–124].
A novel, cationic, water soluble, pyridinium ceramide (Table 8.1) accumulated pre-
dominantly in cellular compartments that are negatively charged such as mitochon-
dria and the nucleus and caused changes in mitochondrial structure and function
and inhibited growth in various human head and neck cancer cell lines [125], while
inducing apoptosis in squamous cell carcinoma (HNSCC) cell lines [126–128].
Experiments to uncover the most efficient means of delivery of these ceramide
analogues have been tried extensively. Pegylated liposomes were very effective in
bringing about ceramide-mediated cell death in breast cancer cell lines (Table 8.1).
Liposomal delivery of ceramide decreased phosphorylated AKT and activation of cas-
pase-3/7 more effectively than non-liposomal ceramide [129]. Vincristine incorporated
in SM-liposomes called sphingosomes (Table 8.1) was found to be effective in animal
models for treatment of acute lymphocytic leukemia (ALL) to the extent that it is cur-
rently in Phase II clinical trials [130]. These studies clearly demonstrate that targeting
ceramide generation might be an effective method to bring about cancer cell death.
8.6.3 Attenuation of the S1P Pathway
Since S1P is found to be involved in angiogenesis and proliferation, it is intuitive to

think that modulation of this pathway offers hope for the treatment of cancer. In fact,
inhibition of SK1 resulted in increased cell death in many forms of cancer [131, 132]
and increased the sensitivity to cell death stimuli such as TNFα and FAS ligand [133].
Dihydroxyaurone, an SK1 inhibitor, exhibits anti-tumor activity in mammary tumors
[37]. Interestingly, in normal tissues of ovary and testis, exogenous S1P treatment
seems to protect cells from chemotherapy-induced apoptosis [134, 135].
FTY720 (Table 8.1), an analogue of sphingosine, has been found to be phos-
phorylated in vivo, and the resulting FTY720 phosphate functioned as a ligand for
sphingosine-1-phosphate receptors. This signaling mechanism enabled sequestration
of lymphocytes in lymphoid tissues thereby causing immunosuppression [136–138].
This compound also induced apoptosis in various cancer cell lines such as lympho-
cyte and bladder cancer (T24, UMUC3 and HT1197), glioma (T98G), and prostate
(DU145) cancer [37, 139–142]. Therefore, it is tempting to hypothesize that S1PR1
and S1PR3 antagonists might have similar anticancer effects. In another study, anti-
S1P mAb greatly reduced tumor progression in murine xenograft and allograft models
by inhibiting capillary formation and angiogenesis [81]. These data strongly support
the candidacy of S1P as a potential therapeutic target for the treatment of cancer.
8.6.4 Dietary SM as a Cancer Therapeutic
Brasitus and co-workers demonstrated that there was a significant increase in SM

levels and activity of SMS in rat colonic mucosa in response to 1, 2-dimethylhydrazine
(DMH), a chemical colonic carcinogen [143]. Merrill and co-workers found that milk
sphingomyelin dietary supplementation reduced the incidence of DMH-induced pre-
malignant lesions of colon tumors in CF1 mice [144]. In addition, mice fed with SM
developed fewer adenocarcinomas. These findings suggest that milk SM might sup-
press advanced malignant tumors in colon [145]. Administration of synthetic SM and
ceramide analogues also suppressed colonic crypt foci formation [146, 147]. In another
study, azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon carcinogen-
esis was modulated by dietary SM in the early stages by activation of peroxisome pro-
liferator-activated receptor γ (PPAR-γ), but its anti-carcinogenic effect was independent
of PPAR-γ [148]. Therefore, dietary SM might modulate the proteins expressed during
early stages of colon carcinogenesis and therefore may be a potential therapeutic candi-
date in the context of colon carcinogenesis.
8.7 Sphingolipids in Drug Resistance
One of the reasons for the failure of chemotherapy in cancer is the development
of tumor cell resistance. Part of the basis for chemoresistance might be attributed
to a re-wiring of sphingolipid metabolism. For instance, in many cases of leuke-
mia, breast cancer, and melanoma, chemotherapeutic agents increase the activity
of GCS which thereby attenuates ceramide levels, resulting in a drug resistance
phenotype [20, 149]. Overexpression of GCS offered increased resistance to doxo-
rubicin whereas siRNA knockdown promoted increased sensitivity to doxorubicin,
paclitaxel, and etoposide in breast cancer cells [150–152]. Mechanistically, GCS
upregulated P-glycoprotein (P-gp) which is an ABC transporter implicated in drug
resistance. Knockdown of GCS inhibited MDR1, a gene that encodes P-gp, revers-
ing drug resistance [153, 154].
Based on the above studies, GCS inhibition has been predicted to improve the
effectiveness of chemotherapeutic drugs. Some studies suggest that this hypothesis
is in fact true. For instance, OGT2378 (Table 8.1), an inhibitor of GCS, inhibited
melanoma growth in a syngeneic orthotopic murine model [155]. In separate stud-
ies, combination of fenretinide, a compound that induces accumulation of dihy-
droceramide [156] through direct inhibition of dihydroceramide desaturase [157],
with GCS inhibitors resulted in synergistic suppression of the growth of various
tumors. Additionally, fenretinide combined with SK inhibitors such as PPMP or
safingol caused growth inhibition [158, 159].
Sphingosine kinase and S1P have also been implicated in drug resistance phe-
notypes. For instance, it has been brought to light that certain drug-resistant mela-
noma cell lines such as Mel-2a and M221 are resistant to Fas-induced cell death
due to a decrease in ceramide and an increase in S1P compared with Fas-sensitive
counterparts such as A-375 and M186. Downregulation of SK1 with siRNA
decreased the resistance of Mel-2a cells to apoptosis [160]. Similar inference was
made in camptothecin resistant prostate cancer cell lines [161]. In a recent study,
SK1 was found to be upregulated in imatinib-resistant chronic myeloid leukemia
cell line concomitant with increased BCR-ABL mRNA and protein levels. The
PI3K/AKT/mTOR pathway was also found to be upregulated. Knocking down
SK1 expression using siRNA reversed the imatinib resistance to apoptosis and
returned BCR-ABL to normal levels [162], suggesting a role for SK1 in conferring
drug resistance.
8.8 Conclusions
There is compelling evidence to suggest that sphingolipid metabolism plays an

integral part of cancer pathogenesis and therapeutic response. Ceramide is a bioac-
tive lipid that activates signaling pathways to induce apoptosis of various cancer
cell lines. S1P, on the other hand, is emerging as a pro-proliferative lipid that is
frequently upregulated in tumors. Therapeutic regimens targeting the balance of
ceramide and S1P may prove useful in the treatment of many carcinomas.
In spite of our understanding of sphingolipid metabolism, and its relevance in
cancer models, we still have a long way to go in understanding the intricacies of
sphingolipid metabolism, how the metabolism proceeds in different cancer sub-
types, how compartmentalization of metabolism may offer unique regulatory
roles, and how different species of individual lipid molecules provide unique sign-
aling functions. With the advent of sophisticated lipidomics and bioinformatic
approaches, more and more sphingolipid functions/signaling mechanisms are
being uncovered, and this area of research holds and will continue to hold promise
as a potential avenue of therapy.
Acknowledgments We thank Benjamin Newcomb for his critical review of this chapter. We
also thank the members of Yusuf Hannun and Lina Obeid laboratory for their helpful discussion.
We apologize to those investigators whose important works were not included in this chapter
because of the space limitations. The Yusuf Hannun laboratory is supported by research grants
from the National Institutes of Health, USA.
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Chapter 9
Leading Small Molecule Inhibitors
of Anti-Apoptotic Bcl-2 Family Members
Victor Y. Yazbeck and Daniel E. Johnson
Abstract Anti-apoptotic members of the Bcl-2 protein family are commonly

overexpressed in human malignancies, where they contribute to tumorigen-
esis and the development of resistance to chemo-, radio-, and immunotherapies.
Considerable effort is being invested in academic and pharmaceutical settings to
identify and design effective small molecule inhibitors of the anti-apoptotic Bcl-2
family members. This chapter will focus on recent advances in the development
and application of three small molecule inhibitors (ABT-737, ABT-263, GX15-
070), with a particular emphasis on progress that has been made in the evaluation
of these compounds in preclinical in vivo models and clinical trials.
9.1 Introduction
As detailed in Chap. 1, the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL,

and Mcl-1 are frequently overexpressed in both hematopoietic and solid tumor
malignancies. Importantly, overexpression of these proteins often correlates
with chemotherapy and radiation resistance and poor clinical prognosis. Small
molecule inhibitors of anti-apoptotic Bcl-2 family proteins have the potential
to restore the sensitivity of tumors to apoptotic stimuli and may be particularly
useful when used in combination with conventional chemotherapeutics or radia-
tion therapy. This has spurred an intensive effort to identify or design small mol-
ecule inhibitors targeting Bcl-2, Bcl-XL, and/or Mcl-1. A number of different
approaches have been utilized to search for these inhibitors, including perfor-
mance of high-throughput screening assays, virtual screening, structure-based
V. Y. Yazbeck (*) · D. E. Johnson
Department of Medicine, University of Pittsburgh
D. E. Johnson
Department of Pharmacology and Chemical Biology, University of Pittsburgh
232 V. Y. Yazbeck and D. E. Johnson
drug design, and fragment-based drug design. To date, several inhibitors have
been isolated from natural sources, while others have been identified in chemi-
cal libraries or developed via medicinal chemistry efforts. Among the inhibitors
that have been reported are peptides derived from BH3 domains of proapoptotic
proteins [1–4], antimycin A3 [5], HA14-1 [6], BH3I compounds [7], several tea
polyphenol compounds [8, 9], chelerythrine [10], sanguinarine [11], (-)-gossypol
[12–14], apogossypolone [15, 16], TW-37 [13], ABT-737 [17], ABT-263 [18], and
GX15-070 [19]. This chapter will focus on three of these inhibitors, ABT-737,
ABT-263, and GX15-070, that have shown particular promise in preclinical stud-
ies and have advanced rapidly to clinical testing. The identification and initial in
vitro evaluation of these compounds will first be described. We will then summa-
rize the current understanding regarding their mechanism of action and the mecha-
nisms which lead to resistance to these inhibitors. Additionally, reported synergies
between these inhibitors and conventional chemotherapeutic agents will be dis-
cussed. Lastly, results obtained from evaluation of the inhibitors in preclinical in
vivo models and human clinical trials will be presented.
9.2 ABT-737: Discovery, Mechanism of Action,

and Mechanisms of Resistance
ABT-737 (see Fig. 9.1a) was developed by Abbott Laboratories using a nuclear

magnetic resonance (NMR)-based and fragment-based approach to rationally
design inhibitors targeting the BH3-binding groove on anti-apoptotic Bcl-XL.
ABT-737 binds with high affinity to Bcl-XL, Bcl-w, and Bcl-2 (Ki ≤ 1 nM), but
with much lower affinity to Bcl-B, A1/Bfl-1, and Mcl-1 (Ki ≥ 0.46 μM) [17]. At
0.1 μM, ABT-737 disrupted nearly 50 % of a Gal4-Bcl-XL/VP-16-Bcl-XS com-
plex in a mammalian two-hybrid system. Also, it competed with the binding of
fluorescently labeled Bad BH3 peptide to Bcl-XL and Bcl-2 with IC50 values of 35
and 103 nM, respectively [17]. In HL-60, an acute myeloid leukemia (AML) cell
line, ABT-737 was capable of activating Bax through disruption of the Bcl-2/Bax
heterodimeric complex [20]. In follicular lymphoma (FL) cells, primary B-cell
lymphocytic leukemia (B-CLL) and multiple myeloma (MM) cells, ABT-737
released Bim from Bcl-2 sequestration, and partial Bim knockdown resulted in
decreased sensitivity to the compound [21]. In an acute lymphoblastic leukemia
(ALL) cell line, ABT-737 was found to induce oxidative stress characterized by
decreased glutathione and increased hydrogen peroxide/superoxide levels, lead-
ing to the activation of caspase proteases [22]. Additionally, ABT-737 induces
autophagy through disruption of physical interactions between the autophagy
regulator Beclin-1 and Bcl-2 or Bcl-XL [23]. As a single agent, ABT-737 demon-
strates cytotoxic activity against a large number of hematological cell lines [2, 20,
24–26], but very weak activity against solid tumor cell lines [27], with the excep-
tion of small-cell lung cancer (SCLC) cell lines which are sensitive to ABT-737
alone (IC50 < 1 μM) [17, 28]. Resistance to ABT-737 is frequently associated with
9 Leading Small Molecule Inhibitors of Anti-Apoptotic Bcl-2 Family Members 233
Fig. 9.1 Chemical structures
of ABT-737, ABT-263, and
GX15-070
overexpression of the anti-apoptotic Bcl-2 family member Mcl-1 [20, 28, 29]. For
example, Mcl-1 is highly expressed in HeLa cells that are resistant to ABT-737,
but reduction of Mcl-1 levels following CDK2 inhibition, DNA-damaging chemo-
therapy, or shRNA treatment, results in enhanced sensitivity to the compound [29].
In solid tumor cell lines, the induction of proapoptotic NOXA following the treat-
ment with chemotherapy or radiation leads to the inhibition of Mcl-1 and result-
ing synergy with ABT-737 [27, 30]. Efficient cell killing by ABT-737 requires
that binding of the compound to anti-apoptotic Bcl-2 family members results in
the release of bound proapoptotic proteins (e.g., Bim, Bax, Bak). Moreover, the
amount of released proapoptotic proteins must be sufficient to saturate empty
Mcl-1 and A1/Bfl-1 that are present in the cell [21].
Deng et al. have studied the mechanism whereby lymphoma cell lines develop
resistance to ABT-737 and have classified three forms of resistance. Class A resist-
ance is observed in cells that express only low levels of BH3-only activator pro-
teins, whereas Class B resistance is due to significant loss or mutation of Bax
or Bak. On the other hand, Class C resistance is due to acute overexpression of

anti-apoptotic proteins (Bcl-2, Bcl-XL, Mcl-1, Bcl-w, A1/Bfl-1) [31]. As noted
above, the importance of Mcl-1 overexpression in conferring resistance to ABT-
737 has been reported by several different groups.
9.3 ABT-737 in Preclinical Models
As previously mentioned, ABT-737 induces apoptosis as a single agent in AML,

diffuse large B-cell lymphoma (DLBCL), FL, MM, ALL, and SCLC cell lines,
as well as primary tumor cells from multiple hematologic malignancies, includ-
ing chronic lymphocytic leukemia (CLL) [17, 20, 21, 24, 25, 28, 32–34]. In these
sensitive cell lines, ABT-737 typically exhibits IC50 values of less than 1 μM and
often in the nanomolar range. By contrast, most solid tumor cell lines that have
been studied are more resistant to the drug [27, 28]. However, ABT-737 has been
found to synergize with chemotherapy in many solid tumor cell line models.
For example, ABT-737 has been shown to synergize with paclitaxel in A549, a non-
small-cell lung cancer (NSCLC) cell line [17]; carboplatin/etoposide in SCLC
cells [28]; dexamethasone and melphalan in MM cells [25]; Ara-C or doxorubicin
in OCI-AML3 leukemic cells that are resistant to single-agent ABT-737 [20]; the
proteasome inhibitor MG-132 in melanoma [35]; MEK inhibitor in BRAF-mutant
solid tumors [36]; transcriptional inhibitors (ARC [37], actinomycin [38]), gem-
citabine [39], methylseleninic acid (second-generation selenium) [40] in multiple
cancer cell lines [37–40]; and cisplatin/etoposide in head and neck cancer [27]; as
well as tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in renal,
prostate, and lung cancer cells [41].
9.3.1 Leukemia
In primary AML specimens, monotherapeutic ABT-737 has been shown to induce

apoptosis at sub-micromolar levels. In AML specimens with activating FLT3
mutations, ABT-737 synergized with FLT3 inhibitors [24]. Synergy against AML
has also been observed with ABT-737 in combination with sorafenib [42], the
protein synthesis inhibitor silvestrol [43], or the MEK inhibitor PD0325901 [44].
Importantly, although AMLs exhibit sensitivity to ABT-737, normal peripheral
blood mononuclear cells (PBMCs) appear to be resistant [20].
Testing of 7 ALL cell lines has revealed synergy between ABT-737 and
L-asparaginase (7/7), vincristine (7/7), and dexamethasone (5/7), without an impact
on normal PBMCs [26]. Synergy with the retinoid N-(4-hydroxyphenyl)retinamide
(4-HPR) has also been observed [45]. In a mouse ALL xenograft model, the com-
bination of ABT-737 and L-asparaginase significantly increased event-free survival
when compared with each agent alone [26]. In pediatric ALL xenograft tumors,
ABT-737 showed single-drug activity and potentiated the anti-leukemic effects of

several clinically relevant agents, including L-asparaginase, topotecan, vincristine,
and etoposide [46]. Similarly, in adult T-cell leukemia, ABT-737 induced signifi-
cant apoptosis as a single agent in cell lines, primary patient tumors, and xenograft
tumors, and synergized with other cytotoxic agents [47].
Analysis of 30 primary CLL specimens demonstrated sensitivity to ABT-737
in 21 of the cases, while the remaining samples demonstrated heightened sensi-
tivities following the addition of at least one cytotoxic agent [48]. However, cells
with deletion of 17p, a poor prognostic indicator for CLL, were less sensitive [49].
In chronic myeloid leukemia (CML), ABT-737 induced apoptosis in cell lines and
primary samples and prolonged the survival of mice harboring CML xenografts
[50]. ABT-737 exhibits synergy with imatinib and INNO-406 in CML [51, 52].
9.3.2 Multiple Myeloma
In MM, ABT-737 demonstrates single-agent activity against a number of cell

line models (IC50 < 1 μM), including those characterized by glucocorticoid
resistance [25, 33, 34], with the highest sensitivity observed in cells expressing
Bcl-2High/Mcl-1Low ratios [53]. An additive effect was seen when ABT-737 was
combined with glucocorticoids, melphalan, and bortezomib [34], while synergy
was observed in combination with Notch pathway inhibitor [54]. Importantly,
ABT-737 did not affect the colony-forming potential of human PBMCs [54] and
bone marrow-derived progenitors [33]. In a myeloma xenograft model, ABT-737
induced dose-dependent tumor regression [25], and combination with Notch inhib-
itor resulted in anti-tumor activity superior to either agent alone [54].
9.3.3 Lymphoma
In several lymphoma cell lines, including those representing MCL and DLBCL,
ABT-737 showed dose-dependent cytotoxicity and synergized with proteasome
inhibitors [55] and the histone deacetylase inhibitor vorinostat [56]. Combination
with bortezomib also demonstrated synergy in primary samples of MCL, DLBCL,
and CLL, but no significant cytotoxic effects were observed in PBMCs from
healthy donors [55]. In Hodgkin’s lymphoma (HL) cell lines, ABT-737 exhibits
dose- and time-dependant cytotoxicity as a single agent and enhanced the activity
of several conventional anti-lymphoma agents [57]. In cutaneous T-cell lymphoma
(CTCL), ABT-737 was found to synergize with the pan-histone deacetylase inhibi-
tor panobinostat [58]. Also, in a murine model of c-myc-driven B-cell lymphoma,
ABT-737 improved overall survival when compared to vehicle alone, even in the
face of Bcl-2 overexpression [29].
9.3.4 Lung Cancer
As mentioned above, ABT-737 exhibits single-agent activity against SCLC cell

lines [28] and synergizes with actinomycin D in these models [59]. In mice har-
boring SCLC xenograft tumors, intraperitoneal administration of ABT-737
(75–100 mg/kg daily) for 3 weeks resulted in complete regression of the tumors [17].
Also, ABT-737 synergizes with actinomycin D [38] and enhances gefitinib-
induced apoptosis [60] in NSCLC cell lines.
9.3.5 Gastrointestinal Malignancies
In hepatoblastoma cell lines, ABT-737 and GX15-070 (see Sects. 9.7, 9.8, 9.9)
exhibit additive effects when combined with standard chemotherapy [61]. ABT-
737 also synergizes with sorafenib against hepatocellular carcinoma (HCC) cell
lines and HCC xenograft tumors [62, 63]. Inhibition of cholangiocarcinoma cell
line proliferation with ABT-737 is observed with IC50 values in the 4–17 μM
range, and synergy with zoledronic acid has also been reported [64]. In pancreatic
cancer cell lines, ABT-737 synergizes with actinomycin D [38] and TRAIL [65].
Similarly, in human colorectal carcinoma (CRC) cell lines, ABT-737 induced dose-
dependent apoptosis and synergized with irinotecan [66] and oxaliplatin [67]. ABT-
737 also enhanced celecoxib-induced apoptosis and autophagy [68], and overcame
resistance to immunotoxin-mediated apoptosis in CRC cell lines [69]. Single-
agent ABT-737 activity against imatinib-sensitive and imatinib-resistant gastro-
intestinal stromal tumor (GIST) cell lines (IC50s of 1–10 μM) has been reported.
Furthermore, potent synergy with imatinib was observed in these models [70].
9.3.6 Breast and Ovarian Cancer
ABT-737 enhances cisplatin-induced apoptosis [71] and synergizes with GSIXII, a

γ-secretase inhibitor [72], as well as GDC-0941, a phosphoinositide 3-kinase (PI3K)
inhibitor, against breast cancer cell lines and xenograft tumors [73]. Additional stud-
ies have shown that ABT-737 alone is ineffective against breast cancer xenograft
tumors exhibiting high Bcl-2 levels, but is effective in combination with docetaxel to
significantly improve anti-tumor effects and overall survival [74]. In ovarian cancer
cell lines and xenograft tumors, ABT-737 enhances sensitivity to carboplatin [75].
9.3.7 Central Nervous System Malignancies
In glioblastoma cells, the cytotoxic activities of vincristine, etoposide, bortezomib, and

TRAIL have been reported to be enhanced by co-treatment with ABT-737 [76, 77].
Prolonged survival in glioblastoma xenograft tumor models was also observed [77].
ABT-737 synergizes with fenretinide in neuroblastoma cell lines, and the combination
increased event-free survival in mice harboring neuroblastoma xenograft tumors [78].
9.3.8 Genitourinary Malignancies
Renal cell carcinoma (RCC) cell lines exhibit very little sensitivity to ABT-737
as a single agent. However, strong super-additive effects were observed when
ABT-737 was combined with etoposide, vinblastine, or paclitaxel [79]. In pros-
tate cancer, ABT-737 synergizes with docetaxel in vitro [80] and with Pim kinase
inhibitors both in vitro and in vivo [81].
9.3.9 Other Malignancies
Single-agent ABT-737 is largely ineffective against melanoma cell lines, but dem-
onstrates synergy when combined with temodar [82], dacarbazine, fotemustine,
imiquimod [83], pseudomonas exotoxin A [84], and p38 MAPK inhibitor [85].
Similarly, ABT-737 alone is ineffective against head and neck squamous cell carci-
noma (HNSCC) cell lines, but produces marked synergy characterized by NOXA
upregulation when combined with cisplatin or etoposide [27]. In human erythroid
cells expressing mutant JAK2, ABT-737 enhances apoptosis induced by JAK2
inhibitor [86]. ABT-737 also enhances the effect of interferon-α (IFN-α) against
JAK2V617F-positive polycythemia vera hematopoietic progenitor cells [87].
Interestingly, ABT-737 induces apoptosis of mast cells in vitro and in vivo [88]
and is effective in treating animal models of arthritis and lupus [89].
9.4 ABT-263 (Navitoclax): Discovery, Mechanism of Action,

and Mechanisms of Resistance
Despite the potency of ABT-737 in targeting Bcl-2 and Bcl-XL, the compound
is not orally bioavailable and exhibits low solubility, hindering intravenous
delivery. Hence, Abbott Laboratories sought to develop second-generation deriv-
atives that would address these issues. Through medicinal chemistry efforts and
further structure-based rational design, an effective ABT-737 derivative named
ABT-263 (see Fig. 9.1b; ABT-263 is also called navitoclax) was developed [18].
ABT-263, like ABT-737, was shown to bind to purified anti-apoptotic Bcl-2 fam-
ily proteins with high affinity (Ki’s < 1 nM for Bcl-2, Bcl-XL, and Bcl-w). The
compound acts by disrupting the interactions of Bcl-2 and Bcl-XL with proap-
optotic Bcl-2 family members, leading to the Bax activation and induction of
the intrinsic, mitochondrial-mediated apoptosis pathway. In preclinical m

odels,
the oral bioavailability of ABT-263 ranged between 20 and 50 %, depending
on the formulation [18]. Also, like ABT-737, the new compound does not bind
and inhibit Mcl-1 [18]. Therefore, overexpression of Mcl-1 represents a primary
mechanism of resistance to single-agent ABT-263.
9.5 ABT-263 in Preclinical Models
Initial reports demonstrated that ABT-263 is cytotoxic for cell lines represent-
ing SCLC and various hematologic malignancies [18]. ABT-263 also induced
complete tumor regression in xenograft models of SCLC [18, 90] and ALL [18].
However, in xenograft models of aggressive B-cell lymphoma and MM, single-
agent ABT-263 exhibited only modest or no activity. On the other hand, ABT-263
significantly enhances the activity of other clinically relevant agents in xenograft
tumor models [18], including enhancement of erlotinib activity and the activities
of several conventional chemotherapy drugs [91, 92].
9.5.1 Solid Tumors
In NSCL adenocarcinoma cell lines, ABT-263 demonstrates synergy with Src

inhibitors in promoting anoikis [93]. In SCLC, the combination of ABT-263 with
an immunotoxin targeting the transferrin receptor was reported to promote syner-
gistic killing of ABT-263-resistant cell lines and to have additive effects against
ABT-263-sensitive lines [94]. ABT-263 also sensitizes HCC cell lines to TRAIL-
induced apoptosis, while sparing normal liver cells [95]. In addition, ABT-263
enhances apoptosis induction when combined with a survivin inhibitor (YM-155)
in HCC cell lines, but not in normal human hepatocytes [96]. When combined
with the glycolysis inhibitor 2-deoxyglucose (2-DG), ABT-263 enhanced apop-
totic death in multiple cell line models [97]. Moreover, the combination of 2-DG
and ABT-263 resulted in improved survival of mice harboring tumors derived
from PPC-1 cells, a highly chemoresistant prostate cancer cell line [97]. Lastly, in
a panel of 27 ovarian cancer cell lines, ABT-263 demonstrated strong synergy in
combination with paclitaxel in roughly half of the cell lines [98].
9.5.2 Hematologic Malignancies
ABT-263 demonstrates single-agent activity against DLBCL cell lines characterized

by poor prognostic features (c-Myc and Bcl-2 overexpression) and additive/syner-
gistic effects in combination with other conventional cytotoxic agents [99]. In MCL
cell lines and primary patient specimens, ABT-263 synergizes with the histone dea-
cetylase inhibitor, vorinostat [100]. The addition of ABT-263 to rapamycin enhances
apoptosis in FL cell lines and promotes tumor regression in vivo [101]. ABT-263
also enhances the activity of bendamustine against DLBCL, MCL, and Burkitt’s
lymphoma in vivo and improves the response to a bendamustine/rituximab regimen
in a subset of these xenograft tumors [102].
9.6 ABT-263 in Clinical Trials
9.6.1 Leukemia
A phase I dose-escalation study of single-agent ABT-263 has been conducted in

29 patients with relapsed or refractory CLL [103]. Patients received ABT-263
daily for 14 or 21 days of a 21-day cycle. At doses of the drug ≥110 mg/d, 35 %
of patients manifested a partial response, while 27 % had stable disease for more
than six months. ABT-263 activity was observed in poor risk patients, including
those with bulky disease, deletion of 17p, and those refractory to fludarabine.
Thrombocytopenia was dose-dependent and a major dose-limiting toxicity (DLT).
The authors concluded that ABT-263 warrants further evaluation as a single agent
or in combination with other agents in this population [103].
9.6.2 Lymphoma
In a phase I trial of relapsed or refractory lymphoid malignancies, 55 patients were

enrolled in a dose-escalation study of single-agent ABT-263 [104]. ABT-263 was
given in an intermittent schedule once daily (14 out of 21 days) or continuously
once daily (21 out of 21 days). Grade 1/2 diarrhea and fatigue were the most com-
mon toxicities observed, while grade 3/4 thrombocytopenia and neutropenia were
the most serious ones. Five DLTs were observed on the intermittent dosing sched-
ule: one grade 3 cardiac arrhythmia, one grade 4 thrombocytopenia, one grade 3
transaminase elevation, and two hospital admissions for pleural effusion and bron-
chitis. Due to the rapid and dose-dependent occurrence of acute thrombocytopenia,
the continuous schedule was preceded by a lead-in dose of 150 mg for 7–14 days
on cycle one. There were three DLTs observed on the continuous schedule: one
grade 4 thrombocytopenia, one grade 3 gastrointestinal bleed, and one grade
3 transaminase elevation. Partial responses were observed in 22 % of patients
(10/46), with clinical responses seen across different histologies and doses. The
authors selected the following regimen for a phase II study: 150 mg daily lead-in
dose given for seven days before the first cycle, followed by 325 mg daily adminis-
tered in a continuous schedule (21/21 days) on subsequent cycles [104].
9.6.3 Solid Tumors
A phase I dose-escalation trial of single-agent ABT-263 in solid tumors enrolled

47 patients, including 29 with SCLC or pulmonary carcinoid [105]. Thirty-five
patients were enrolled in the intermittent cohort where ABT-263 was started on
day minus 3, and 12 patients were enrolled in the continuous dosing cohort where
ABT-263 was given as a 1-week lead-in dose of 150 mg daily followed by continu-
ous daily administration. Common toxicities were grade 1/2 gastrointestinal side
effects and fatigue. Dose- and schedule-dependent thrombocytopenia was observed
in all patients. A confirmed partial response lasting more than two years was seen
in one patient with SCLC, and stable disease was seen in eight patients with SCLC/
carcinoid tumor. The MTD of 325 mg was reached. The authors concluded that
ABT-263 was safe and well tolerated, with encouraging preliminary results in
SCLC and dose-dependent thrombocytopenia as a side effect [105]. In the phase IIa
trial, 36 patients received ABT-263 with an initial lead-in dose of 150 mg daily for
seven days, followed by 325 mg daily for a 21-day cycle. Thrombocytopenia was
the most common toxicity (41 % with grade 3/4). A partial response was observed
in one (2.6 %) patient, while 9 (23 %) patients had stable disease. The authors con-
cluded that ABT-263 exhibits limited single-agent activity in advanced and recur-
rent SCLC and future studies should focus on combination with other agents [106].
9.7 GX15-070 (Obatoclax): Discovery and Mechanism

of Action
A screen of natural compounds for Bcl-2 family inhibitors by Gemin X

Pharmaceuticals led to the identification of a chemotype from the polypyrrole class
of molecules [107] that was further developed as a non-prodigiosin compound and
given the name GX15-070 (Fig. 9.1c; also called obatoclax). The clinical formu-
lation of GX15-070 is labeled obatoclax mesylate [108]. Unlike ABT-737, which
binds to selected anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, and Bcl-w),
GX15-070 binds and inhibits all anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-
XL, Bcl-w, Mcl-1, Bfl-1, Bcl-B) [109]. However, despite the broadened inhibitory
activity of GX15-070, it exhibits somewhat reduced affinity for Bcl-2 and Bcl-XL
relative to ABT-737/ABT-263 (Ki = 220 nM for Bcl-2 and ~500 nM for Bcl-XL,
Mcl-1, and Bcl-w) [110]. None the less, the ability of GX15-070 to inhibit Mcl-1
represents a distinct advantage of this compound, since expression of Mcl-1 has
been shown to mediate resistance to ABT-737/ABT-263. GX15-070 efficiently
inhibits the interactions between Mcl-1 and proapoptotic proteins, including Bak,
leading to the activation of the intrinsic apoptosis pathway [110]. Kidney epithe-
lial cells derived from mice deficient in Bax and Bak expression fail to activate
caspases when treated with GX15-070, underscoring the importance of these
proteins and the intrinsic pathway in the mechanism of GX15-070 action [108].
In addition to inhibiting anti-apoptotic Bcl-2 family members, GX15-070 has also

been reported to inhibit the activation of NFκB and to upregulate expression of the
TRAIL receptor DR5 [111].
9.8 GX15-070 in Preclinical Models
9.8.1 Leukemia
Using AML cell lines, Konopleva et al. [20] determined that GX15-070 induces
time- and dose-dependent cell death through activation of the intrinsic apoptotic
pathway, with IC50 values in the low micromolar range (1.1–5.0 μM). At lower
concentrations, GX15-070 induced cell cycle arrest at S-G2 phase. Treatment of
AML progenitor cells with GX15-070 resulted in potent induction of apoptosis
(IC50 = 3.6 ± 1.2 μM), and clonogenicity was inhibited by concentrations in
the 75–100 nM range. GX15-070 synergized with ABT-737 against AML cell
lines and synergized with Ara-C against leukemia cell lines and primary AML
specimens [20].
In other studies, GX15-070 demonstrated synergy with a histone deacetylase
inhibitor [112], and the multi-kinase inhibitor sorafenib against AML cell lines
and primary AML cells, but not against normal CD34+ cells [113]. Combination
of GX15-070 and sorafenib markedly reduced AML tumor growth in a xenograft
model and significantly enhanced the survival of mice bearing these tumors when
compared with either agent alone [113]. Single-agent GX15-070 was found to
inhibit growth and activate both apoptosis and autophagy in ALL cell lines that
were either sensitive or resistant to dexamethasone [114]. In primary CLL cells
derived from patients who had not received treatment in the past 3 months, GX15-
070 demonstrated killing activity in most specimens, including those characterized
by poor prognostic features such as deletion of 11q or 17p [115]. However, ZAP-
70-positive cases, associated with poor prognosis, were less sensitive to GX15-070
than were ZAP-70 negative cases. Additionally, inhibition of extracellular signal-
regulated kinase (ERK)-1/2 was found to inhibit Bcl-2 phosphorylation in CLL
cells, increasing the sensitivities of these cells to GX15-070 alone or in combina-
tion with proteasome inhibitors [115].
9.8.2 Lymphoma
In B-cell lymphoma cell lines, GX15-070 has been reported to enhance ritux-
imab activity [116]. Moreover, GX15-070 synergized with chemotherapy
to induce apoptosis in both rituximab/chemotherapy-sensitive (RSCL) and
rituximab/chemotherapy-resistant cell lines and primary tumor cells from B-cell
non-Hodgkin’s lymphoma patients [116]. Via NFκB inhibition and inhibition
of the DR5 repressor Yin Yang 1 (YY1), GX15-070 induced DR5 expression
and sensitized B-cell lymphoma cells to TRAIL-induced apoptosis [111].
In Hodgkin’s lymphomas, ABT-737 and GX15-070 demonstrated synergy with
the histone deacetylase inhibitor SNDX-275 [117], while in MCL cell lines and
primary cells, GX15-070 demonstrated single-agent activity and synergy with
bortezomib [118].
9.8.3 Multiple Myeloma
As a single agent, GX15-070 reduced viability in 15 human MM cell lines,

including those resistant to melphalan and dexamethasone, with a mean IC50 at
246 nM [19]. GX15-070 also induced cell death in primary patient specimens
and demonstrated an additive effect with the myeloma drugs melphalan, dexa-
methasone, and bortezomib [19]. Recently, the pan-CDK inhibitor flavopiridol
was found to synergize with GX15-070 in both drug-naïve and drug-resistant
MM cells [119].
9.8.4 Solid Tumors
Single-agent GX15-070 is largely ineffective against melanoma cell lines and

primary cells, although enhanced killing is observed when the drug is combined
with ER stress inducers such as tunicamycin or thapsigargin [120]. By contrast,
combination of ABT-737 with ER stress inducers resulted in only a very mod-
est induction of apoptosis in melanoma cells [120]. In breast cancer cell lines,
GX15-070 showed synergy in combination with the EGFR-HER-2/neu inhibitors
lapatinib and GW2974 [121]. In CNS tumor cell lines, the combination of GX15-
070 with lapatinib is also effective, except in cells lacking PTEN [122]. These
studies further showed that pre-treatment with GX15-070 prior to addition of
lapatinib resulted in improved killing compared with simultaneous treatment. The
effectiveness of the GX15-070/lapatinib combination has also been demonstrated
in vivo [122].
Treatment of esophageal cancer cell lines with GX15-070 alone results in dose-
dependent growth inhibition, with IC50 values between 1.0 and 3.1 μM. Synergy
was observed when the drug was combined with carboplatin or 5-fluorouracil
[123]. In pancreatic cells, GX15-070 significantly reduced tumor viability and
synergized with TRAIL [124]. Single-agent GX15-070 was reported in clono-
genic survival assays of cholangiocarcinoma cell lines, with IC50s ranging from
5 to 100 nM [125]. Experiments in a syngeneic rat orthotopic model confirmed the
single-agent activity of GX15-070 in promoting overall survival when compared
with vehicle control (44 versus 23 days) [125].
9.9 GX15-070 in Clinical Trials
9.9.1 Leukemia
The first phase I study of single-agent obatoclax mesylate (the clinical formulation of
GX15-070; hereafter referred to as obatoclax) was undertaken in patients with refrac-
tory hematological malignancies [126]. A total of 44 patients with refractory AML,
ALL, CLL, myelodysplasia (MDS), or CML in blast crisis were given 24-h infusions
of the drug, with a median of five infusions per patient. The drug was found to be
well tolerated, aside from grade 1/2 CNS symptoms, and the highest planned dose
was reached without any DLT. One AML patient obtained complete remission for
eight months, but subsequently relapsed, causing the authors to suggest that GX15-
070 induced proliferation arrest, as opposed to differentiation. The final recom-
mended dose for phase II was 28 mg/m2, given as an infusion for 24 h up to four
consecutive days. The authors concluded that obatoclax was well tolerated and sug-
gested further investigation in patients with leukemia and myelodysplasia [126].
A subsequent phase I trial of single-agent obatoclax enrolled 26 CLL patients
[127]. Patients were treated with single-agent obatoclax doses ranging from 3.5
to 14 mg/m2 as a 1-h infusion and from 20 to 40 mg/m2 as a 3-h infusion every
3 weeks. The observed neurological dose-limiting reactions (euphoria, ataxia, som-
nolence) were dose related, but quickly resolved after cessation of drug infusion.
Only one patient achieved partial remission at an obatoclax dose of 3.5 mg/m2. This
patient had been pre-treated with fludarabine, Rituxan, and alemtuzumab but was
the only patient in the trial who was naïve to alkylating agents. In other patients,
there was reduction in transfusion dependence. The MTD was determined to be
28 mg/m2 over 3 h every three weeks and was recommended for phase II studies.
The authors concluded that the biologic activity of obatoclax is not only dependant
on the dose, but also on tumor biology. The activity was modest in this population
and suggested investigation of the drug in a less heavily pre-treated population and
in combination with other agents [127].
9.9.2 Lymphoma
In another series of trials, 35 patients with previously treated lymphoma and solid
tumors, who were not candidates for standard therapies, were enrolled in two
phase I trials (GX001 and GX005) evaluating the safety and tolerability of single-
agent obatoclax given as 1-h (GX001) or 3-h (GX005) weekly infusions [128].
With the 1-h infusion schedule, neurological DLT (somnolence) was observed at
5 and 7 mg/m2. The MTD was 1.25 mg/m2. Patients on the 3-h infusion schedule
showed better tolerability, with an MTD of 20 mg/m2. Stable disease by RECIST
criteria was achieved in 25 % (2/8) of patients with the 1-h infusion and in 18 %
(5/27) of patients with the 3-h infusion. A partial response was observed in one
patient with large cell lymphoma stage IV assigned to the 28 mg/m2 dose group,
who received a total of 32 weeks of therapy. The authors concluded that the 3-h
weekly infusion of obatoclax was better tolerated with an MTD of 20 mg/m2 and
evidence of clinical activity [128].
Recently, Oki et al. [129] reported a phase II trial in patients with relapsed or
refractory classical Hodgkin’s lymphoma (cHL) treated with single-agent obato-
clax administered intravenously at 60 mg over 24 h and given every two weeks.
Thirteen patients received at least one dose of obatoclax, with a median of four
cycles per patient (range 1–24). The drug was found to be well tolerated, with
grade 1 toxicities: dizziness (n = 5), euphoria (n = 3), and hypotension (n = 1).
There were no objective responses; 38 % (5/13) of patients had stable disease. The
authors concluded that obatoclax showed limited activity in this heavily pre-treated
population of cHL. They recommended investigation of more potent Bcl-2 family
inhibitors with pharmacodynamic studies to ensure target inhibition and biomarker
analysis for plausible patient selection [129].
9.9.3 Myelofibrosis
Twenty-two patients who had previously been treated were enrolled in a multi-
center, open-label, non-comparative phase II study of single-agent obatoclax
administered as a 24-h infusion every 2 weeks at a fixed dose of 60 mg [130].
Patients received a median of 7 cycles. No objective responses were observed.
Only one patient had clinical improvement (a decrease in transfusion require-
ment). The most common side effects were low-grade ataxia and fatigue, observed
in 50 % of the patients. One patient had a dose reduction secondary to toxicity,
and two patients were taken off the study due to grade 3 toxicity (ataxia and heart
failure). The authors concluded that obatoclax showed no clinical activity in this
patient population at the dose and schedule used in the study [130].
9.9.4 Solid Tumors
A phase I trial studied the safety, MTD, and early anti-tumor activity of the com-
bination of topotecan and obatoclax in patients (n = 14) with solid tumors who
would benefit from treatment with topotecan. Obatoclax was given at a start-
ing dose of 14 mg/m2 over a 3-h intravenous infusion on a 3-week cycle, while
topotecan was given at 1.25 mg/m2 on days 1–5 on a every 3-week cycle [131]. Of
the observed toxicities, 88 % were grade 1 and 2, mainly neurologic, in the form
of ataxia, somnolence, mood alterations, and cognitive dysfunction. Two-fifths
of the patients developed grade 3 neurological DLTs at the 20 mg/m2 dose. Two
patients (SCLC) exhibited partial responses, and four (three with SCLC and one
with pulmonary carcinoid) had stable disease. The recommended dose for phase
II trial was obatoclax at 14 mg/m2 on days 1–3, in combination with topotecan

at 1.25 mg/m2 on days 1–5 of a 3-week cycle [131]. Given the preliminary effi-
cacy data seen in SCLC, an open-label, single-arm, phase II extension trial was
undertaken [132]. A total of 9 patients with recurrent SCLC were enrolled. They
received a median of two cycles. The most common grade 3/4 toxicities were
hematologic and ataxia. There were no objective responses, with 56 % (5/9) of
patients exhibiting stable disease. The authors concluded that the combination did
not achieve a better response rate than was previously seen with topotecan alone
in relapsed SCLC after first-line platinum therapy [132].
In another phase I trial, the safety of obatoclax in combination with standard
chemotherapy was evaluated in patients with extensive stage SCLC [133]. Twenty-
five patients were treated with carboplatin (area under the curve of 5) on day 1
and etoposide (100 mg/m2 on days 1–3) in combination with escalating doses
of obatoclax given either as a 3- or 24-h infusion on days 1–3 of a 21-day cycle.
Compared with the 24-h infusion cohort, there were more neurological DLTs
(somnolence, euphoria, and disorientation) in the 3-h infusion cohort. However,
these neurological toxicities were transient, resolving after the end of the infusion
and without sequelae. The MTD for the 3-h cohort was 30 mg/day, while it was
not reached for the 24-h cohort. The response rate was higher in the 3-h cohort
(81 versus 44 %). The authors concluded that the 3-h infusion was associated with
more neurological side effects, but better efficacy than the 24-h cohort. For further
studies in SCLC, they recommended an obatoclax daily dose of 30 mg given intra-
venously over 3 h on three consecutive days [133].
Acknowledgments We apologize to those authors whose works have not been cited due to
space limitations, or our oversight. This work was supported by National Institutes of Health
grants R01 CA137260 and P50 CA097190.
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Chapter 10
SMAC IAP Addiction in Cancer
Matthew F. Brown, Kan He and Jian Yu
Abstract Deregulation of apoptosis is a hallmark of human cancer. Inhibitor of

apoptosis proteins (IAPs) were first identified as inhibitors of caspases and apop-
tosis. They were later shown to play important roles in signal transduction to
promote cell survival and proliferation beyond apoptosis, a function conserved
in lower eukaryotes. Genetic alterations of cIAPs, as well as widespread altered
expression in IAPs and their endogenous inhibitors such as SMAC, have been
reported to be associated with disease progression and chemoresistance in can-
cer. Several strategies have been devised to target IAPs in cancer including ration-
ally designed small-molecule IAP antagonists based on the conserved interaction
between IAPs and SMAC. Known as SMAC mimetics, these agents are showing
promise as novel cancer therapeutics and help reveal novel functions of IAPs.
10.1 Introduction
Apoptosis is an evolutionally conserved process essential for normal develop-

ment, maintenance of tissue homeostasis and removal of unwanted or damaged
cells following stress or injury. Deregulated cell death is a hallmark of cancer
and contributes to chemoresistance [1, 2]. The inhibitor of apoptosis (IAP) fam-
ily of proteins was originally identified as inhibitors of caspases in model organ-
isms. Endogenous inhibitors of IAPs such as SMAC (second mitochondria-derived
activator of caspase) and HrtA2 (high temperature requirement protein A2, also
known as Omi) can directly bind to a conserved BIR (baculovirus interact-
ing repeat) within IAPs. The binding of SMAC to IAPs is mediated through a
M. F. Brown · K. He · J. Yu (*)
Department of Pathology, University of Pittsburgh School of Medicine,
University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
M. F. Brown · J. Yu
Department of Pathology and Molecular and Cellular Pathology Graduate Training Program,
University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute,
Pittsburgh, PA, USA
256 M. F. Brown et al.
conserved tetrapeptide. Cumulative evidence in the last decade using biochemical,

molecular and cellular, pharmacological, structural approaches, and genetically
modified model systems has revealed a multitude of IAP functions in cell death,
survival, proliferation, and immunity. Furthermore, several IAPs play a key role
in regulating NF-wB (nuclear factor-кB) activation and non-apoptotic cell death.
Overexpression and genetic alterations of IAPs, as well as reduced expression of
their endogenous inhibitors, have been reported in solid tumors and hematologi-
cal malignancies [3, 4]. In rare cases, loss of IAPs is associated with the develop-
ment of certain types of hematological malignancies, suggesting a potential role as
a tumor suppressor [5].
Agents targeting IAPs have been developed, with the most promising class
being small molecules mimicking the IAP-binding domain in SMAC, also known
as SMAC mimetics. These compounds have limited toxicity to cancer cells as a
single agent, while synergizing with different classes of anticancer agents in a
wide array of cancer cell types. The use of SMAC mimetics has helped elucidate
signaling functions of IAPs, particularly cIAP1 and cIAP2, independent of their
ability to suppress caspase activation or apoptosis.
This chapter provides an overview of the biology of IAP family members and
the attempts to target them in cancer. Extensive studies have provided strong evi-
dence that XIAP and cIAPs are key regulators of apoptosis, which are the focus of
this book chapter. The roles of other IAPs such as survivin in apoptosis and cancer
are less understood and will only be discussed briefly. More information can be
found elsewhere [6, 7].
10.2 The IAP Family
Modern virology has produced some of the most valuable insights into the mecha-
nisms of cell death. Many viruses encode gene products that suppress cell death
by disabling tumor suppressor proteins and producing viral versions of anti-apop-
totic Bcl-2 proteins and/or caspase inhibitors. This is critical for viral replication
and carcinogenesis in certain patients [8–10]. The baculovirus protein, IAP, was
the first inhibitor of apoptosis protein (IAP) discovered and shown to promote
viral replication [11, 12]. Through sequence homology-based screens and other
methods, eight IAP family members have been discovered in human, including
NAIP, XIAP, cIAP1, cIAP2, Survivin, BRUCE (Apollon), ILP2, and ML-IAP.
Seven of them have homologs in mice (Fig. 10.1 and Table 10.1). All IAP fam-
ily members in viruses and metazoans contain at least one signature baculovirus
IAP repeat (BIR) domain (Fig. 10.1 and Table 10.1), which is capable of interact-
ing with caspases and various IAP antagonists. Several of these, including XIAP,
cIAP1, cIAP2, survivin, and ILP2, directly bind to and suppress caspases through
their BIR domains [13]. XIAP, cIAP1, and cIAP2 also contain a RING domain
(Fig. 10.1), a signature of E3 ligases [13], and play critical regulatory roles in
NF-κB signaling and necrosis. Certain IAP family members, including XIAP and
10 SMAC IAP Addiction in Cancer 257
Fig. 10.1 Human IAP family members. Eight human IAP proteins have been identified and
all contain at least one baculoviral IAP repeat (BIR) domain. Several IAPs contain a carboxy-
terminal RING domain with E3 ubiquitin ligase activities, as well as ubiquitin-binding domains
(UBA/C). Other domains such as leucine-rich repeats (LRR), nucleotide oligomerization domain
(NOD) and caspase activation and recruitment domain (CARD) are present in some IAPs, which
mediate protein–protein interactions
cIAP1/2, BRUCE and ILP-2 contain UBA/C (ubiquitin binding or conjugating)

domains allowing them to bind or add mono- or poly-ubiquitin (Ub) to various
substrates. Other domains such as LRR (leucine-rich repeats), NOD (nucleo-
tide oligomerization domain), and CARD (caspase activation and recruitment
domains) mediate protein–protein interactions and are found in IAPs and various
proteins regulating apoptosis and inflammation. However, their roles in IAP regu-
lation of apoptosis are not fully understood [7].
10.3 Apoptotic Pathways
Apoptosis is initiated and executed by a series of well-ordered biochemical events

and regulated by complex signaling networks. There are two major apoptotic path-
ways, termed as the extrinsic and intrinsic pathways, which are responsible for
processing stress signals and executing cell demise [14, 15]. Although with dis-
tinct regulatory processes, there is significant cross-talk in many situations among
these two pathways.
The extrinsic apoptotic pathway was first found to be used by immune cells
to kill infected or damaged cells and subsequently shown to operate in many
cell types. This pathway is engaged upon binding of pro-apoptotic ligands to
cell surface death receptors of the TNFR family. All death receptors contain a
cysteine-rich extracellular domain, which is responsible for ligand binding, and
Table 10.1 IAPs and their endogenous inhibitors

Chr. Chr. Domains
Other location AA location AA
Protein names (Human) (Human) (Mouse) (Mouse) BIR RING Other
NAIP BIRC1 5q13 1403 13 D1–D3 1403 3 0 NOD,
LRR
cIAP1 BIRC2, 11q22 618 9 A1 612 3 1 CARD,
API1 UBA
cIAP2 BIRC3, 11q22 604 9 A2 602 3 1 CARD,
API2 UBA
XIAP BIRC4, Xq25 497 X A3–A5 496 3 1 UBA
API3
Survivin BIRC5, 17q25 142 11 E2 140 1 0 N/A
API4
BRUCE BIRC6, 2p22 4857 17 E3 4854 1 0 UBC
Apol-
lon
LIVIN BIRC7 20q13 298 2 H4 285 1 1 N/A
ILP-2 BIRC8, 19q13 236 N/A N/A 1 1 CARD,
Ts-IAP UBA
BIM RING Other
SMAC DIABLO 12q24 239 5F 237 1 0 N/A
HtrA2 Omi 2p12 458 6 C3 458 1 0 Trans-
mem-
brane
XAF1 BIRC4- 17p13 301 11 B4 273 0 0 Zinc
bind- finger
ing
protein
an intracellular death domain for transmitting signals through the recruitment of

effectors. Pro-apoptotic ligands belong to the extended cytokine TNF superfamily
and are either present on the cell surface or secreted into the extracellular space
[14, 15]. Upon binding of pro-apoptotic ligands to their respective receptors, each
receptor can independently form a death-inducing signaling complex (DISC) by
recruiting the adapter protein FADD, along with pro-caspase-8 and pro-caspase-10
(Fig. 10.2) [15]. FADD recruitment and DISC formation lead to proximity-
induced processing and activation of caspase-8 and caspase-10, and their release
into the cytoplasm triggers subsequent activation of effector caspases to execute
apoptosis (Fig. 10.2).
The intrinsic apoptotic pathway is triggered by stresses such as DNA dam-
age, deregulated oncogenes or nutrient/growth factor deprivation and is largely
regulated by the Bcl-2 family of proteins and mitochondria [16, 17] (Fig. 10.2).
For example, following DNA damage and p53 activation, death signals are trans-
mitted to BH3-only proteins to allow for the activation of Bax and Bak, which
leads to mitochondrial outer membrane permeabilization (MOMP) [18–20] and
Fig. 10.2 IAP-mediated cell survival and signaling. IAP proteins serve as a signaling hub for
cell survival. IAPs are negative regulators of programmed apoptosis and necrosis. XIAP and
cIAPs can inhibit both the initiator and effector caspases to block apoptosis. In addition, cIAPs
positively regulate canonical NF-κB signaling to allow TNFα production and inflammation,
while negatively regulating non-canonical NF-κB signaling, necrosis and caspase-8 activation,
all through complex assembly. SMAC or SMAC mimetics suppress XIAP and cIAP-dependent
cell survival by relieving IAP caspase inhibition through competitive binding, as well as by
depletion of cIAPs via activation of their E3 ligase activity (see text for details). (P)-phosphoryl-
ated and (Ub)-ubiquitinated
release of several mitochondrial apoptogenic proteins, including cytochrome c,

SMAC/Diablo, Omi/HtrA2, AIF (apoptosis-inducing factor), and EndoG (endo-
nuclease G) [21]. The release of cytochrome c promotes the assembly of the
apoptosome and subsequent activation of caspase-9 [21]. Additionally, SMAC/
Diablo and HtrA2/Omi facilitate apoptosis by binding to IAPs and relieving
their inhibition of caspases [22, 23]. The release of AIF and Endo G promotes
DNA degradation [24, 25]. Notably, in some cells where there are low levels
of DISC, caspase-8-dependent cleavage of the BH3-only protein Bid gener-
ates truncated Bid (tBid) to amplify apoptotic signaling via the mitochondria
(Fig. 10.2) [26, 27].
10.4 IAPs as Caspase Inhibitors
IAPs can inhibit caspases through two major mechanisms. Some IAPs bind
directly to active sites of caspases to prevent processing or activity. Among all
IAPs, mammalian XIAP is the only IAP that has high-affinity interactions with
caspase-3, caspase-7, and caspase-9, and functions as a direct caspase inhibi-
tor [28]. XIAP inhibits caspases-3, caspase-7, and caspase-9 via its BIR2 and
BIR3 domains [29–33]. Specifically, insertion of the linker region between
BIR1 and BIR2 into the catalytic pocket of active effector caspases (such as
caspase-3 or caspase-7) prevents substrate entry. In addition, XIAP can prevent
initiator caspases such as caspase-9 from dimerization and subsequent activa-
tion, by hindering a conformational change required for a functional catalytic
pocket [34, 35].
IAPs can also induce ubiquitin-mediated degradation of caspases. The RING
finger domain in DIAP1 regulates Ub conjugation of caspases, and therefore,
apoptosis in Drosophila, though the precise role of mono- versus poly-ubiquityla-
tion in this process, is not clear [36–39]. Consistent with these findings, cIAP1 and
cIAP2, or their homologs in lower eukaryotes do not inhibit caspases efficiently in
vitro [40]. Conversely, in living cells, XIAP promotes the degradation of active-
form caspase-3 [30], but not pro-caspase-3 [38]. Interestingly, the RING domain
in XIAP is not required for its inhibition of caspase-3, caspase-7, or caspase-9 in
vitro or when overexpressed [41, 42]. These findings suggest multiple mechanisms
are involved in IAP-mediated caspase inhibition.
10.5 cIAPs as Signaling Molecules in Cell Survival

and Non-Apoptotic Cell Death
cIAP1 and cIAP2 were initially identified as interacting proteins of the scaf-
fold protein TRAF2 (tumor necrosis factor receptor-associated factor 2). They
are now known to play crucial roles in regulating NF-кB signaling, DNA dam-
age response, and necrosis by regulating the assembly and stability of distinct
signaling complexes through their E3 ligase function [4, 43]. This knowledge
was largely obtained by using small molecules that deplete both cIAPs. In the
canonical NF-кB pathway, cIAP1 and cIAP2 regulate Ub-dependent activation
of NF-кB downstream of TNFR1, which in turn drives transcriptional programs
important for cell survival and inflammation [4, 43]. In addition, cIAPs sup-
press non-canonical NF-кB signaling, necrosis, and the assembly of caspase-8
activation complexes in resting cells. Rapid degradation of cIAP1 and cIAP2
triggered by SMAC mimetics, or DNA damage can lead to activation of the
non-canonical NF-кB pathway, apoptosis, or necrosis if caspase activation is
blocked [44–49].
10.5.1 cIAPs in NF-κB Pathways
NF-κB regulates a wide variety of cellular functions, such as survival, prolif-

eration, migration, and immune response. Constitutive activation of NF-κB and
chronic inflammation plays a major role in tumor progression [50–52]. The NF-κB
family consists of five transcription factors, RELA (also known as p65), RELB,
CREL and the precursor proteins NF-κB1/p105 and NF-κB2/p100. NF-κB signal-
ing is activated in response to receptor stimulation and various stresses [53, 54]
(Fig. 10.2) and is regulated by Ub-dependent signal transduction cascades and
assembly of protein complexes that lead to the processing of NF-κB1/p105 to p50
and NF-κB/p100 to p52, which is required for the transcription of target genes
[53–55] (Fig. 10.2). NF-κB signaling can be divided into canonical or non-canon-
ical pathways depending on the transcription factors used, either RELA/NF-κB1
(p50) or RELB/NF-κB2 (p52) (Fig. 10.2).
cIAPs are required for the activation of the canonical NF-κB pathway in
response to TNFα. Binding of TNFα to TNFR1 triggers receptor trimerization and
the recruitment of the adaptor protein TNFRSF1A-associated via death domain
(TRADD), the Ub ligases TRAF2/5, cIAP1, and cIAP2, and the protein kinase
RIP1 to a membrane-associated complex, referred to as complex-I (Fig. 10.2) [56].
Upon cIAP-mediated ubiquitination, RIP1 recruits additional components that
promote poly-ubiquitination of IKKγ in complex-I. This leads to a phosphoryla-
tion cascade and degradation of the inhibitor of transcription factor NF-κB (IκB),
allowing NF-κB to translocate to the nucleus. In Drosophila, DIAP2 is required
for NF-κB activation in the immune deficiency (Imd) signaling cascade [57–60].
On the contrary, cIAPs negatively regulate non-canonical NF-κB signaling via
the degradation of NF-κB-inducing kinase (NIK; also known as MAP3K14) [44]
(Fig. 10.2). Non-canonical activation of NF-κB is activated upon receptor binding
to the ligands of the TNF receptor superfamily, including CD40L, B-cell activating
factor (BAFF) and TWEAK/TNFRSF12A [54, 61]. This pathway is suppressed in
resting cells by the constitutive degradation of NIK through an Ub ligase complex
consisting of TRAF2/3–cIAP1/2 and the proteasome (Fig. 10.2). NIK degrada-
tion requires the E3 ligase activity of cIAP1 and cIAP2 while TRAF2/3 serves as
adaptors to recruit NIK to cIAP1/2. Depletion of IAPs or other components of the
TRAF2/3–cIAP1/2 Ub ligase complex leads to stabilization of NIK and spontane-
ous activation of the non-canonical pathway and TNFα production [62, 63].
10.5.2 cIAPs and Necrosis
Necrosis is characterized by ATP depletion, swelling of organelles, and spilling

of cellular contents. This form of cell death had long been thought to be a pas-
sive process until specific pathways came into light in recent years [10, 64]. In
the absence of cIAPs, TNFα stimulates the formation of a secondary cytoplasmic
complex, complex-II, which contains RIP1, Fas-associated via death domain

(FADD) and caspase-8 [46, 65] (Fig. 10.2). Complex-II formation leads to a rapid
activation of caspase-8 and subsequently apoptosis. In most cases, this form of
cell death can be completely blocked by caspase inhibitors. In some cells, cas-
pase inhibitors, or lack of caspase-8 activation can switch the apoptotic response
to RIP1/RIP3-dependent necrosis [47, 48, 66–68]. Some evidence suggests that
cIAPs can block necrosis triggered by CD95 stimulation perhaps in a fashion simi-
lar to TNFR1 stimulation [69].
10.5.3 cIAPs and DNA Damage Response
Radiation and common chemotherapeutics cause DNA damage and activation

of NF-κB, ATM/p53 and other signaling pathways [70]. Extensive DNA damage
depletes XIAP, cIAP1, and cIAP2 and promotes the assembly of a large ∼2MDa
cytoplasmic cell death-inducing platform referred to as the “ripoptosome”. This
complex contains RIP1, FADD, and caspase-8, and its formation can be stimulated
by SMAC mimetics [49, 71] (Fig. 10.2). Ripoptosome assembly requires RIP1
kinase activity and can stimulate caspase-8-mediated apoptosis as well as caspase-
independent necrosis without involvement of autocrine TNFα, a process similar
to complex-II formation in response to TNFα (Fig. 10.2). In some cells, following
excessive DNA damage, ATM employs NEMO (NF-κB essential modulator) and
RIP1 kinase through autocrine TNFα signaling to switch on cytokine production
and caspase activation [72]. These results illustrate novel p53-independent mecha-
nisms of cell killing after DNA damage in cancer cells, which are negatively regu-
lated by IAPs via both TNFα-dependent and TNFα-independent mechanisms.
10.6 Endogenous Inhibitors of IAPs
Several cellular proteins can antagonize the anti-apoptotic activities of IAPs. The
best studied endogenous IAP inhibitor is second mitochondria-derived activator
of caspase (SMAC), also known as direct inhibitor of apoptosis protein (IAP)-
binding protein with low pI (Diablo) or SMAC/Diablo. Upon apoptotic induction,
SMAC is released into the cytosol and binds to BIRs in IAPs through its amino
(N)-terminus to facilitate caspase activation [22, 73]. The Drosophila death pro-
teins Hid, Grim, and Reaper have limited homology with SMAC, in the BIR bind-
ing motif [22, 73, 74] (Fig. 10.3a), suggesting that regulation of caspase activation
is an ancient function of these proteins [41, 75].
SMAC/Diablo functions as a general IAP inhibitor and binds to XIAP, cIAP1,
cIAP2, survivin, livin, and BRUCE, but not NAIP [22, 73, 76–79]. The four
amino-terminal residues of mature SMAC/DIABLO, Ala-Val-Pro-Ile (AVPI) are
both necessary and sufficient for SMAC/DIABLO–IAP interaction [3, 80–83].
Fig. 10.3 IAP-binding motif (IBM) and SMAC mimetics. a The conserved BIR binding motif.
This tetrapeptide motif has the consensus sequence of A-(V/T/I)-(P/A)-(F/Y/I/V) and is found in
SMAC, the drosophila proteins Reaper, Grim, and Hid [41]. b The chemical structure of SMAC
AVPI [151]. c Structures of several small-molecule (IAP) antagonists: monovalent SMAC mimet-
ics GDC-0152 (Genentech) [200] and AT-406 (Aegera Therapeutics) [201], and the bivalent
SMAC mimetic TL32711 [4] and natural product Embelin [151]
This tetrapeptide motif is exposed following SMAC release from the mitochon-
dria after proteolytical removal of a 55-residue N terminal mitochondria-targeting
sequence. Crystallography data revealed that SMAC/DIABLO homodimerizes
through an extensive hydrophobic interface, which is essential for its high-affin-
ity binding to IAPs and its pro-apoptotic functions [81]. Several SMAC isoforms
have been reported to regulate IAPs through ubiquitination-dependent or caspase-
dependent mechanisms to potentiate apoptosis in vitro [3].
Additional pro-apoptotic IAP-binding proteins have been identified and are
capable of binding to several IAP members, including serine protease HtrA2
(high-temperature-requirement protein A2) also known as Omi [23, 84–86], and
XIAP-associated factor 1 (XAF1) [87, 88]. However, a SMAC AVPI-like element
is only found in HtrA2/Omi [86], and the role of these proteins as selective IAP
antagonists in the regulation of apoptosis is much less understood compared with
SMAC.
10.7 Phenotypes Associated with Gain-

and Loss-of-Functions in IAPs and Their Inhibitors
The functions of IAPs, including XIAP, cIAPs, and their inhibitors have been
examined extensively in human cells, mice, and other model organisms using both
loss-of-function and overexpression systems (reviewed in [7, 60, 89]). In general,
the findings support that IAP overexpression inhibits caspase activation and apop-
tosis mediated by both intrinsic and extrinsic apoptotic pathways. Overexpression
of SMAC, HtrA2/Omi, or XAF1 increases apoptosis in a variety of cancer cell
lines when combined with other anticancer agents, particularity with death recep-
tor ligands such as TRAIL (TNF-related apoptosis-inducing ligand).
Knockout of IAP in mice or human cancer cell lines results in a range of phe-
notypes associated with altered apoptosis or cell survival, and also supports func-
tion of IAPs in regulating NF-κB activation and cell proliferation [7]. IAPs appear
to regulate apoptosis with a great deal of redundancy. Total knockout of XIAP in
mice resulted in highly elevated apoptosis in response to brain injury, increased
NF-κB activation, and an overall reduced survival [90, 91]. XIAP knockout
HCT116 colon cancer cells are sensitized to TRAIL-induced apoptosis [92]. Mice
with total knockout of cIAP1 have no discernable phenotype due to redundant
pathway activation by cIAP2 [13]. Knockout of cIAP2 in mice leads to increased
macrophage death and resistance to LPS-induced sepsis [93]. Double knockout of
cIAP1/cIAP2 or XIAP/cIAP1 is embryonic lethal due to apoptotic defects, while
XIAP/cIAP2 double-knockout mice showed little change in apoptosis due to com-
pensation by cIAP1 [94]. Conditional cIAP1/2 double-knockout mice show uncon-
trolled B-cell proliferation independent of growth factor [95].
Endogenous IAP inhibitors also exhibit redundant functions in apoptosis regula-
tion [3, 7]. SMAC deficiency results in apoptosis resistance to a selective group of
agents, including TRAIL and non-steroidal anti-inflammatory drugs (NSAID) in
HCT116 colon cancer cells and mice, yet has little to no impact on apoptosis induced
by most DNA-damaging agents [96–99]. HtrA2/Omi knockout mice or cells, or
SMAC/HtrA2 double-knockout mice have minimal changes in apoptosis [100].
10.8 IAPs and Their Endogenous Inhibitors in Human

Cancer
Extensive studies have examined genetic and expression alterations in IAPs and
their inhibitors in cancer, and their correlation with clinical outcomes (reviewed
in [3, 4, 43, 101]). Genetic alterations in cIAP1/2 have been reported in multiple
cancers. Overexpression of IAP members, including XIAP, survivin, and cIAP1/2
and reduced expression of SMAC, HtrA2/Omi, or XFA1 are common in cancer
and suggested to correlate with chemoresistance, disease progression, and poor
prognosis. Although these results support targeting IAPs in cancer, they should be
interpreted with caution for several reasons. For example, some studies reported
nuclear but not cytosolic expression as having prognostic value. In certain cases,
either a lack of correlation or a reverse correlation is reported in a different cohort
of the same tumor type. Notably, the sample size was limited in several studies.
Lastly, genetic evidence supports that loss of cIAPs can contribute to tumor initia-
tion or progression in some cases.
10.8.1 IAPs and Cancer
While IAP overexpression can result from genetic changes such as gene ampli-
fication or chromosomal aberrations, the reason for their overexpression is not
known in most cases. To date, genetic alterations have been reported for cIAPs,
but not survivin, XIAP or ML-IAP. Amplification and translocation of cIAPs
have been found in both solid tumors and hematological malignancies. The
11q21–q23 amplification, encompassing both cIAP1 and cIAP2 loci, has been
reported in esophageal cancer [102], cervical cancer [103], liver cancer [104],
lung cancer [105], pancreatic cancer [106], medulloblastoma [107], and glioblas-
toma [108]. Interestingly, recurrent amplification of cIAP1 and cIAP2 is found
in Myc-driven liver cancer [104] as well as spontaneous osteosarcomas in mice
[109]. Additionally, the cIAP2–MALT1 fusion protein resulting from t(11;18)
(q21;q21) translocation is found in mucosa-associated lymphoid tissue lymphoma
[110–114].
In rare cases, cIAPs can function as tumor suppressors. For example, 20 %
of patients with multiple myeloma have activated non-canonical NF-κB sign-
aling associated with frequent genetic alterations, including biallelic deletions
of cIAP1, cIAP2, or TRAF2, TRAF3, CYLD, as well as enhanced expression of
CD40, lymphotoxin-β receptor (LTβR), TNFRSF13B, NFKB2, and NIK [5, 115].
Mutational changes leading to increased NIK and non-canonical activation of NF-
κB have also been reported in breast [116] and pancreatic cancers [117].
Elevated IAP expression has been reported to be a predictor of poor progno-
sis in many cancers. For example, elevated XIAP levels are correlated with dis-
ease progression, metastasis, and poor survival in colon cancer [118], liver cancer
[119], gastric cancer [120], breast cancer [121], and melanoma [122]. High lev-
els of cIAP1 are associated with poor overall survival and local recurrence-free
survival in cervical squamous cell carcinomas [103], and nodal metastasis in
squamous cell carcinoma of the tongue [123]. Furthermore, high levels of cIAP2
expression are correlated with reduced survival in colorectal cancer [124] and pan-
creatic ductal adenocarcinomas [125]. Increased ML-IAP expression in mRNA or
protein is found in melanoma and renal cancer [126–129] and is associated with
disease progression or poor prognosis in superficial bladder cancer [130], adult
ALL [131], osteosarcoma [132], and a subset of N-Myc-amplified neuroblastoma
[133]. Lastly, elevated survivin expression is prevalent in cancer and associated
with poor prognosis [101, 134–136].
10.8.2 SMAC and Other Endogenous IAP Inhibitors

in Cancer
No genetic alterations in SMAC, Omi/HtrA1, or XFA1 have been reported in human

cancer. A significant inverse correlation between SMAC expression with either
stage or grade has been reported in a variety of cancers, including colon [137],
esophageal [138], lung, renal cell, prostate, hepatocellular, and testicular germ
cancers [139]. Moreover, reduced expression of HtrA2/Omi and XFA1 in cancer
or cancer cell lines has been reported [7, 140]. While it is possible that reduced
SMAC or XFA1 contributes to IAP upregulation, direct evidence is still lacking.
Reports on SMAC expression and prognosis in cancer are more divergent.
Reduced or lack of SMAC expression was associated with poor prognosis, shorter
survival, disease progression, and metastasis in colon [137], lung [141], breast
[142], bladder [143], renal [144, 145], and endometrial cancers [146]. However,
high SMAC expression was found to be associated with a worse prognosis in
patients with cervical cancer [147] and acute myeloid leukemia [148].
10.9 IAP Antagonists as Anticancer Agents
Based on their widespread overexpression in human cancer and roles in promot-

ing cell survival and chemoresistance, several strategies have been devised to tar-
get IAP-addicted cancer cells. These include siRNA or antisense targeting XIAP
expression, small molecules targeting survivin expression, and peptidomimetics
and small-molecule SMAC mimetics targeting several IAPs [149, 150]. Among
these, small-molecule SMAC mimetics have garnered the most attention. As sin-
gle agents, SMAC mimetics exert little or no toxicity in non-malignant human
cells and can induce apoptosis in a limited number of cancer cell lines via TNFα
production. However, most cancer cell lines are resistant to treatment with SMAC
mimetics alone. On the other hand, the combination of enhanced expression of
SMAC or SMAC mimetics sensitize human cancer cells to apoptosis induced
by many classes of anticancer agents, including chemotherapeutics, radiation,
death receptor agonists and kinase inhibitors, via both XIAP and cIAP-dependent
mechanisms.
10.9.1 SMAC Mimetics
Detailed structural information on the binding between SMAC and IAPs prompted
the development of peptidomimetics and small-molecule SMAC mimetics, pro-
viding an excellent example of rational drug design [151]. SMAC AVPI-derived
peptides were first tested and found to effectively block IAP–caspase interactions
and sensitize a variety of cancers to pro-apoptotic stimuli [76], as well as synergize

with TRAIL in glioma xenografts [152]. However, SMAC peptidomimetics lacked
favorable pharmacological properties for further development, as they have poor
cell permeability, bind with low affinity to the XIAP BIR3 domain, and are sen-
sitive to proteolytic degradation [153]. These early studies provided both critical
rationale and tools for the subsequent development of small-molecule IAP antago-
nists [76, 152, 154, 155], which can overcome many of the mentioned limitations.
A number of small molecular SMAC mimetics have been developed using
structure-based designs and synthetic and medicinal chemistry (Fig. 10.3) [151].
Most SMAC mimetics are dimers, containing two SMAC APVI-like structures
connected by a chemical linker, resembling the higher-order structure of SMAC
in solution [82]. The first bivalent SMAC mimetic was developed in 2004 [153],
followed by development of many mono- and divalent SMAC mimetics [151].
Compared to the AVPI domain [156] or monovalent compounds, divalent SMAC
mimetics have higher affinity to IAPs and are much more potent in inducing cyto-
toxicity in cancer cells or mouse xenografts [65, 157–159]. The increased potency
of bivalent compounds may be attributable to better inhibition of XIAP via bind-
ing to BIR2 and BIR3 regions, allowing for prominent caspase-3 and caspase-7
activation [160, 161], and release of endogenous SMAC [162]. Furthermore, biva-
lent IAP mimetics effectively promote cIAP dimerization [44, 159] and facilitate
conformational changes and activation of cIAP1 E3 ligase activity [163–165],
leading to enhanced auto-ubiquitination of cIAPs and perhaps cross-ubiquitination
of other signaling molecules such as RIP1 [157, 158].
10.9.2 Mechanisms of SMAC Mimetic-Induced Cell Killing
Induction of apoptosis is the major mechanism of IAP antagonists-mediated

cell killing, and both XIAP- and cIAP-related mechanisms are involved [161,
166–170]. SMAC mimetics have been a particularly useful tool for probing cIAP
functions, especially due to the difficulties in simultaneous and acute ablation of
cIAP1 and cIAP2 in cells. The single agent sensitivity can be explained by produc-
tion of TNFα following rapid loss of cIAP1/2 and activation of the non-canon-
ical NF-κB pathway. SMAC mimetics can promote either apoptosis or necrosis
of cancer cells in response to TNFα. In the absence of cIAPs and a suppressed
canonical NF-κB pathway, TNFα/TNFR1 further stimulates the formation of a
cytoplasmic RIP1/FADD/caspase-8 (complex-II), and apoptosis [45, 65, 157–159]
(Fig. 10.2), or RIP1/RIP3-dependent necrosis when caspase activation is blocked
[46–48] (Fig. 10.2). SMAC mimetics do not appear to sensitize normal primary
cells to TNFα-induced killing [65, 159]. However, the underlying reasons why
only certain cells produce TNFα and the selectivity against tumors cells are not
well understood.
SMAC mimetics alone have limited toxicity to most cancer cell lines [65, 167].
The antitumor effects of SMAC mimetics have therefore been extensively examined
in combination with other cytotoxic agents, including chemotherapeutic agents, radia-

tion, death receptor ligands, kinase and proteasome inhibitors [4]. Synergistic or addi-
tive responses have been reported in numerous studies using in vitro and mouse
xenograft models (reviewed in [4]). Perhaps, the most significant synergy observed is
when SMAC mimetics are combined with death receptor ligands such as TRAIL [153,
167, 171–177], or with CD95 ligand or agonistic CD95-specific antibodies to trigger
apoptotic cell death [69, 178]. This synergy might be explained by enhanced caspase-8
activation [96] as well as reduced c-FLIP (cellular FLICE-like inhibitory protein)[167],
which can bypass the need for mitochondrial amplification to execute apoptosis in cer-
tain cells [167, 179]. The synergy with DNA-damaging agents is at least two-fold: the
activation of the mitochondrial pathway [16] amplifying the death receptor signal, and
DNA damage-induced cIAP depletion and subsequent caspase-8 action via TNFα-
dependent [71] and TNFα-independent mechanisms [71, 180] (Fig. 10.2). Suppression
of cancer cell invasion and metastasis might also contribute to the antitumor activities of
SMAC mimetics without increased pro-apoptotic death receptor signaling in response
to TRAIL [181].
10.9.3 IAP Antagonists in Clinical Development
Over 50 patents have been filed on IAP antagonists as potential anticancer agents,
and some of these agents have entered clinical development [4, 43]. Currently, a
large number of clinical trials are underway to assess their safety, pharmacologi-
cal properties and efficacy in patients with advanced solid tumor or hematological
malignancies (Table 10.2).
10.9.3.1 SMAC Mimetics in Clinical Development
Most of the IAP antagonists in clinical development are small-molecule SMAC

mimetics, including both mono- and bivalent compounds that are currently in
Phase I and Phase II trials (Table 10.2) [4, 43]. These include monovalent IAP
antagonists GDC-0152 (Genentech) and AT-406 (Ascenta Therapeutics), as
well as bivalent IAP antagonists LCL161 (Novartis Pharmaceuticals), TL32711
(TetraLogic Pharmaceuticals), and HGS1029 (Aegera Therapeutics/Human
Genome Sciences). The structures of several of them have been published (exam-
ples in Fig. 10.3c). Preliminary data indicate that both monovalent compounds
HGS1029 and bivalent compounds LCL161 and TL32711 are well tolerated
and show predicted biomarker modulation, such as cIAP1 downregulation and
increased levels of processed caspase-3 and caspase-7 in the serum. Manageable
side effects such as transient lymphopaenia and neutrophilia have been observed
with the use of bivalent IAP antagonists HGS1029 and TL32711 in some patients
[4]. Despite more potent cytotoxicity in cancer cells and mouse xenograft models,
divalent SMAC mimetics require intravenous administration due to their large size
and present a challenge for administering in combination with agents taken orally
Table 10.2 IAP antagonists in clinical development

Compound
Target Type Name developer Cancer Phase
cIAP1/2, XIAP Monovalent GDC-0152 Genentech Locally advanced I
and others SMAC or meta-
mimetic static solid
tumors, non-
Hodgkin’s
lymphoma,
breast cancer
[200]
Bivalent SMAC LCL161 Novartis Advanced solid I
mimetic pharmaceu- tumors [202]
ticals
Bivalent SMAC TL32711 TetraLogic Solid tumors, II
mimetic pharmaceu- lymphoma
ticals [203]
Monovalent AT-406 Ascenta Advanced solid I
SMAC therapeutics tumors, lym-
Mimetic phomas
Bivalent SMAC HGS1029 Human genome Advanced solid I
mimetic sciences tumors [204]
XIAP Antisense AEG35156 Aegera Advanced solid I/II
therapeutics tumors
[169], AML
[205],Lym-
phocytic,
chronic,
B-Cell,
advanced
hepatocellu-
lar, pancre-
atic [206],
mammary
carcinoma,
non-small cell
lung cancer
[4]. Therefore, additional studies are required to determine which classes of agents
have more desirable pharmacodynamic or pharmacokinetic properties for clinical
application.
10.9.3.2 XIAP Antagonists in Clinical Development
Two major strategies have been used to target XIAP by preventing it from binding
to caspase-3 [182] or downregulation with antisense. A number of small-molecule
XIAP antagonists have shown efficacy in vitro and are in preclinical development,
including dTWX-024 [183], TPI-1396-34 [184], XAC 1396-11 [185], and the natu-
ral product embelin [186]. The XIAP antisense oligonucleotides AEG35156 (Aegera
Therapeutics) displays potent antitumor activities in vitro as a single agent and syn-
ergizes with various chemotherapeutic compounds, such as the death receptor ligand,
TRAIL, and radiation to induce apoptosis in cell lines and mouse xenograft models
[187–190]. Results from Phase I and II clinical trials indicated that AEG35156 is
well tolerated and exhibits predictable pharmacokinetic properties, dose-dependent
changes in circulating biomarkers of cell death, as well as some antitumor activities
(Table 10.2) [191, 192], consistent with in vitro data on the suppression of XIAP
mRNA and protein levels [187–190]. Future studies will be needed to determine
whether modalities containing this agent improve efficacy over standard therapies.
10.10 Conclusions
During transformation, neoplastic cells become resistant to apoptosis due to

acquired genetic and epigenetic alterations, which can in turn drive additional
tumorigenic events and contribute to therapeutic resistance [1, 2, 17]. Mechanistic
dissection of apoptotic pathways has stimulated intensive efforts to restore apopto-
sis in cancer cells for disease control [14–16]. Cancer cells appear to be addicted
to overexpression of IAPs, which promotes carcinogenesis and drug resistance by
inhibiting caspases and stimulating cell survival and inflammation. The interac-
tion between IAP and SMAC guided the development of small-molecule SMAC
mimetics and furthered the biology of IAPs.
IAP signaling has several prominent features; it is highly dependent on protein–
protein, protein–ligand interactions such as BIR/IAP/and AVIP/SMAC, and ubiq-
uitination mediated by E3 ligase complexes [193]. IAPs regulate distinct signaling
complexes, including NF-κB, RIP1-associated death complex and Toll-like recep-
tor (TLR) signaling [194] via competitive utilization of shared signaling or adaptor
proteins, including cIAP1/2, FADD, TRADD, TRAFs, and RIP1 (Fig. 10.2). This
mode of regulation is in contrast to p53-mediated apoptosis following DNA dam-
age, which selectively activates the transcription of pro-apoptotic BH3 proteins,
and death receptors, and ligands [18, 19].
A better understanding of IAP biology will certainly help the development and
application of IAP antagonists in the clinic (Fig. 10.3), as a number of impor-
tant questions still remain. For example, what is the relative contribution of
TNFα/TNFR1-dependent and TNFα/TNFR1-independent mechanisms in SMAC
mimetics and IAP depletion-induced cell killing? TNFα dependence is certainly
worth exploration in inflammation-associated cancers, such as colon and liver can-
cer [52, 195, 196]. What influences the decision between distinct signaling com-
plexes and whether there is a more predominant role of cIAP1 or cIAP2 [197]?
What is the significance of necrosis induction in cancer treatment? Lastly, block-
ade in the mitochondrial apoptotic pathway is common in cancer due to frequent
alterations in p53 and the Bcl-2 family of proteins [1, 16, 17]. Therefore, rational
combination of IAP antagonists with BH3 mimetics [198, 199] might bring new
hopes to patients whose cancers are addicted to IAPs.
Acknowledgments We are grateful to Dr. Lin Zhang for critical reading and comments, and
Mrs. Laurice Vance-Carr for excellent secretarial assistance. The work in authors’ laboratory is
supported in part by NIH grants CA129829, UO1-DK085570, American Cancer Society grant
RGS-10-124-01-CCE, and Flight Attendant Medical Research Institute (FAMRI).
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Oncol
Chapter 11
Harnessing Death Receptor Signaling
for Cancer Treatment
Simone Fulda
Abstract Apoptosis, the cell’s intrinsic cell death program, is a key regulator of

tissue homeostasis. Accordingly, tilting the balance between cell death on one side
and cell proliferation on the other side toward survival promotes tumor forma-
tion. The death receptor (extrinsic) pathway represents one of the major apopto-
sis signaling cascades, which links exogenous stimuli via transmembrane surface
receptors to the intracellular signaling machinery that mediates and executes the
death signal. Since defects in death receptor signaling can confer resistance to
apoptosis, a better understanding of the regulation of the signaling events and their
perturbation in human cancers may lead to the identification of new molecular tar-
gets that can be exploited for therapeutic purposes. This strategy is expected to
open new perspectives to target the death receptor pathway for cancer therapy.
11.1 Introduction
Programmed cell death (apoptosis) represents an evolutionary highly conserved

intrinsic cell death program that is critically involved in the regulation of various
physiological and pathological processes [1]. For example, tissue homeostasis
is maintained by a delicate balance of cell growth on one side and cell death on
the other side [2]. Tipping this balance toward one or the other side results in too
little or too much apoptosis and can foster either tumor formation or tissue loss
[3]. In addition, the efficacy of most current cancer treatments including chemo-,
radio- or immunotherapy, largely depends on intact cell death programs in cancer
cells [4–6]. Therefore, defective apoptosis signaling pathways can lead to treat-
ment resistance, one of the major challenges nowadays in clinical oncology. The
identification of the molecular mechanisms that are responsible for cancer cell’s
evasion of apoptosis is expected to open new perspectives to specifically exploit
S. Fulda (*)
Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt,
Frankfurt, Germany
282 S. Fulda
cell death pathways for therapeutic purposes. The current review focuses on the
opportunities to target death receptor signaling in order to develop new therapeutic
strategies for the treatment for cancer.
11.2 Death Receptors
Death receptors belong to the superfamily of tumor necrosis factor (TNF) recep-
tors, which consist of more than 20 members with a wide spectrum of biologi-
cal functions including regulation of cell death, survival, differentiation, and
immune regulation [7]. All TNF receptor family members share a similar, cyto-
plasmic motif of about 80 amino acids, the so-called death domain, which is
critical for transmitting the death signal from the cell’s surface to intracellular
signaling pathways. In addition, death receptors harbor cysteine-rich extracellu-
lar domains for ligand binding. Among the death receptors CD95 (APO-1/Fas),
TNF receptor 1 (TNFR1) and TNF-related apoptosis-inducing ligand (TRAIL)
receptors have been extensively studied in the last two decades [7]. The corre-
sponding death receptor ligands of the TNF superfamily comprise among others
CD95 ligand, TNFα, lymphotoxin-α (the latter two bind to TNFR1), TRAIL and
TWEAK, a ligand for DR3 [7]. The CD95 receptor/CD95 ligand system repre-
sents a major signaling pathway that mediates apoptosis in several different cell
types, for example in the immune system [8]. TRAIL was identified in 1995 based
on its sequence homology to other members of the TNF superfamily and is consti-
tutively expressed in a wide range of tissues [7]. There are two agonistic TRAIL
receptors, that is, TRAIL-R1 and TRAIL-R2, that contain a death domain, and
therefore a signal to cell death upon ligand binding, whereas TRAIL-R3 to R-5
are antagonistic decoy receptors, which bind TRAIL, but are not able to transmit a
death signal [7].
11.3 Apoptosis Pathways
Two major apoptosis signaling pathways have been identified, that is the death
receptor (extrinsic) and the mitochondrial (intrinsic) pathway [4]. Stimulation
of death receptors such as CD95, TRAIL-R1 or TRAIL-R2 by CD95 ligand,
TRAIL or agonistic antibodies results in receptor oligomerization and recruit-
ment of FADD and caspase-8 to activated death receptors to form the death-
inducing signaling complex (DISC) [7]. This multimeric complex drives the
activation of caspase-8, which in turn transmits the apoptosis signal. To this end,
caspase-8 can directly activate effector pathways of apoptosis by cleaving cas-
pase-3. Alternatively, caspase-8 can initiate a crosstalk to the mitochondrial path-
way of apoptosis by processing Bid into its active form tBid [9]. Bid belongs to the
proapoptotic proteins of the Bcl-2 family that contains a BH3-only domain [10].
11 Harnessing Death Receptor Signaling for Cancer Treatment 283
tBid translocates from the cytosol to the mitochondria and triggers mitochondrial
outer membrane permeabilization. This results in the release of mitochondrial pro-
teins from the intermembrane space of the mitochondria into the cytosol, for exam-
ple of cytochrome c or second mitochondrial activator of caspases (Smac) [10].
Cytochrome c forms a complex in the cytosol together with Apaf-1 and caspase-9
to trigger caspase-9 and subsequently caspase-3 activation [10]. Smac antagonizes
Inhibitor of Apoptosis (IAP) proteins, which function as endogenous inhibitors of
caspases [11]. The release of caspases from the inhibition of IAP proteins by Smac
promotes caspase activation and apoptosis. The engagement of either the extrinsic
or the intrinsic apoptosis pathway results in activation of caspases, a family of pro-
teases that function as executioners in multiple modes of cell death [12].
Cell death pathways are tightly regulated by pro- and antiapoptotic factors.
This should ensure that they are rapidly activated upon stimulation, for example
upon death receptor ligation. Vice versa, this tight control should prevent their
accidental engagement, which could have detrimental effects on cellular survival.
Importantly, cancer cells have adopted many of these antiapoptotic mechanisms to
escape programmed cell death. Accordingly, evasion of apoptosis represents one of
the hallmarks of cancer cells. This also implies that targeting defective cell death
pathways bears the potential to tackle one of the key properties of m alignant cells.
11.4 Apoptosis and Cancer Biology
One of the hallmarks of human cancers is their ability to evade apoptosis in order
to survive environmental or oncogenic stress signals. This favors the progressive
growth of a tumor and, as such, cooperates with proliferative signals to foster
tumor development as well as its progression. On a theoretical ground, apoptosis
programs can be disrupted either via a reduction in the apoptosis promoting fac-
tors or via the dominance of processes that block apoptosis. Both genetic as well
as epigenetic events can cause inactivation of apoptosis pathways, implying that at
least a proportion of these events is in principle reversible and amenable for thera-
peutic interventions.
11.4.1 Aberrant Death Receptor Signaling in Cancers
11.4.1.1 Alterations in the CD95 Pathway
Signaling via the death receptor pathway of apoptosis may be disturbed at multiple
levels in human cancers. Along the signaling cascade, the surface levels of death
receptors have been described to be downregulated in human cancers. CD95 was
reduced in CD95-resistant tumor cells [13, 14] as well as in cells that were refrac-
tory to various anticancer drugs [13, 14], indicating that CD95 expression also
284 S. Fulda
regulates drug responsiveness. Genetic alterations of CD95 have also been impli-
cated in tumorigenesis. Hematological malignancies, as well as solid tumors, were
reported to harbor CD95 gene mutations [15]. In addition to genetic lesions, epige-
netic alterations including hypermethylation of the CD95 promoter have also been
implicated as an underlying cause for reduced CD95 expression in cancers [16, 17].
This mechanism may contribute to tumor immune escape, as cancer with epigeneti-
cally inactivated CD95 displayed low sensitivity to immune cell-mediated killing.
Consequently, restoration of CD95 expression by treatment with epigenetic drugs
such as histone deacetylase inhibitors concomitantly enhanced NK cell-dependent
tumor cell killing as well as the response to chemotherapy [18].
11.4.1.2 Alterations in the TRAIL Pathway
Along the same lines, resistance toward TRAIL has been linked to low or absent
surface expression of one of the two agonistic TRAIL receptors TRAIL-R1 and
TRAIL-R2. Interestingly, the chromosomal localization of these TRAIL receptors
on chromosome 8p falls within a region that is often genetically altered in human
cancers, for example by the loss of heterozygosity (LOH) [19]. Furthermore, the
loss of both copies of TRAIL-R1 or TRAIL-R2 due to deletions or mutations has
been detected in a small percentage of various cancers, for example several car-
cinomas (colorectal, breast, head and neck, lung), non-Hodgkin’s lymphoma and
osteosarcoma [20, 21]. In addition to these genetic events, aberrations in the sub-
cellular distribution of TRAIL receptors may account for the evasion of TRAIL-
induced apoptosis. In this respect, it has been reported in colon carcinoma cells
that the apoptosis-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2, are
retained in intracellular stores, for example the endoplasmic reticulum, and are not
properly transported to the cell surface, the location where they usually engage
with their ligand TRAIL to initiate proapoptotic signaling [22].
Another mechanism to block death receptor signaling resides in the rela-
tive abundance of decoy receptors. Both the CD95 and TRAIL cascade can be
impaired by such decoy receptors. Decoy receptor 3 (DcR3) has been shown to
competitively bind to CD95 ligand, thereby blocking CD95-induced apoptosis
[23, 24]. Of note, high expression levels of DcR3 or genetic amplification of
CcR3 were detected in lung or colon cancer and in glioblastoma [23, 24], indi-
cating that DcR3 may contribute to CD95 resistance in these cancers. TRAIL-R3
and TRAIL-R4 represent the decoy receptors that bind TRAIL, but are not able to
transmit a death signal, since they are devoid of a functional death domain. The
death domain is the intracellular region of the receptor that is required for the
recruitment of signaling proteins to activated death receptors and the subsequent
activation of initiator caspases. Interestingly, it was described that TRAIL-R3
and TRAIL-R4 inhibit TRAIL-R1 and TRAIL-R2-mediated apoptosis upon
treatment with the soluble ligand TRAIL via distinct mechanisms [25]. While
TRAIL-R3 inhibits the assembly of the DISC complex by sequestrating TRAIL
within lipid rafts, TRAIL-R4 is co-recruited together with TRAIL-R2 into the
DISC and interferes with the activation of initiator caspases [25]. Overexpression
of TRAIL-R3 was found in several cancers, for example in gastrointestinal can-
cers or leukemia [26–28]. In colorectal cancer, concomitant high TRAIL-R3 and
low/medium TRAIL-R1 expression was shown to correlate with a poor response
to 5-FU-based first-line chemotherapy and with shorter progression-free survival
[27]. AML blasts were recently reported to express TRAIL-R3 in a substantial
proportion of patients in addition to the proapoptotic TRAIL receptors [26]. Of
note, co-expression of this decoy receptor correlated with a significant shortened
overall survival. [26]. Knockdown of TRAIL-R3 resulted in TRAIL-induced
cell death confirming the decoy function of TRAIL-R3 on AML blasts [26].
Also, treatment with TRAIL-R2-specific antibodies resulted in higher cell death
rates [26]. This underlines that specific targeting of agonistic TRAIL receptors
is required in cancers that express TRAIL decoy receptors in order to exploit the
apoptosis-inducing activities of TRAIL [26].
Furthermore, DNA-damaging events such as anticancer drugs or ionizing
radiation were found to transcriptionally activate expression levels of TRAIL-R3
[28, 29]. This p53-stimulated transactivation occurred via a p53 consensus ele-
ment located within the first intron of the human TRAIL-R3 gene [29]. Similarly,
TRAIL-R4 was found to be induced by p53 upon ectopic expression of p53 [30].
This indicates that genotoxic drugs not only induce proapoptotic TRAIL recep-
tors [31], but may also stimulate antiapoptotic genes such as TRAIL-R3 and
TRAIL-R4.
TRAIL-R4 has recently been demonstrated to stimulate activation of signaling
pathways in an Akt-dependent manner in addition to its ability to inhibit TRAIL-
mediated signaling and cell death at the membrane by forming a heteromeric com-
plex with the agonistic receptor TRAIL-R2 [32]. Overexpression of TRAIL-R4
triggered morphological changes such as cell rounding, loss of adherence,
increased cell proliferation in vitro, and promoted tumor growth in vivo, indicating
that it contributes to carcinogenesis [32].
11.4.1.3 Overexpression of Death Domain-Containing Proteins
In addition to blocking death receptor signaling at the level of surface expression

of the receptors, the cascade can also be blocked by factors that interfere with the
formation of the DISC complex upon ligand binding by preventing the recruitment
of signaling molecules to activated death receptors. c-FLIP or phosphoprotein
enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15)
are two death domain-containing proteins that bind to the cytoplasmatic domains
of CD95 or TRAIL receptors and subsequently impair the recruitment of procas-
pase-8 or procaspase-10 to the DISC [33, 34]. High expression levels of c-FLIP
occur in a variety of cancers and have been shown to mediate resistance to death
receptor- and also to chemotherapy-induced apoptosis [35, 36]. For example, in
pancreatic cancer, c-FLIP was shown to be expressed in pancreatic intraepithe-
lial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas
286 S. Fulda
c-FLIP was not detected in normal pancreatic ducts [37]. Concomitant knockdown
of both c-FLIPL and c-FLIPS isoforms and individual silencing of either c-FLIPL
or c-FLIPS by RNA interference significantly increased TRAIL- and CD95-
induced apoptosis [37]. In addition, downregulation of c-FLIP by pretreatment
with chemotherapeutic drugs, for example 5-fluorouracil (5-FU), sensitized pan-
creatic carcinoma cells to death receptor-mediated apoptosis [37]. Of note, pri-
mary cultured pancreatic cancer cells were similarly primed for TRAIL-triggered
apoptosis by pre-exposure to anticancer drugs [37].
11.4.1.4 Silencing of Caspase-8
The notion that caspase-8, a key factor of death receptor-triggered apoptosis, may
restrict tumor development is underlined by a study, showing that a deficiency in
caspase-8 can facilitate cellular transformation [38]. Furthermore, expression of
caspase-8 can be downregulated by epigenetic inactivation. Caspase-8 expres-
sion was shown to be silenced by hypermethylation of a regulatory sequence
of the caspase-8 gene mapped to the boundary between exon 3 and intron 3 in
several malignancies, for example neuroectodermal tumors, sarcoma, and lung
carcinoma [39–43]. While this regulatory region of caspase-8 is not a classical
CpG island and is devoid of promoter activity, its methylation status was associ-
ated with caspase-8 expression levels in cell lines and primary tumor specimens
[11, 13, 14, 44–46]. Interestingly, co-methylation for caspase-8 and FLIP was
identified in neuroblastoma in one study, suggesting that caspase-8 is epigeneti-
cally silenced in a non-random fashion [47]. Hypermethylation of caspase-8 was
recently shown to be associated with relapse susceptibility in neuroblastoma [48].
In medulloblastoma, the loss of caspase-8 expression correlated with unfavorable
outcome in childhood medulloblastoma [42]. The loss of caspase-8 protein expres-
sion was identified in the majority of neuroblastoma tumor samples and was not
restricted to advanced disease stages [46]. No correlation was observed between
caspase-8 expression and MYCN amplification or other variables of high-risk dis-
ease (e.g., 1p36 aberrations, disease stage, age at diagnosis, or tumor histology)
[46]. Also, the loss of caspase-8 protein had no effect on event-free or overall sur-
vival in the overall study population or in distinct subgroups of patients [46], indi-
cating that inactivation of caspase-8 is not a characteristic feature of aggressive
neuroblastoma.
Moreover, caspase-8L is a dominant-negative variant of caspase-8 that is pro-
duced by alternative splicing, for example in CD34+ progenitor cells as well as in
leukemia and neuroblastoma [49–52]. Caspase-8L was found to block the bind-
ing of caspase-8 to FADD [49]. In addition, caspase-8L was recruited to the DISC
after CD95 stimulation instead of caspase-8, thereby interfering with CD95 signal-
ing at the receptor level by preventing caspase-8 activation [52].
Further, the tyrosine kinase Src was identified as a kinase that p hosphorylates
caspase-8 on tyrosine 308 in the linker loop of caspase-8, resulting in the sup-
pression of the proapoptotic activity of caspase-8 [53]. Phosphorylation of
caspase-8 occurred constitutively in cells with aberrant Src activity or alterna-

tively in response to receptor tyrosine kinase stimulation, following the growth
factor ligand binding [53]. In addition, integrin-mediated adhesion was described
to increase the phosphorylation of caspase-8 on tyrosine 380 [54]. Interestingly,
this phosphorylation step resulted in increased cell migration independent of
its protease activity [54]. The linker region of caspase-8 encompassing T380
was shown to function as a Src homology 2 binding site that is required for the
interaction of caspase-8 with Src homology 2 domains and the recruitment of
caspase-8 to lamella of migrating cells [54]. In addition, phosphorylation of cas-
pase-8 on T380 was shown to promote its interaction with the p85 alpha subunit
of phosphatidylinositol 3-kinase to regulate cell adhesion and motility [55]. Also,
caspase-8 was reported to be necessary for efficient adhesion-induced stimula-
tion of the extracellular signal-regulated kinase (Erk)-1/2 pathway independently
of its proteolytic activity [44]. This function of caspase-8 was demonstrated to
require specific residues within the caspase-8 “RXDLL motif” that mediates com-
plex formation with Src [44]. In addition, caspase-8 was found to interact with
the focal adhesion complex, thereby promoting cleavage of focal adhesion sub-
strates and subsequently cell migration [56]. Rab5 has been implicated as an
important integrator of caspase-8-mediated signal transduction downstream of
integrins during cell migration [57]. Together, these data demonstrate that pro-
caspase-8 exerts important functions in cell adhesion and motility independently
of its catalytic activity. Phosphorylation of caspase-8 that prevents the conversion
of procaspase-8 into its mature catalytically active cleavage fragments consti-
tutes an important switch between these non-apoptotic and apoptotic functions of
caspase-8.
11.5 Therapeutic Strategies to Target the Death Receptor

Pathway in Cancers
Since the death receptor pathway of apoptosis represents a signaling cascade that
directly connects to an intrinsic cell death machinery and that is amenable to ther-
apeutic targeting from the outside, it has attracted much attention in the last dec-
ade for the development of molecular cancer therapeutics. Most strategies focused
on targeting the two agonistic TRAIL receptors as discussed in more detail in the
following paragraphs.
11.5.1 TRAIL Receptor Agonists
Death receptors represent promising targets to directly engage the apoptotic

machinery. Most approaches for cancer therapy have so far focused to develop
agents directed against the proapoptotic TRAIL receptors. Tumor selectivity is
288 S. Fulda
one of the characteristics of TRAIL receptor agonists, as they have been shown
to predominantly trigger apoptosis in malignant cells with little effect on normal
cells [7].
There is now a large body of evidence from preclinical studies that recombinant
soluble TRAIL or antibodies against the proapoptotic TRAIL receptors TRAIL-R1
or TRAIL-R2 trigger apoptosis in a wide range of cancer cell lines and human
cancer xenograft models [19, 58, 59]. Of note, TRAIL-R2 antibodies were found
to trigger tumor-specific T-cell memory besides their cytotoxic effects against can-
cer cells, thereby protecting from tumor recurrence [60]. In addition to soluble
recombinant TRAIL ligand or TRAIL receptor specific antibodies, gene therapy
approaches have been launched to deliver TRAIL to the tumor site. An adenoviral
vector-based system based on the hTERT promoter yielded high levels of TRAIL,
leading to tumor-specific induction of apoptosis, suppression of tumor growth in a
xenograft model of breast cancer, and increased tumor-free survival of mice [61].
Furthermore, the apoptosis-inducing activity of TRAIL has been combined with
the ability of mesenchymal stem cells (MSCs) to infiltrate tumors as well as lym-
phatic tissues in order to deliver TRAIL to the primary tumor site as well as to
disseminated cancer cells [62]. In a lung cancer model, MSCs expressing TRAIL
were shown to cause tumor growth inhibition by triggering apoptosis [63].
11.5.2 TRAIL-Based Combination Therapies
Since a substantial proportion of tumors displays low sensitivity or even resistance

toward TRAIL, although at least one of the agonistic TRAIL receptors was found
to be expressed on the surface, a range of different TRAIL-based combination
therapies have been designed. They comprise chemo-, radio- or immunotherapy
as well as various targeted therapeutics. Both hypothesis-driven evaluations and
exploratory screening approaches have led to the identification of synergistic inter-
action between TRAIL receptor agonists and other cytotoxic principles in vari-
ous human cancers [64–71]. The cooperative interaction of TRAIL and anticancer
drugs has been attributed to various mechanistic events, for example transactiva-
tion and increased surface expression [72–74] or enhanced aggregation of proap-
optotic death receptors [75]. Recently, chemotherapeutic drugs have been reported
to restore the sensitivity to TRAIL-induced apoptosis in TRAIL-R4-expressing
cells by enhancing the recruitment of caspase-8 to the TRAIL DISC, thereby pro-
moting caspase-8 activation [76].
Inhibition of the proteasome presents another strategy to increase the sen-
sitivity of malignant cells toward TRAIL. For example, Bortezomib (PS-341,
VELCADE), a dipeptidyl boronic acid, is a reversible inhibitor of the proteolytic
activity of the proteasome that has been shown to synergistically induce apoptosis
together with TRAIL in a large variety of human cancers. This cooperative inter-
action has been attributed to a number of molecular events, including increased
TRAIL-R1 or TRAIL-R2 expression; enhanced formation of the TRAIL DISC,
reduced expression of c-FLIP or XIAP; reduced degradation of caspase-8, Bid,

Bax or Bim; upregulation of Bim, Bik, Puma, Bax or p53; enhanced release of
Smac from the mitochondria; accumulation of p21; or inhibition of NF-κB
[77–107].
Moreover, HDAC inhibitors have been shown in a set of different human malig-
nancies to prime cancer cells toward TRAIL-induced apoptosis [108]. This HDAC
inhibitor-mediated sensitization toward TRAIL may involve modulation of death
receptor (extrinsic) or mitochondrial (intrinsic) signal transduction pathways. For
example, HDAC inhibitors have been reported to cause upregulation of TRAIL-R1
or TRAIL-R2 surface expression [109–117], redistribution of TRAIL receptors
into lipid rafts [118], increased expression of caspase-8 [119], downregulation of
c-FLIP [120–124], enhancement of caspase-9 activation [125], downregulation of
antiapoptotic Bcl-2 family proteins [112, 126–130] accompanied by the upregula-
tion of proapoptotic Bcl-2 family members [116, 127, 130]. There is also growing
evidence in a variety of cancers that the inhibition of IAP proteins, for example by
small-molecule inhibitors, may present a particularly promising approach to sensi-
tize cancer cells to TRAIL-induced apoptosis [47, 62, 131–140].
11.5.3 Upregulation of Caspase-8
Furthermore, several therapeutic strategies have been developed to restore the

function of caspase-8 in cancer cells by upregulating its expression levels. Since
caspase-8 is frequently silenced by epigenetic mechanisms, several approaches
have been developed to reverse these events. For example, the demethylating agent
5-aza-2-deoxycytidine (5-AZA) caused demethylation of the regulatory sequence
of caspase-8 and enhanced caspase-8 promoter activity and caspase-8 expression
in several cancers with epigenetically inactivated caspase-8 [39, 141–143]. Since
the caspase-8 promoter was found to harbor several interferon-sensitive response
elements that can regulate caspase-8 gene expression, interferon-γ was used to
stimulate re-expression of caspase-8 [144–146]. Pretreatment with interferon-γ
resulted in enhanced TRAIL-, chemotherapy- or radiotherapy-induced apoptosis
[42, 145–158]. Interferon-γ-mediated upregulation of caspase-8 was mediated,
at least in part, via activation of signal transducer and activator of transcription
1 (STAT-1) [144, 146, 159]. This transcription factor transactivates interferon-γ-
inducible genes via interferon-γ activation sites (GAS) that also occur in the cas-
pase-8 promoter [144, 146, 159]. Overexpression of dominant-negative STAT-1
prevented interferon-γ-stimulated upregulation of caspase-8 [150], underscor-
ing that STAT-1 acts as a mediator of interferon-γ-stimulated re-expression of
caspase-8. Administration of interferon-γ also caused upregulation of caspase-8
expression in an in vivo model of Ewing’s sarcoma [156]. Of note, results from
a phase I clinical trial in neuroblastoma showed that treatment with interferon-γ
within an immunotherapeutic regimen resulted in upregulation of caspase-8 pro-
tein levels in tumor cells in vivo [160]. Combining interferon-γ together with
290 S. Fulda
demethylating agents (i.e., 5-Aza) proved to be even more effective compared

with single agents to stimulate re-expression of caspase-8, resulting in enhanced
apoptosis [45]. Further, interferon-γ was recently shown to cooperate with histone
deacetylase inhibitors to restore caspase-8 expression resistance in cancers with
silencing of caspase-8, thereby overcoming resistance to TRAIL [119]. Of note,
interferon-γ acted in concert with histone deacetylase inhibitors to re-express cas-
pase-8 expression and to enhance sensitivity to TRAIL-induced apoptosis also
in primary medulloblastoma samples and in a medulloblastoma model in vivo
[119]. In addition to interferon-γ, interferon-α has been shown to stimulate cas-
pase-8 expression [161]. Moreover, several cytotoxic drugs including methotrexate
and 5-fluorouracil were described to sensitize resistant tumor cells for apoptosis
by p53-mediated upregulation of caspase-8 [162]. Experiments using p53 decoy
oligonucleotides as well as p53 or caspase-8 RNA interference vectors underlined
the requirement of p53 and caspase-8 for this chemotherapy-mediated sensitiza-
tion toward TRAIL [162]. Upregulation of caspase-8 and sensitization to TRAIL-
induced apoptosis was observed in a panel of tumor cell lines with caspase-8
silencing as well as in TRAIL-resistant primary acute leukemia cells [162], under-
scoring both the general implications and the clinical relevance of these findings.
Retinoic acid represents another agent that has been shown to trigger caspase-8
expression in neuroblastoma cells [163]. This involves transcription via an intronic
region of the caspase-8 gene through a CREB binding site, leading to sustained
increase in caspase-8 levels for several days [163]. As a consequence, retinoic acid
in combination with doxorubicin or TNFα resulted in enhanced apoptosis [163].
Together, these reports demonstrate that demethylating agents, interferons, retinoic
acid, or anticancer drugs can be used to stimulate caspase-8 re-expression, result-
ing in the sensitization to apoptotic cell death.
11.5.4 Clinical Studies with TRAIL Receptor Agonists
TRAIL receptor agonists have been evaluated in a number of early clinical tri-
als over the last years. This includes protocols with recombinant human TRAIL
(e.g., dulanermin) [164–169] that is directed against both agonistic TRAIL recep-
tors TRAIL-R1 and TRAIL‐R2 as well as regimens with agonistic monoclonal
antibodies targeting selectively either TRAIL‐R1, for example mapatumumab
[170–178], or TRAIL‐R2, for example lexatumumab [179–182], conatumumab
[166, 169, 183–189], drozitumab [190–194], tigatuzumab [195], and LBY135
[196]. Initially, these trials were conducted with TRAIL or TRAIL receptor anti-
bodies as single agents. Subsequently, combination studies were launched to
exploit additive or even synergistic interactions by simultaneously targeting both
death receptor and additional signaling pathways. The design of these combina-
tion protocols was based on a large body of data from preclinical studies show-
ing that the incorporation of additional agents that concomitantly trigger, for
example, mitochondrial apoptosis such as chemotherapeutic drugs significantly
enhances the antitumor activity of TRAIL receptor agonists. In addition, targeted

therapies, for example kinase inhibitors or antiangiogenic drugs, have been com-
bined with TRAIL receptor agonists. Ongoing trials are testing TRAIL recep-
tor antibodies in different combination protocols, for example, together with the
proteasome inhibitor Bortezomib, the histone deacetylase inhibitor vorinostat or
interferon-γ (see clinicaltrials.gov).
One of the key challenges for the successful application of TRAIL recep-
tor agonists for the treatment for cancer is the identification of suitable biomark-
ers that can be used to rationally select patients for the inclusion in clinical trials
that most likely benefit from TRAIL-based protocols. In addition, biomarkers are
required to guide the treatment course with TRAIL receptor agonists, for example
to monitor treatment response and to adjust the dosing. While there has been a dis-
cussion whether or not expression levels of TRAIL receptors may serve as a suit-
able biomarker, several preclinical studies could not detect a correlation of TRAIL
receptor surface levels with TRAIL sensitivity. Similarly, immunohistochemi-
cal detection of TRAIL receptors in tumor tissue samples did not correlate with
outcome in clinical studies [170, 172, 173, 177, 178]. Thus, additional studies are
required to identify and validate suitable biomarkers for TRAIL-based regimens.
11.6 Conclusion
Targeting death receptor pathways represents a promising strategy to exploit

endogenous cell death programs for the treatment for cancer. This approach has
been extensively studied in preclinical cancer models and has more recently also
been transferred to the phase of clinical evaluation. Harnessing the possibilities to
engage the cell’s intrinsic death program will hopefully pave the avenue for inno-
vative treatment options for patients suffering from cancer.
Acknowledgments Work in the author’s laboratory is supported by grants from the Deutsche
Forschungsgemeinschaft, the Deutsche Krebshilfe, the Bundesministerium für Forschung und
Technologie (01GM0871, 01GM1104C), Wilhelm-Sander-Stiftung, Else Kröner- Fresenius-
Stiftung, Novartis Stiftung für therapeutische Forschung, the European Community (ApopTrain,
APO-SYS), and IAP6/18.
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J Clin Oncol 26:3538 (Meeting Abstracts)
Chapter 12
Proteasome Inhibition as a Novel Strategy
for Cancer Treatment
Min Shen and Q. Ping Dou
Abstract The proteasome is a multi-subunit protease complex, responsible for

the degradation of misfolded, damaged, or short-lived proteins. Indeed, more than
90 % of intracellular proteins are degraded through the ubiquitin–proteasome sys-
tem (UPS). The UPS is extensively involved in various cellular events, including
cell cycle, cell signaling, stress response, and apoptosis. Inhibition of proteasome
function could result in growth arrest and/or cell death. The involved molecular
mechanisms include dysregulation of the cell cycle, inactivation of NF-κB pathway,
disturbance of the pro- and anti-apoptotic balance, induction of unfolded protein
response, and induction of oxidative stress. The observation that suppression of pro-
teasome function by small chemical inhibitors was able to induce apoptosis in cancer
cells but not in normal cells supports the hypothesis that the proteasome could be
a valuable target for cancer treatment. This idea has been validated from benchtop
to bedside. In 2003, the first proteasome inhibitor anticancer drug, bortezomib, was
approved in the United States for the treatment of multiple myeloma and mantle cell
lymphoma. While bortezomib achieved great success in clinical applications, prob-
lems such as resistance, dose-limiting toxicities, unsatisfied efficacy in solid tumors,
and interaction with some natural compounds have been observed. Therefore, it is
necessary to further investigate the proteasome inhibition-mediated mechanism of
cell death as well as the development of novel, new-generation proteasome inhibitors
with lower toxicity and wider applications.
12.1 Introduction
Cellular protein homeostasis is precisely regulated by a series of cellular processes

including protein synthesis, folding, trafficking, and degradation. Protein
degradation by the UPS was initially underestimated as a “garbage disposal” sys-
tem, but its role has since been appreciated, as it plays an important regulatory
M. Shen · Q. P. Dou (*)

Karmanos Cancer Institute and Department of Pharmacology,
Wayne State University School of Medicine, Detroit, MI, USA
304 M. Shen and Q. P. Dou
role in various cellular events, such as protein quality control, cell cycle progres-
sion, cell differentiation, embryonic development, apoptosis, signal transduction,
gene expression, circadian clocks, as well as immune and inflammatory responses
[1, 2]. In all eukaryotic cells, protein degradation is executed by two major path-
ways, namely the lysosomal pathway, which mainly degrades extracellular and
transmembrane proteins, and the ubiquitin–proteasome pathway (UPP), which
mainly degrades intracellular proteins [2]. Dysregulation of proteasome function
is implicated in several pathological processes, including neoplastic disease and
autoimmune disease which exhibit increased proteasome function. Furthermore,
neurodegenerative disease has been found to be associated with decreased protea-
some function. These properties grant the proteasome potential to be a therapeu-
tic target. Indeed, the first proteasome inhibitor bortezomib was launched in the
United States in 2003 and subsequently in Europe in 2004 for the treatment of
multiple myeloma (MM) and mantle cell lymphoma (MCL) [3].
12.2 Ubiquitin–Proteasome System
The ubiquitin–proteasome system (UPS) was discovered in the late 1970s and
early 1980s. The importance of this discovery was acknowledged through the
award of the 2004 Nobel Prize in Chemistry to its discoverers, Aaron Ciechanover,
Avram Hershko, and Irwin Rose.
12.2.1 Ubiquitin–Proteasome Pathway
Protein degradation by the ubiquitin–proteasome pathway (UPP) usually includes

two steps, ubiquitin conjugation and proteasomal degradation. Ubiquitin (8.5 kDa)
is a highly conserved small regulatory protein. Only proteins conjugated with a
Lys48-linked poly-ubiquitin chain can be recognized and processed by the protea-
some for degradation. Ubiquitin conjugation occurs through an enzymatic cascade
that involves three distinct enzymes, Ub-activating (E1), Ub-conjugating (E2),
and Ub-ligating (E3) enzymes. The poly-ubiquitinated proteins with four or more
Lys48-linked ubiquitin are then directed to the 26S proteasome complex where the
poly-ubiquitin chain will be removed and recycled, while the protein substrates
will be progressively degraded into oligopeptides that are 3–25 amino acids long
[4, 5] (Fig. 12.1).
12.2.2 Proteasome
The 26S proteasome (2.4 MDa) is a multi-subunit protease complex composed

of the 20S catalytic core (700 kDa) and two 19S regulatory particles (700 kDa).
12 Proteasome Inhibition as a Novel Strategy for Cancer Treatment 305
AMP+Pi
Ub
Ub
Ub
E1
ATP Repeated Ub
Ub Ub
E2
ubiquitination
Ub Target
Ub
E1 E3
Target
E2
E3
Target ATP
lid base
AMP+Pi
T
A
G
T Ub
R Ub
E Ub
Ub
19S 20S 19S Recycled
26S
Fig. 12.1 A schematic diagram of the UPP and the structure of the 26S proteasome
The 20S core is formed by two identical α rings and two identical β rings stacked
in a symmetric manner with the outside α rings surrounding the inner β rings
(Fig. 12.1). Each α or β ring contains seven different subunits, named α1–α7
or β1–β7, among which only β1, β2, and β5 possess proteolytic activity. The
β1 subunit possesses caspase-like or peptidyl-glutamyl peptide-hydrolyzing-
like (PGPH-like) activity which preferentially cleaves after acidic residues such
as aspartate and glutamate; the β2 subunit possesses trypsin-like activity which
preferentially cleaves after basic residues such as arginine and lysine; and the
β5 subunit possesses chymotrypsin-like activity which preferentially cleaves
after hydrophobic residues such as tyrosine and phenylalanine [3, 6]. The pro-
teasome also exhibits two other enzymatic activities, the “branched chain amino
acid–preferring” (BrAAP) activity and the “small neutral amino acid–prefer-
ring” (SNAAP) activity [7]. The 19S regulatory particle binds to both ends of the
20S core proteasome and is composed of a “lid” and a “base” (Fig. 12.1). The
“lid” contains at least nine non-ATPase subunits, which remove the poly-ubiq-
uitin chain from the substrate by a process called deubiquitylation. The “base”
contains six ATPase and four non-ATPase subunits and opens the gate of the 20S
proteasome, unfolds the substrate proteins, and promotes their entry into the 20S
proteasome [3].
In addition to the 19S regulatory particle, the 20S core can alternatively interact
with other regulatory particles at one or both ends, resulting in the formation of
asymmetric or symmetric isoforms of the proteasome. These regulatory particles
include PA28 (also referred to as the 11S regulatory particle) and PA200 [5, 8].
12.2.3 Immunoproteasome
In addition to the constitutive proteasome described thus far, which contains cat-
alytic subunits β1, β2, and β5, there also exists the immunoproteasome, which
harbors different sets of catalytic subunits designated as β1i, β2i, and β5i, corre-
spondingly. Upon cytokine stimulation, especially interferon-γ and tumor necrosis
factor-α, the expression of β1i, β2i, and β5i dramatically increases and coopera-
tively assembles into nascent 20S core particles to form immunoproteasomes.
Interferon-γ stimulation also induces the expression of regulatory subunits PA28α
and PA28β, both of which are non-ATPase subunits. Three PA28α and four PA28β
comprise the heteroheptameric PA28αβ regulatory particle, which facilitates the
assembly of the immunoproteasome and enhances its activity. The immunopro-
teasome is a key component of antigen processing. Compared with the constitu-
tive proteasome, the immunoproteasome possesses enhanced chymotrypsin- and
trypsin-like activities and reduced caspase-like activity. This specialized enzymatic
property endows the immunoproteasome the ability to generate peptides that are
suitable for MHC class I-mediated antigen presentation [9, 10].
12.3 Proteasome Inhibitors
12.3.1 Synthetic Proteasome Inhibitors
Based on their chemical structures, the known synthetic proteasome inhibitors

fall into three classes: peptide aldehydes, peptide boronates, and peptide vinyl
sulfones.
Peptide aldehydes are widely used proteasome inhibitors. The representatives
of this class, MG132 (A-LLL-al, Fig. 12.2a) and PSI, were among the first protea-
some inhibitors developed. This class of compounds inhibits the proteasome by
forming a covalent hemiacetal bond with the hydroxyl group of the N-terminal
threonine (Thr1) in the catalytic β subunits. This inhibition is reversible, and the
dissociation rate is relatively fast. MG132 is an analog of Calpain Inhibitor I,
which shows a 10-fold higher preference toward the proteasome over calpains and
cathepsins. Although it is a well-known research tool in protesome studies in vitro
and in cellulo, the fact that MG132 is rapidly oxidized into inactive carbonic acid
in vivo largely prevents its development as a therapeutic drug [3, 11, 12].
A. Aldehydes C. Vinyl sulfones

I
O O
H HO
N O O O
O N N H
H H N
O O 2N N N S
H H O O
O
MG132 NLVS
B. Boronates
O O
OH Cl O O OH
O O OH H
H OH H N B
N N B N N B N O
N OH N OH H
H O H O
N O HO O
Cl
Bortezomib CEP-18770 MLN9708
D. -lactones
O O
Cl NH NH
O OH O OH
O O
Marizomib Omuralide
E. Epoxyketones
O O
H H O
N N O O O
N N N H
H H N O
O O O O N N
N H H
S O O
O
Carfilzomib ONX-0912
F. Cyclic peptides
O S
H
HO NH O N O
HO N
NH
O N
O
N O O NH
H O O
HO O NH NH2 H O
O HN N
N
H
O O HN
N
H
O
TMC-95A Argyrin A
Fig. 12.2 Representatives of the major classes of proteasome inhibitors. Electrophilic functional

groups that directly interact with the proteasome are depicted in red. a Aldehydes, b Boronates,
c Vinyl Sulfones, d β-lactones, e Epoxy Ketones, f Cyclic Peptides
Peptide boronates are aldehyde surrogates with improved potency and specific-
ity for the proteasome. For example, MG262 (Z-LLL-Boronate), the boronate surro-
gate of MG132, was shown to be 100-fold more potent than MG132. Besides MG262,
Table 12.1 A summary of proteasome inhibitors in clinical use or clinical trials [11, 135]
Binding Route of
Chemical mode and Target selec- administra- Development
Inhibitor Developer nature kinetics tivity tion stage
Bortezomib Millennium Boronate Covalent, β5 > β1 IV/SC FDA
(PS341) slowly approved
reversible
Carfilzomib ONXY Epoxyketone Covalent, β5, β5i IV Phase III
(PR-171) irrevers-
ible
Marizomib Nereus β-lactone Covalent, β5 > β2 > β1 IV Phase I
(NPI- irrevers-
0052) ible
MLN-9708 Millennim Boronate Covalent, β5 > β1 IV/oral Phase I-II
rapidly
reversible
Delanzomib Cephalon Boronate Covalent, β5 > β1 IV/oral Phase I-II
(CEP- slowly
18770) reversible
Oprozomib ONXY Epoxyketone Covalent, β5, β5i Oral Phase I-II
(ONX- irrevers-
0912) ible
IV intravenous; SC subcutaneous; FDA Food and Drug Administration
bortezomib (PS341), delanzomib (CEP-18770), and MLN9708 are the most success-
ful representatives in this class (Fig. 12.2b). Peptide boronates form tetrahedral adducts
with Thr1 in catalytic β subunits, which are further stabilized by two extra hydrogen
bonds. Therefore, although the inhibition is reversible, the dissociation rate is very slow.
Furthermore, unlike peptide aldehydes, peptide boronates have increased bioavailability
and are metabolically stable under physiological conditions. These characteristics make
peptide boronates suitable for therapeutic development. Indeed, bortezomib (Velcade®)
was the first proteasome inhibitor launched in 2003 (Table 12.1). Both delanzomib and
MLN9708 are currently under clinical trials. MLN9708 is sophisticatedly designed as a
prodrug in the form of a boronic ester so that it is orally bioavailable [11, 12].
Peptide vinyl sulfones are irreversible proteasome inhibitors. They were first
described as inhibitors of cysteine proteases. Not surprisingly, they exhibit less
potency and specificity toward the proteasome. Nevertheless, this feature of irre-
versible inhibition offers them certain advantages of being activity-based pro-
teasome probes in basic research. The representatives of this class are ZLVS
(Z-LLL-vs), NLVS (NIP-LLL-vs) (Fig. 12.2c), and AdaAhx3-LLL-vs [3, 11].
12.3.2 Natural Proteasome Inhibitors
A large portion of proteasome inhibitors were initially derived from natural prod-
ucts, and the number of such is still growing. This large family can be further
divided into four groups based on their mechanisms of action: covalent inhibi-
tors, non-covalent inhibitors, non-proteasome-specific inhibitors, and allosteric
inhibitors.
12.3.2.1 Covalent Inhibitors
β-lactones are non-peptide natural products showing appreciable proteasome-

inhibitory activity. The representatives of this class are lactacystin, marizomib
(salinosporamide A, NPI-0052) (Fig. 12.2d), and belactosins. The carbonyl group
of the β-lactones can interact with the hydroxyl group of Thr1 in catalytic β subu-
nits, leading to the formation of an acyl-ester adduct. Although this is a reversible
process, the dissociation rate of the covalent adduct is extremely slow. Lactacystin,
is a metabolite produced by the bacteria Streptomyces, it was the first identified
natural proteasome inhibitor. Lactacystin itself is an inactive precursor, which
will be spontaneously hydrolyzed into clasto-lactacystin-β-lactone (omuralide)
(Fig. 12.2d), the active form, in aqueous solutions at pH 8. Marizomib, a second-
ary metabolite produced by the bacteria Salinispora tropica, is a very promising
compound, which is currently under investigation into a phase I clinical trial. In
addition to the acyl-ester bond, marizomib also forms a cyclic tetrahydrofuran ring
with the proteasome, making the reaction irreversible [11, 13].
Peptide epoxyketones are the most specific and potent proteasome inhibitors
thus far. The representatives of this class are epoxomicin, carfilzomib (PR-171),
and oprozomib (ONX-0912 or PR-047) (Fig. 12.2e). The α,β-epoxyketone moi-
ety interacts with both the hydroxyl group and the free α–amino group of Thr1
in the catalytic β subunits, leading to the formation of the morpholino adduct in
an irreversible way. Since the free α–amino group required for adduct formation
is not present in serine and cysteine proteases, these proteases are not affected by
epoxyketones. Epoxomicin was originally isolated from an unidentified strain of
Actinobacteria. Since its identification as a proteasome inhibitor, little, if any off-
target effects have been reported. Carfilzomib and oprozomib are derivatives of
epoxomicin, both currently under clinical development. As irreversible proteasome
inhibitors, peptide epoxyketones are, like peptide vinyl sulfones, also widely used
as activity-based proteasome probes in basic research [11, 13].
12.3.2.2 Non-covalent Inhibitors
Some natural cyclic peptides have been found to inhibit proteasome func-
tion in a non-covalent manner. The representatives of this class are TMC-95A
(Fig. 12.2f), argyrin A (Fig. 12.2f), and more recently scytonemides [14].
The TMC-95 family is a group of cyclic tripeptides produced by the fungus
Apiospora montagnei. They interact with the active sites of catalytic β subunits
through five hydrogen bonds, thereby reversibly inhibiting proteasome func-
tion [15]. TMC-95A is able to selectively and competitively inhibit all three
catalytic β subunits in the low nanomolar range [15]. The shortcoming for the
development of these large inhibitors is that their de novo synthesis is very dif-
ficult and expensive. Argyrin A is a cyclic octapeptide produced by the bacteria
Archangium gephyra. It also reversibly inhibits proteasomal activity in the low
nanomolar range, making itself a promising candidate [16]. However, given its
large size, the exact mechanism by which argyrin A interacts with the protea-
some remains unclear.
12.3.2.3 Non-specific Proteasome Inhibitors
Besides the natural proteasome inhibitors discussed above, most of which are
metabolites of bacteria or fungus, a rapidly growing group of natural proteasome
inhibitors has also been isolated from the plant kingdom. These natural products
include some flavonoids, triterpenoids, and others such as curcumin [17] and shi-
konin [18]. Unlike the proteasome inhibitors discussed hitherto that mainly target
the proteasome, plant-derived natural products usually have multiple cellular tar-
gets including the proteasome.
Flavonoids are a group of natural products with a common structure of two
aromatic rings linked by three carbons (C6–C3–C6) [19]. They are abundant in
various vegetables and fruits. Flavonoids that have been found to possess pro-
teasome-inhibitory activity include (-)-epigallocatechin-3-gallate [(-)-EGCG]
(Fig. 12.3a), apigenin, quercetin, and genistein. Among them, (-)-EGCG, the
major catechin in green tea, is most potent in terms of proteasome inhibition.
It inhibits the proteasomal β5 subunit irreversibly through covalent binding.
Notably, (-)-EGCG is able to interact with not only the β5 subunit of the constitu-
tive proteasome but also the β5i subunit of the immunoproteasome with an even
greater affinity [20]. However, (-)-EGCG has been found to have the potential
to decrease the efficacy of bortezomib and other boronate proteasome inhibitors.
This neutralizing effect is mainly due to the direct interaction between the pyro-
catechol moieties on (-)-EGCG and the boronic acid moiety on bortezomib [21].
Whether patients receiving bortezomib should consume green tea needs a clarifi-
cation by further clinical studies.
Triterpenoids are a group of compounds comprised of six isoprene units with
30 carbons. They occur naturally as complex cyclic structures, with the pentacy-
clic structure being most common. Some have been reported to have proteasome-
inhibitory activity, including celastrol [22] (Fig. 12.3b), withaferin A [23], and
pristimerin [24]. These compounds are present in many herbal medicines.
12.3.2.4 Allosteric Inhibitors
Allosteric inhibitors refer to inhibitors that bind to the allosteric site (a site other than
the enzyme’s active site) of an enzyme and cause its inactivation. The advantage of
A. Flavonoids B. Triterpenoids
OH O
OH OH
HO O
OH
O O
OH OH
O
HO
OH
(-)-EGCG OH
Celastrol
C. Hydroxyquinolines D. Dithiocarbamates
NH2
S
S N
N S
N
S
OH
5AHQ Disulfiram
Immunoproteasome - Specific Inhibitors

E. Aldehydes F. Epoxyketones
O O O O
H H
N N N O
O N N N
H H H
O O O
O
O
IPSI-001 ONX-0914
Fig. 12.3 Chemical structures of some natural compounds with proteasome-inhibitory activity

and immunoproteasome-specific inhibitors. Electrophilic functional groups that directly interact
with the immunoproteasome are depicted in red. a Flavonoids, b Triterpenoids, c Hydroxyquino-
lines, d Dithiocarbamates, e Aldehydes, f Epoxyketones
allosteric inhibitors lies in their ability to overcome some particular types of resist-
ance, for example, resistance caused by mutations at the active site. A number of
different metal-binding organic compounds have been shown to be allosteric protea-
some inhibitors in vitro and in cellulo. However, like proteasome inhibitors derived
from plants, most of the organometallic compounds have multiple cellular targets
with the proteasome being one of them.
Two biggest groups of metal-binding compounds that have proteasome-inhibi-
tory activity are hydroxyquinolines [such as 5-amino-8-hydroxyquinoline (5AHQ)
(Fig. 12.3c) and clioquinol] and dithiocarbamates [such as disulfiram (Fig. 12.3d),
diethyldithiocarbamate, and pyrrolidine dithiocarbamate] [25–27]. NMR data indi-
cated that 5AHQ might bind to proteasomal α subunits [27]. Copper is the metal
most commonly used to form complex with these ligands. Other metals that have
been studied include zinc, gold, and gallium.
12.3.3 Immunoproteasome-Specific Inhibitors
In recent years, the immunoproteasome has been attracting increasing attention,

as are immunoproteasome-specific inhibitors. To date, the immunoproteasome-
specific inhibitors IPSI-001 (Fig. 12.3e), ONX-0914 (PR-957) (Fig. 12.3f), and
PR-924 are the most advanced developed.
IPSI-001 belongs to the peptide aldehydes class of compounds. It selectively
inhibits β5i and β1i subunits of the immunoproteasome. It has a 100-fold prefer-
ence for the immunoproteasome (Kis of 1.03 and 1.45 μM against β5i and β1i,
respectively) over the constitutive proteasome (Kis of 105 and 239 μM against β5
and β1, respectively) in vitro. Moreover, IPSI-001 potently inhibits proliferation
and induces apoptosis in MM cell lines as well as in patient samples. More impor-
tantly, it was able to overcome conventional and novel drug resistance, including
resistance to bortezomib [28].
ONX-0914 and PR-924 belong to the class of epoxyketones. Like other epox-
yketones, the inhibition caused by ONX-0914 and PR-924 is irreversible. Both of
the inhibitors selectively inhibit the β5i subunit. ONX-0914 is 20- to 40-fold more
selective toward the β5i subunit than both the β5 and β1i subunits [29].
12.4 Mechanisms of Proteasome Inhibition-Induced

Growth Arrest and/or Cell Death
Proteasome function can be suppressed or inhibited by either endogenous protein

inhibitors or exogenous chemical inhibitors. Inhibition of proteasomal activity will
result in the accumulation of substrate proteins and disruption of normal cellular
function.
12.4.1 Proteasome Inhibition and Cell Cycle Arrest: p27 and p53
Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs),

cyclin-dependent kinase inhibitors (CDKIs), and other proteins involved in cell
cycle checkpoints. The UPS is critical in the regulation of the cell cycle. Basically,
the cell cycle is driven by CDKs whose activity is determined by the level of cyc-
lins present in the cell. Oscillations in the level of cyclins are achieved by periodic
protein synthesis and proteasome-dependent degradation. Inhibition of proteasome
function can result in the accumulation of two cyclins at the same time, which
results in two contradictory signals. These conflicting signals are only resolved
through cellular self-destruction. Proteasome inhibition stabilizes two important
negative regulators of cell cycle, p27KIP1 and p53, both of which are known protea-
some substrates. P27KIP1 is an inhibitor of CDK2 and CDK4, and during cell cycle
progression, it is degraded by the proteasome to release CDK2 and CDK4 for G1/S
phase transition, whereas accumulation of p27KIP1 by proteasome inhibition results
in G1 phase arrest [30]. Tambyrajah et al. [31] reported that p27KIP1 is degraded
by the proteasomal β1-mediated activity, which is upregulated as cells enter the
cell cycle without concomitant changes in the protein levels of β1, β2, or β5 subu-
nits. Furthermore, one of the novel proteasome inhibitors discussed above, argyrin
A, was actually discovered in a screen for compounds that prevent degradation of
p27KIP1 [16]. P53 is a bona fide tumor suppressor. As a transcription factor, accu-
mulation of p53 by proteasome inhibition could lead to increased expression of its
target genes such as p21WAF1, another inhibitor of CDK2 [32, 33], and PUMA,
a proapoptotic Bcl-2 family member [34]. However, p53 is not indispensable in
proteasome inhibitor-induced apoptosis, at least in certain types of cancer cells
[35–37].
12.4.2 Proteasome Inhibition and Suppression of the Cell

Survival NF-κB Pathway
The transcription factor NF-κB is a critical player against apoptosis in cells and
proteasome function is essential to two proteolytic processes required for the acti-
vation of NF-κB. First, the NF-κB dimer is trapped in the cytoplasm by inhibitor of
NF-κB (IκB) protein. Degradation of IκB by the proteasome is required to release
the NF-κB complex for its nuclear translocation and activation. Inhibition of pro-
teasome function stabilizes IκB and thereby prevents NF-κB activation [38, 39].
Secondly, p105 and p100, two precursors of NF-κB subunits p50 and p52, respec-
tively, need to be processed by the proteasome; otherwise they function like IκB
and confine their dimeric partners to the cytoplasm [39–41]. In addition, it is well
established that conventional cytotoxic drugs as well as inflammatory factors such
as TNFα and IL-1β often activate NF-κB, which is at least partially responsible
for the development of drug resistance. Moreover, constitutive activation of NF-
κB was observed in a large fraction of advanced cancers that are very resistant to
most therapies. Pioneering work done by Delic et al. [42] demonstrated that a low
dose of proteasome inhibitor lactacystin was sufficient to prevent NF-κB activation
and sensitize chemo- and radio-resistant cancer cells to TNFα-induced apoptosis.
Synergistic apoptosis-inducing effects were also observed when combining a pro-
teasome inhibitor with TNF-related apoptosis-inducing ligand (TRAIL) [43, 44],
paclitaxel [45], or etoposide [46]. Therefore, combining a proteasome inhibitor
with other chemotherapeutic agents may represent an effective way to overcome
NF-κB-mediated drug resistance through resensitizing cancer cells to apopto-
sis [47]. However, a study by Hideshima et al. [48] pointed out that under some
experimental conditions, bortezomib actually induced canonical NF-κB activa-
tion in MM cells through phosphorylation of IκB kinase (IKKβ) and its upstream
receptor-interacting protein 2. More intriguingly, Amschler et al. [49] reported that
NF-κB inhibition by either proteasome inhibition or IKKβ blockade sensitized
melanoma cells to camptothecin through distinct pathways. Therefore, more

mechanistic studies are needed to unveil the regulation of NF-κB pathway by the
proteasome.
12.4.3 Proteasome Inhibition and Mitochondrial Apoptotic

Pathway: The Bcl-2 Family Members
The Bcl-2 family plays a pivotal role in the mitochondrial apoptotic pathway (the
intrinsic pathway). It is comprised of both pro-apoptotic (e.g., Bax, Bak, Bid,
Bim, Bik, NOXA, and PUMA) and anti-apoptotic (e.g., Bcl-2, Bcl-XL, A1, and
Mcl-1) members, the ratio of which is tightly regulated by different factors. The
proteasomal activity influences the level of Bcl-2 family members both directly
and indirectly. Inhibition of proteasome function by different types of inhibitors
was reported to directly induce the accumulation of its substrates Bax [50, 51],
Bik [52], and Bim [53–55]. However, in other studies, conformational change
and mitochondrial translocation, instead of accumulation, of Bax was observed
under proteasome-inhibitory conditions [56, 57]. Proteasome inhibition was
also reported to indirectly induce the transcriptional upregulation of NOXA,
PUMA, and Mcl-1. Although both NOXA and PUMA are target genes of p53,
only upregulation of PUMA, but not NOXA, upon proteasome inhibition was
found to be p53 dependent [34, 36, 51, 58]. A study by Nikiforov et al. [59] sug-
gested that the oncogene c-Myc, another transcription factor, was responsible for
tumor cell-selective upregulation of NOXA in response to proteasome inhibition.
Dysregulation of c-Myc was listed among the most pivotal events by a genome-
wide siRNA screen looking for modulators of cell death induced by bortezomib
[60]. Intriguingly, the anti-apoptotic protein Mcl-1 was also upregulated upon
proteasome inhibition and was found to be mediated by activating transcription
factor-4 (ATF4), an important effector of the unfolded protein response (UPR)
[61]. However, the effect of Mcl-1 was mostly counteracted by the upregulation
of NOXA, which binds to and functionally represses Mcl-1 [62, 63]. Moreover,
regardless of its accumulation, proteasome inhibition caused Mcl-1 cleavage in a
caspase-3-dependent manner [63–65].
12.4.4 Proteasome Inhibition and Unfolded Protein Response
Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER)

lumen causes ER stress and subsequently the activation of unfolded protein response
(UPR). ER stress is sensed, and the activation of UPR is controlled by three ER
transmembrane proteins, PERK, IRE1 and ATF6. Initially, the UPR can support
cell survival through ATF6-mediated or IRE1- and XBP1-mediated induction of
ER chaperone proteins and proteins involved in ER-associated protein degradation
(ERAD), both of which help to relieve ER stress. However, prolonged UPR can
eventually lead to apoptosis through the PERK-, ATF4-, and CHOP/GADD153-
mediated pathway or the IRE1- and JNK-mediated pathway [66]. Although the
proteasome does not directly reside in the ER lumen, it participates in the UPR in
multiple levels. First, increased expression of the ER chaperones GRP78/Bip and
GRP94/gp96 was observed upon proteasome inhibition, suggesting activation of
UPR [67]. Secondly, proteasome inhibitor treatment was found to suppress the acti-
vation of IRE1- and XBP1-mediated survival pathways during UPR [68]. Thirdly,
proteasome inhibitors were reported to induce the proapoptotic/terminal UPR medi-
ated by PERK, ATF4, and CHOP/GADD153 [69]. However, the exact mechanism
by which proteasome inhibition induces ER stress remains unclear and debatable.
12.4.5 Proteasome Inhibition and Oxidative Stress
Generation of reactive oxygen species (ROS) upon proteasome inhibition has been
observed in different cell types including glioma, thyroid cancer, head and neck
squamous cell carcinoma, and non-small cell lung cancer [70–73]. Pre-treating
cells with an antioxidant was able to relieve oxidative stress and suppress apop-
tosis induction by proteasome inhibition [71, 74]. Consistently, Du et al. [72]
reported that high-level intracellular glutathione protected cells from proteasome
inhibition-induced oxidative stress, suggesting that a glutathione-dependent redox
system might play an important role in the sensitivity of certain cells to protea-
some inhibition-induced apoptosis. More intriguingly, Lee et al. [75] reported that
low-dose lactacystin led to increased oxidative/nitrosative protein damage and
lipid peroxidation with little cell death, while high-dose lactacystin further caused
oxidative DNA damage and induced apoptotic cell death. Similar effects were
observed by using another proteasome inhibitor epoxomicin [75]. A comprehen-
sive proteomic and transcriptomic analysis by Bieler et al. [76] further revealed
that low-dose proteasome inhibition induced a transcriptional profile reminiscent
of a physiological stress response that preconditions and protects cells from oxida-
tive stress, while high-dose proteasome inhibition induced massive transcriptional
dysregulation and pronounced oxidative stress, triggering apoptosis.
12.5 Clinical Development of Bortezomib
12.5.1 Clinical Development of Bortezomib for Treatment

of Multiple Myeloma and Mantle Cell Lymphoma
Bortezomib (Velcade®) is the first and currently the only proteasome inhibitor
approved by the U.S. Food and Drug Administration (FDA). Based on two phase
II trials, the CREST trial [77, 78] and the SUMMIT trial [79], bortezomib received
a fast-track approval from the FDA in 2003 for the treatment of patients with
relapsed or refractory MM who had received at least two prior lines of therapy and
progressed on their last therapy. In 2005, bortezomib was promoted by the FDA to
treat patients with MM who had received at least one prior therapy based on the
phase III APEX trial [80, 81]. In 2008, bortezomib successfully became the front-
line therapy for MM patients who are newly diagnosed or previously untreated
based on the phase III VISTA trial [82, 83]. Bortezomib was initially approved for
intravenous injection, but very recently, in 2012, it received FDA approval for sub-
cutaneous administration based on the results from a phase III, non-inferiority trial
conducted in bortezomib-naive patients with relapsed MM [84].
The rational combinations of bortezomib with other chemotherapeutic agents
have been explored. High-dose dexamethasone is a mainstay of therapy for MM.
The combination of bortezomib and dexamethasone was studied in a phase II
trial, where it was used as the first-line treatment in untreated MM patients. The
results suggest that bortezomib with or without dexamethasone is an effective and
well-tolerated induction regimen for the frontline treatment of MM [85, 86]. In
a global phase IIIb trial, the similar regimen was extended to heavily pre-treated
patients with relapsed or refractory MM and proved to be safe and effective in
these patients as well [87]. Adding a third drug to the combination of bortezomib
and dexamethasone has also been studied. For example, a combination of bort-
ezomib, doxorubicin, and dexamethasone used as induction therapy before stem-
cell transplantation induced >90 % of responses in newly diagnosed MM patients
with well-tolerated and manageable toxicities [88, 89]. Likewise, a combination
of bortezomib, thalidomide, and dexamethasone also achieved favorable outcome
when used as induction regimen in newly diagnosed MM patients [90].
For patients ineligible for stem-cell transplantation, melphalan plus prednisone
has long been the standard treatment with response rates around 50 % and median
survival around 3 years [91, 92]. The previously mentioned pivotal VISTA trial
compared the use of melphalan and prednisone with or without bortezomib in
untreated MM patients who were ineligible for high-dose therapy and eventually
supported the approval of bortezomib as frontline therapy in these patients. In
this trial, adding bortezomib to melphalan and prednisone (VMP) achieved much
higher complete response (CR) rate and partial response (PR) rate as well as sig-
nificantly prolonged overall survival [82, 83]. Replacing melphalan with thalido-
mide in this triple-drug regimen resulted in comparable efficacy but more serious
adverse events and discontinuations, suggesting that the VMP regimen should
remain the upfront treatment for untreated MM patients who are ineligible for
stem-cell transplantation [93]. Rather than replacing melphalan with thalidomide,
adding thalidomide to the VMP regimen as a fourth drug was found to be superior
to VMP alone [94, 95].
In 2006, the FDA extended the application of bortezomib to the treatment
of mantle cell lymphoma (MCL) based on the results from the pivotal phase II
PINNACLE trial where 155 patients with relapsed or refractory MCL received
bortezomib monotherapy [96, 97]. The overall response rate (ORR) was 32 %.
The median time to progression and overall survival time in responders were much
longer than those in the entire patient population, suggesting that single-agent
bortezomib therapy is associated with lengthy responses and notable survival in
relapsed or refractory MCL patients. In another phase II trial, the combination of
bortezomib and gemcitabine was investigated and found to be active in patients
with relapsed or refractory MCL with the ORR being 60 %. This regimen also
offers a chemotherapy backbone to which other less myelosuppressive agents may
be added [98].
12.5.2 Clinical Trials of Bortezomib in Other Hematological

Malignancies and Solid Tumors
Dozens of clinical trials are currently undergoing in order to further expand the
application of bortezomib to other hematological malignancies such as different
types of B-cell lymphoma as well as some advanced-stage solid tumors [99].
The application of bortezomib in different B-cell lymphomas has been
explored. Besides MCL, which accounts for 5 % of all B-cell lymphomas, two
major types of B-cell lymphomas are diffuse large B-cell lymphoma and follicu-
lar lymphoma, accounting for 30–40 and 20 %, respectively [100]. In a phase II
trial, a regimen of weekly or twice-weekly bortezomib plus rituximab was evalu-
ated in patients with relapsed or refractory follicular or marginal-zone B-cell lym-
phoma [101]. Based on the encouraging results, the regimen of weekly bortezomib
plus rituximab was further evaluated in patients with relapsed follicular lymphoma
in a phase III trial. However, comparing with rituximab alone, the improvement
elicited by adding bortezomib was not as great as expected, suggesting that this
regimen might be useful for only some subgroups of patients [102]. In the phase
II VERTICAL trial, a combination of bortezomib, bendamustine, and rituximab
was evaluated and proved to be highly active (ORR of 88 % and CR of 53 %)
in patients with relapsed or refractory follicular lymphoma [103]. In a phase I/II
study, bortezomib was introduced to the standard CHOP (cyclophosphamide, dox-
orubicin, vincristine, and prednisone) plus rituximab regimen (R-CHOP). In dif-
fuse large B-cell lymphoma patients, the evaluable ORR was 100 % with 86 %
CR/unconfirmed CR. The numbers were 91 and 72 % in MCL patients, respec-
tively. These results strongly support the bortezomib plus R-CHOP regimen to
enter phase III trial [104].
The application of bortezomib in solid tumors has also been extensively inves-
tigated. Unfortunately, in contrast to its potent efficacy in hematological malignan-
cies, the efficacy of bortezomib in solid tumors is disappointing. In patients with
castration resistant metastatic prostate cancer, neither bortezomib alone nor bort-
ezomib plus prednisone, exhibited significant antitumor effects [105]. Similarly,
docetaxel plus bortezomib used as first-line treatment showed no improved effi-
cacy versus docetaxel alone [106]. In heavily pre-treated metastatic breast cancer
patients, bortezomib plus pegylated liposomal doxorubicin was well tolerated but
had minimal activity [107]. A trial testing the bortezomib monotherapy in chemo-
therapy-naïve patients with advanced-stage non-small cell lung cancer was termi-
nated in the first stage due to lack of response in all patients [108]. The bortezomib
monotherapy was inactive in patients with unresectable or metastatic gastric and
gastroesophageal junction adenocarcinoma, either [109]. A first-line regimen of
bortezomib, paclitaxel, and carboplatin also failed in the treatment of patients with
metastatic esophageal, gastric, and gastroesophageal cancer [110].
Therefore, continuous efforts are required both on the benchtop and at the
bedside. A mechanistic explanation regarding the failure of bortezomib in solid
tumors will help in designing a rational regimen and eventually lead to clinical
success.
12.6 Advantages and Disadvantages of Bortezomib

and the Development of Second Generation
Proteasome Inhibitors
12.6.1 Advantages of Bortezomib
According to the preclinical and clinical studies comparing proteasome inhibitors

with other anticancer drugs, proteasome inhibitors, represented by bortezomib,
possess several advantages with respect to cancer treatment.
First, as a proteasome inhibitor, bortezomib exhibits favorable selectivity
toward tumor cells over normal cells, which is an important criterion for being a
good anticancer drug. Numerous studies have proved the selectivity of bortezomib
in tumor cells versus normal cells. Multiple factors have been implicated in con-
tributing to this tumor cell selectivity. (1) Cancer cells are rapidly dividing cells
compared with non-cancerous cells. Therefore, they more heavily rely on protea-
somal turnover of cell cycle regulatory proteins to promote cell cycle progression
than normal cells. (2) Cancer cells appear to generate misfolded or damaged pro-
teins much faster than non-cancerous cells due to their uncontrolled cell prolif-
eration. (3) The amount of proteasomes and/or the proteasomal activity have been
implicated to be upregulated in many types of cancers and this upregulation is
important for the maintenance of the malignant phenotype [111, 112].
Secondly, the outstanding efficacy of bortezomib in the treatment of MM and
MCL has been observed in clinical trials and applications. More importantly, clini-
cal trials suggest that bortezomib holds activity in about one third of patients who
were heavily pre-treated. This could be attributed to the fact that bortezomib is tar-
geting a unique, previously unaffected target, the proteasome; therefore, the mech-
anism of action of bortezomib differs from any existing chemotherapeutic agents
[113, 114].
Thirdly, bortezomib is capable of enhancing the sensitivity of cancer cells to
conventional chemotherapeutic agents or radiation. In several previously discussed
clinical trials, bortezomib was introduced into the standard therapy and largely
improved the outcome in terms of ORR, time to progression, etc. The chemo- and
radio-sensitizing effect of bortezomib could also be attributed to its unique target
and mechanism of action. It is conceivable that two drugs with different targets or
mechanisms of action may have better efficacy and less toxicity than two drugs
with the same target or similar mechanism of action [115, 116].
12.6.2 Disadvantages of Bortezomib
On the other hand, some disadvantages or limitations of bortezomib have been

observed during its preclinical and clinical development.
First, although it is generally well tolerated, bortezomib still generates some
toxicity and in some cases, even causes discontinuation of the regimen. The most
frequently occurring side effects are nausea, diarrhea, and fatigue. More serious
adverse drug reactions include peripheral neuropathy, thrombocytopenia, neutro-
penia, and lymphopenia. It was estimated that more than 40 % of patients were
afflicted with peripheral neuropathy [117]. The newly approved subcutaneous
administration was found to significantly alleviate peripheral neuropathy com-
pared with intravenous injection [84].
Another shortcoming of bortezomib is its narrow therapeutic window.
According to a phase I trial, the therapeutic dose of bortezomib is 1.3 mg/m2,
while dose-limiting toxic effects appear at 1.5 mg/m2. However, the latest results
from the phase II CREST trial, which compared two doses of bortezomib (1.3 vs.
1.0 mg/m2), indicate that if bortezomib dose reduction is required, the 1.0 mg/m2
dose still offers patients a substantial survival benefit [78].
In addition, despite the appreciable therapeutic outcome of bortezomib, like
almost all anticancer drugs, drug resistance occurs after a certain period. Even
bortezomib resistance was observed in patients who were newly diagnosed and
received bortezomib treatment as monotherapy for the first time. These clini-
cal observations indicate that bortezomib resistance could be either acquired
or inherent. The results from cell-based studies suggest that bortezomib resist-
ance could occur either at the level of the proteasome itself or its downstream.
Increased mRNA and protein expression of proteasomal β5 subunit was observed
in bortezomib-resistant leukemic cell lines as well as patient samples [118–120].
Mutations in β5 subunits which impair bortezomib binding were also reported
[120, 121]. In addition, constitutively active NF-κB pathway, located down-
stream of the proteasome pathway, was found in some bortezomib-resistant cell
lines [122].
Moreover, during the clinical application of bortezomib, it was noticed that
some natural products, including green tea polyphenols, are able to reduce the
efficacy of bortezomib. This was due to the direct interaction between the boronic
acid structure of bortezomib and the catechol structure of green tea polyphenols,
resulting in the formation of a borate ester and inactivation of bortezomib [21].
Further clinical studies are needed for evidence-based recommendations of green

tea consumption in patients receiving bortezomib treatment. Fortunately, this issue
only affects boronic acid-based proteasome inhibitors.
Finally, as discussed earlier, the efficacy of bortezomib, either alone or in a
multi-drug regimen, is unsatisfactory in the treatment of solid tumors. Addressing
this issue will largely extend the application of proteasome inhibitors in cancer
treatment.
12.6.3 Second Generation Proteasome Inhibitors
At least five second generation proteasome inhibitors are currently under clinical
investigation (Table 12.1).
Carfilzomib is an irreversible proteasome inhibitor, with new protein syn-
thesis being required for recovery of proteasome activity, which gives it greater
potency. Whether it has decreased side effects compared with bortezomib needs
to be determined. In addition to targeting the β5 subunit in the constitutive pro-
teasome, carfilmozib also targets the correlated β5i subunit in the immunoprotea-
some, which appears to be preferentially expressed in MM. Moreover, carfilzomib
is shown to be more specific than borzetomib, with little or no off-target activity
outside of the proteasome [123]. However, like bortezomib, the administration of
carfilzomib is intravenous twice weekly, which is inconvenient for patients.
Carfilzomib has been evaluated in two phase II trials, PX-171-003-A1 [124]
and PX-171-004 [125, 126], as monotherapy for the treatment of relapsed and
refractory MM patients. Both trials observed durable response to carfilzomib
with well-tolerated and manageable side effects, regardless of prior exposure to
bortezomib. Analysis of 136 patients in the above two trials indicated that periph-
eral neuropathy occurred in 15 % of patients, among which 9 % was attributed to
carfilzomib. None of the patients required discontinuation or dose adjustments due
to neurotoxicity, allowing long-term treatment and prolonged disease control by
carfilzomib [127]. These data also indicate that peripheral neuropathy is not a class
effect of proteasome inhibitors [127]. A recent study comparing bortezomib and
carfilzomib further pointed out that the neurotoxicity of bortezomib was related
to the off-target inhibition of HtrA2/Omi by bortezomib, a factor known to be
involved in neuronal survival [128].
Carfilzomib has also been evaluated in combination with lenalidomide, and
low-dose dexamethasone (CRd) in both relapsed and/or refractory MM patients
[129] and newly diagnosed MM patients [130] in two separate phase II trials.
CRd regimen is highly active and well tolerated in both trials. In newly diagnosed
patients, the responses are rapid and improve over time reaching 100 % very good
PR or better, which compare favorably with the best frontline regimens in MM
therapy [130]. In relapsed and/or refractory patients, the ORR was 78 % [129].
Based on these encouraging results, the phase III ASPIRE trial of this regimen is
underway for previously bortezomib-treated MM patients.
Marizomib is another irreversible proteasome inhibitor administrated intra-

venously twice weekly. Compared with other proteasome inhibitors, marizomib
produces rapid, broad, and prolonged inhibition of all three 20S proteasome cata-
lytic activities. Data from a phase I trial suggest that responses to marizomib were
found in patients with bortezomib-refractory MM. The safety profile of marizomib
clearly differs from bortezomib, with no significant treatment-emergent peripheral
neuropathy, myelosuppression or thrombocytopenia reported and dose-limiting
toxicities being transient hallucinations, cognitive changes, and loss of balance. In
addition, marizomib exhibited interesting pharmacokinetic and pharmacodynamic
properties and tissue distribution, supporting a possible role for marizomib in
patients with different disease characteristics such as extramedullary spread [131].
MLN-9708, a boronate-based reversible proteasome inhibitor, has the advan-
tage of being the first oral proteasome inhibitor to enter clinical investigation in
MM patients. Oral administration is not only convenient but also seems to produce
milder side effects. Data from a phase I trial suggest that single-agent MLN-9708
may result in clinical activity in heavily pre-treated relapsed and/or refractory
MM patients including durable disease control and is generally well tolerated with
infrequent peripheral neuropathy [132].
Delanzomib (CEP-18770) is another boronate-based reversible proteasome
inhibitor with oral bioavailability. However, current phase I/II clinical investiga-
tions are still using intravenous administration. Delanzomib has shown proteas-
ome-inhibitory activity similar to that of bortezomib in hematological and solid
tumor cell lines, as well as in primary cells from MM patients [133].
Oprozomib (ONX-0912) is an epoxyketone-based irreversible proteasome
inhibitor with oral bioavailability. It showed similar potency to carfilzomib in
cytotoxicity assays. More excitingly, orally administered oprozomib had equiva-
lent antitumor activity to intravenously administered carfilzomib in human tumor
xenograft and mouse syngeneic models [134]. It is currently being investigated in
early clinical trials via oral administration twice daily.
12.7 Conclusion
The proteasome has been shown to be a validated, novel and valuable target for
cancer treatment. Suppression of proteasome function by synthetic or natural com-
pounds is proven to be an effective way to induce cancer cell death with minimal
effects on normal healthy cells. The first FDA-approved proteasome inhibitor bort-
ezomib has achieved great success in the treatment of MM and MCL. However,
some limitations and side effects have been noted during clinical application, such
as the occurrence of bortezomib resistance, dose-limiting toxicities, and interac-
tion with some natural compounds, all of which compromise the clinical benefits
of bortezomib. Furthermore, the efficaciousness of bortezomib in solid tumors
is disappointing. Therefore, the development of novel, new-generation protea-
some inhibitors is necessary to improve the efficacy, reduce toxicity, and expand
the application of proteasome inhibition-based strategies in cancer therapy.

Meanwhile, continuous exploration of the effective combination of a proteasome
inhibitor with other chemotherapeutic agents is essential to optimize the clini-
cal outcome. A better understanding of the mechanism of proteasome inhibition-
induced cell death will greatly promote the development and clinical application
of novel anticancer drugs which will help illuminate the bright future of cancer
treatment.
Acknowledgments The authors thank Sara Schmitt and Daniela Buac for critical reading of the
manuscript. This work was partially supported by the National Cancer Institute (1R01CA120009,
3R01CA120009-04S1, and 5R01CA127258-05, to QPD).
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Chapter 13
New Agents and Approaches
for Targeting the RAS/RAF/MEK/ERK
and PI3K/AKT/mTOR Cell Survival
Pathways
James A. McCubrey, Linda S. Steelman, William H. Chappell, Stephen L.
Abrams, Richard A. Franklin, Giuseppe Montalto, Melchiorre Cervello,
Ferdinando Nicoletti, Graziella Malaponte, Clorinda Massarino, Massimo
Libra, Jörg Bäsecke, Agostino Tafuri, Michele Milella, Francesca Chiarini,
Camilla Evangelisti, Lucio Cocco and Alberto M. Martelli
Abstract The Ras/Raf/MEK/ERK and PI3K/Akt/mTOR cascades are often a ctivated

by genetic alterations, either by mutations in upstream signaling molecules or by muta-
tions in intrinsic pathway components. Upstream mutations in one signaling pathway
or even in downstream components of the same pathway can alter the sensitivity of the
J. A. McCubrey (*) · L. S. Steelman · W. H. Chappell · S. L. Abrams · R. A. Franklin

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina
University, Greenville, NC, USA
G. Montalto
Department of Internal Medicine and Specialties University of Palermo, Palermo, Italy
M. Cervello
Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare
“Alberto Monroy”, Palermo, Italy
F. Nicoletti · G. Malaponte · C. Massarino · M. Libra
Department of Biomedical Sciences, University of Catania, Catania, Italy
J. Bäsecke
Department of Medicine, University of Göttingen, Göttingen, Germany
A. Tafuri
Sapienza, University of Rome, Department of Cellular Biotechnology and Hematology,
Rome, Italy
M. Milella
Regina Elena National Cancer Institute, Rome, Italy
F. Chiarini · C. Evangelisti · A. M. Martelli
Institute of Molecular Genetics, National Research Council-Rizzoli Orthopedic Institute,
Bologna, Italy
L. Cocco · A. M. Martelli
Department of Biomedical and Neuromotor Sciences, Università di Bologna, Bologna, Italy
332 J. A. McCubrey et al.
cells to certain small molecule inhibitors. These pathways have profound effects on
proliferative, apoptotic, and differentiation pathways. Dysregulation of components of
these pathways can contribute to: malignant transformation, resistance to other pathway
inhibitors, and chemotherapeutic drug resistance. This chapter will first briefly describe
these pathways and then evaluate potential uses of Raf, MEK, PI3K, Akt, and mTOR
inhibitors that have been investigated in preclinical and clinical investigations.
13.1 Introduction
Since the discovery of the RAS, RAF, MEK1, PIK3CA, and AKT oncogenes and neu-
rofibromin 1 (NF1), PTEN, TSC1, and TSC2 tumor suppressor genes, the Ras/Raf/
MEK/ERK, and Ras/PI3K/PTEN/Akt/mTOR signaling cascades have been extensively
investigated with the ultimate goal of determining how these genes become activated/
inactivated and whether it is possible to suppress their activity in human cancer and
other diseases [1]. Furthermore, these pathways are also implicated in the resistance and
sometimes sensitivity to therapy [2]. There have been breakthroughs in the discovery of
complex interacting pathway components, and their genetic and epigenetic regulation.
Furthermore, elucidation of the mechanisms by which mutations of components of the
pathways can lead to aberrant signaling, uncontrolled proliferation, and in some cases
confer sensitivity to targeted therapy has greatly advanced the field. This chapter will
review some of the current inhibitors, their targets, and how they are being used to treat
cancer and overcome therapeutic resistance.
Usually signaling commences upon ligation of a growth factor/cytokine/interleu-
kin/mitogen (ligand) to its cognate receptor at the cell surface. This event can result
in the activation of many downstream signaling cascades including the Ras/Raf/
MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways. These pathways can fur-
ther transmit their signals to different subcellular components, namely to the nucleus
to control gene expression, to the translational apparatus to enhance the translation of
“weak” mRNAs, to the apoptotic machinery to regulate apoptosis, or to other events
involved in the regulation of cellular proliferation (e.g., interactions with the p53 path-
way to regulate cell cycle progression). Regulation of the Ras/Raf/MEK/ERK and Ras/
PI3K/PTEN/Akt/mTOR pathways is mediated by a series of kinases, phosphatases,
GTP:GDP exchange, and scaffolding proteins. There are also many tumor suppressor
proteins which interact with these cascades which frequently serve to fine tune or limit
activity (e.g., NF1, PTEN, RKIP, PP2A, TSC1, and TSC2). Mutations occur in many of
the genes in these pathways leading to uncontrolled regulation and aberrant signaling.
13.2 The Ras/Raf/MEK/ERK Pathway
An overview of the Ras/Raf/MEK/ERK pathway and the sites where small mol-
ecule inhibitors act is presented in Fig. 13.1. This figure serves to illustrate the flow
of information through this pathway from a growth factor to a specific receptor
13 New Agents and Approaches for Targeting 333
Fig. 13.1 Overview of the Ras/Raf/MEK/ERK cascade and small molecule inhibitors used for
targeting this pathway. Activation of this pathway can occur by mutations in upstream Growth
factor receptors (GFR) or by stimulation by the appropriate growth factors (GF). In addition,
mutations can occur in intrinsic members of the pathway (RAS, RAF, MEK1, or the tumor sup-
pressor Neurofibromin (NF1)). Sites where NF1, protein phosphatase 2A (PP2A), Raf kinase
inhibitory protein (RKIP), kinase suppressor of Ras (KSR) interact with this pathway are on the
right hand side of the Ras/Raf/MEK/ERK pathway. NF1, PP2A, and RKIP are depicted in black
rectangles as they normally serve to dampen the activity of this pathway. Sites where various
small molecule inhibitors function are in black octagons on the left hand side of the pathway.
Representative inhibitors are listed in boxes next to the octagons
to phosphorylation of appropriate transcription factors as well as affect proteins

involved in translation and apoptosis. Following the stimulation of a receptor with
a growth factor/cytokine/mitogen, a Src homology 2 domain-containing protein
(Shc) adaptor protein becomes associated with the C-terminus of the activated
growth factor receptor (GFR), for example, epidermal growth factor receptor
(EGFR), insulin-like growth factor-1 receptor (IGF-1R), vascular endothelial
growth factor receptor (VEGFR) and many others [1, 2]. Shc recruits the growth
factor receptor–bound protein 2 (Grb2) protein and the son of sevenless (SOS)
homolog protein [a guanine nucleotide exchange factor (GEF)], resulting in the
loading of the membrane-bound GDP:GTP exchange protein (GTPase). GEFs
promote Ras activation by displacing GDP from Ras which leads to GTP binding.
Ras activation is suppressed by the GTPase-activating proteins (GAPs) that stimu-
late the GTPase activity of Ras. There are two prominent GAP proteins, p120GAP
and NF1. Ras can also be activated by growth factor receptor tyrosine kinases
(GFRTK), such as insulin receptor (IR), via intermediates like insulin receptor sub-
strate (IRS) proteins that bind Grb2 [3]. Ras:GTP then recruits the serine/threonine
(S/T) kinase Raf to the membrane where it becomes activated [1, 2].
Both RAS and RAF are members of multi-gene families, and there are three
RAS members (KRAS, NRAS, and HRAS) and three RAF members (BRAF, RAF1
(a.k.a c-Raf), and ARAF) [1, 2]. Raf-1 and A-Raf are activated, in part, by a Src-
family kinase, while B-Raf does not require the Src-family kinase for activation.
Raf-1 can be regulated by dephosphorylation by the protein serine/threonine phos-
phatase 2A (PP2A). PP2A has been reported to positively and negatively regulate
Raf-1 [4, 5].
Raf is responsible for S/T phosphorylation of mitogen-activated protein kinase
kinase-1 (MEK1) (a dual specificity kinase (T/Y) [1, 2]. Other proteins such as
kinase suppressor of Ras (KSR) have recently been shown to phosphorylate
MEK1 [6]. KSR has scaffolding properties and interacts with Raf, MEK, and
ERK which regulates ERK activation. KSR can form dimers with various Raf pro-
teins which alter the effects of Raf inhibitors. KSR competes with Raf-1 for Raf
inhibitor–induced binding to B-Raf which decreases the normal ERK activation
observed after Raf-inhibitor treatment [7].
MEK1 phosphorylates extracellular signal-regulated kinases 1/2 (ERK1 and 2)
at specific T and Y residues [1, 2]. MEK1 was originally not thought to be mutated
frequently in human cancer. However, recent large-scale mutation screening stud-
ies and studies aimed at determining mechanisms of resistance to small molecule
inhibitors have observed that MEK1 is mutated in certain human cancers and also
is mutated in certain inhibitor-resistant cells [8].
Activated ERK1 and ERK2 serine S/T kinases phosphorylate and activate a
variety of substrates, including p90 ribosomal six kinase-1 (p90Rsk1) [2]. ERK
also phosphorylates MAPK signal-integrating kinases (Mnk1/2) which can in turn
phosphorylate eukaryotic translation initiation factor 4E (eIF4E), a key protein
involved in the translation of difficult mRNAs [9].
p90Rsk1 can activate the cAMP-response element-binding protein (CREB) tran-
scription factor as well as proteins involved in regulation of protein translation
(e.g., Mnk-1, p70 ribosomal S6 kinase (p70S6K), eukaryotic translation initiation
factor 4B, (eIF4B), and ribosomal protein S6 (rpS6) [10].
The number of ERK1/2 substrate/targets is easily in the hundreds. These
substrates/targets include different types of molecules including other kinases,
transcription factors, or proteins involved in protein translation or apoptosis.

Suppression of MEK and ERK can have profound effects on cell growth, inflam-
mation, and aging. Activated ERK can also phosphorylate “upstream” Raf-1 and
MEK1 which alter their activity. Depending upon the site phosphorylated on Raf-
1, ERK phosphorylation can either enhance [11] or inhibit [12] Raf-1 activity.
In contrast, some studies have indicated that when MEK1 are phosphorylated by
ERK, their activity decreases [13]. Recent studies indicate that ERK does not neg-
atively feedback-inhibit B-Raf [14].
These phosphorylation events induced by ERK serve to alter the stability and/
or activities of the proteins. These examples of feedback loops become important
in consideration of whether to just target MEK or to target both Raf and MEK in
various cancers. It is important that the reader realize that certain phosphorylation
events can either inhibit or repress the activity of the affected protein. This often
depends on the particular residue on the protein phosphorylated which can confer
a different configuration to the protein or target the protein to a different subcel-
lular localization that may result in proteasomal degradation or association with
certain scaffolding proteins.
13.3 The Ras/PI3K/PTEN/Akt/mTOR Pathway
An introductory overview of the Ras/PI3K/PTEN/Akt/mTOR pathway is pre-

sented in Fig. 13.2. Also outlined in this diagram are common sites of intervention
with signal transduction inhibitors. Many of these inhibitors have been evaluated
in various clinical trials, and some are currently being used to treat patients with
specific cancers. Extensive reviews of many inhibitors targeting these pathways
have been recently published [1, 15, 16].
Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric protein with an 85 kDa
regulatory subunit and a 110 kDa catalytic subunit (PIK3CA) [1, 2, 66–69].
PIK3CA is frequently mutated in certain cancers such as breast, ovarian, colorec-
tal, endometrial, and lung [2, 17].
PI3K serves to phosphorylate a series of membrane phospholipids including
phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bis-
phosphate (PtdIns(4,5)P2), catalyzing the transfer of ATP-derived phosphate
to the D-3 position of the inositol ring of membrane phosphoinositides, thereby
forming the second messenger lipids phosphatidylinositol 3,4-bisphosphate
(PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisPhosphate (PtdIns(3,4,5)P3)
[2, 15]. Most often, PI3K is activated via the binding of a ligand to its cognate
receptor, whereby p85 associates with phosphorylated tyrosine residues on the
receptor via a Src homology 2 (SH2) domain. After association with the receptor,
the p110 catalytic subunit then transfers phosphate groups to the aforementioned
membrane phospholipids [15]. It is these lipids, specifically PtdIns [3–5] P3, that
attract a series of kinases to the plasma membrane, thereby initiating the signaling
cascade.
Fig. 13.2 Overview of the PI3 K/Akt/mTOR cascade and small molecule inhibitors used for
targeting this pathway. Activation of this pathway can occur by mutations in upstream growth
factor receptors (GFR) or by stimulation by the appropriate GF. In addition, mutations can occur
in intrinsic members of the pathway (RAS, PIK3CA, AKT, or the tumor suppressors (NF1, PTEN,
TSC1, TSC2). Sites where NF1, PTEN, TSC1, TSC2 are depicted in black rectangles as they nor-
mally serve to dampen the activity of this pathway. Sites where various small molecule inhibitors
function are in black octagons. Representative inhibitors are listed in boxes next to the octagons
Downstream of PI3K is the primary effector molecule of the PI3K signaling

cascade, Akt/protein kinase B (PKB) which is a 57 kDa S/T kinase that phospho-
rylates many targets on RxRxxS/T (R = Arginine) consensus motifs [18]. Akt was
discovered originally as the cellular homolog of the transforming retrovirus AKT8
and as a kinase with properties similar to protein kinases A and C [19]. Akt con-
tains an amino-terminal pleckstrin homology (PH) domain that serves to target the
protein to the membrane for activation [15]. Within its central region, Akt has a
large kinase domain and is flanked on the carboxyl-terminus by hydrophobic and
proline-rich regions [15]. Akt-1 is activated via phosphorylation of two residues:
T308 and S473. Akt-2 and Akt-3 are highly related molecules and have similar
modes of activation. Akt-1 and Akt-2 are ubiquitously expressed, while Akt-3
exhibits a more restricted tissue distribution and is found abundantly in nervous
tissue [20].
The phosphatidylinositol-dependent kinases (PDKs) are responsible for the

activation of Akt. PDK1 is the kinase responsible for phosphorylation of Akt-1
at T308 [18]. Akt-1 is also phosphorylated at S473 by the mammalian target of
rapamycin (mTOR) complex referred to as (Rapamycin-insensitive companion
of mTOR/mLST8 complex) mTORC2 [15]. Therefore, phosphorylation of Akt is
complicated as it is phosphorylated by a complex that lies downstream of activated
Akt itself [15]. Thus, as with the Ras/Raf/MEK/ERK pathway, there are feedback
loops that serve to regulate the activity of the Ras/PI3K/PTEN/Akt/mTOR path-
way. Once activated, Akt leaves the cell membrane to phosphorylate intracellular
substrates.
After activation, Akt is able to translocate to the nucleus [15] where it affects
the activity of a number of transcriptional regulators. Some examples of molecules
which regulate gene transcription that are phosphorylated by Akt include CREB
[21], E2F [22], nuclear factor kappa from B cells (NF-κB) via inhibitor kappa B
protein kinase (Iκ-K) [23], and the forkhead transcription factors [24]. These are
all either direct or indirect substrates of Akt and each can regulate cellular prolif-
eration, survival, and other important biologic processes. Besides transcription fac-
tors, Akt targets a number of other molecules to affect the survival state of the cell
including the proapoptotic molecule Bcl-2-Associated Death promoter (BAD) [25]
and glycogen synthase kinase-3β (GSK-3β) [26].
Negative regulation of the PI3K pathway is primarily accomplished through
the action of the phosphatase and TENsin homolog deleted on chromosome 10
(PTEN) tumor suppressor protein. PTEN encodes a lipid and protein phosphatase
whose primary lipid substrate is PtdIns(3,4,5)P3 [27]. The purported protein
substrate(s) of PTEN are more varied, including focal adhesion kinase (FAK), the
Shc exchange protein, the transcriptional regulators E-twenty six-2 (ETS-2) [28]
and Sp1 and the platelet-derived growth factor receptor (PDGF-R) [29].
Next, we discuss some of the key targets of Akt that can also contribute to
abnormal cellular growth by the regulation of protein translation. Akt-mediated
regulation of mTOR activity is a complex multi-step phenomenon. Akt inhibits
tuberous sclerosis 2 (TSC2 or tuberin) function through direct phosphorylation
[30]. TSC2 is a GTPase-Activating protein (GAP) that functions in association
with the putative tuberous sclerosis 1 (TSC1 or hamartin) to inactivate the small
G protein Ras homolog enriched in brain (Rheb) [31]. TSC2 phosphorylation by
Akt represses GAP activity of the TSC1/TSC2 complex, allowing Rheb to accu-
mulate in a GTP-bound state. Rheb-GTP then activates, through a mechanism not
yet fully elucidated, the protein kinase activity of mTOR which complexes with
Raptor (Regulatory-associated protein of mTOR) adaptor protein, DEP domain-
containing mTOR-interacting protein (DEPTOR) and mLST8, a member of the
Lethal-with-Sec-Thirteen gene family, first identified in yeast, FK506-binding
protein 38 (FKBP38) and proline-rich Akt Substrate 40 kDa protein (PRAS40).
mTORC1 is sensitive to rapamycin and, importantly, inhibits Akt via a negative
feedback loop which involves, at least in part, p70S6K [31]. This is due to the neg-
ative effects that p70S6K has on IRS1. DEPTOR may be a tumor suppressor gene
as decreased expression of DEPTOR results in increased mTORC1 activity [32].
The mechanism by which Rheb-GTP activates mTORC1 has not been fully
elucidated yet; however, it requires Rheb farnesylation and can be blocked by
Farnesyl transferase (FT) inhibitors. It has been proposed that Rheb-GTP would
relieve the inhibitory function of FKBP38 on mTOR, thus leading to mTORC1
activation [33].
As stated previously, TSC1 and TSC2 have important roles in the regulation of
mTORC1. Two additional molecules important in this regulation are liver kinase B
(LKB1 also known as STK11). LKB1 is an upstream activator of 5′AMP-activated
protein kinase (AMPK) which activates TSC2 that negatively regulates mTORC1
[34]. LKB1 mediates the effects of the diabetes drug metformin [35]. Metformin
has also been shown to be effective in suppressing the developments of certain
cancers [36–38].
Akt also phosphorylates PRAS40, an inhibitor of mTORC1, and by doing
so, it prevents the ability of PRAS40 to suppress mTORC1 signaling (recently
reviewed in [15]). Thus, this could be yet another mechanism by which Akt acti-
vates mTORC1. Moreover, PRAS40 is a substrate of mTORC1 itself, and it has
been demonstrated that mTORC1-mediated phosphorylation of PRAS40 prevents
the inhibition of additional mTORC1 signaling [31].
Ras/Raf/MEK/ERK signaling positively impinges on mTORC1. Both p90Rsk−1
and ERK 1/2 phosphorylate TSC2, thus suppressing its inhibitory function [31].
Moreover, mTORC1 inhibition resulted in ERK 1/2 activation, through p70S6K/
PI3K/Ras/Raf/MEK [39].
The relationship between Akt and mTOR is further complicated by the exist-
ence of the mTOR/Rictor complex (mTORC2), which, in some cell types, displays
rapamycin-insensitive activity. mTORC2 is comprised of rapamycin-insensitive
companion of mTOR (Rictor), mTOR, DEPTOR, mLST8, stress-activated pro-
tein kinase interacting protein 1 (SIN1), and protein observed with Rictor (Protor).
mTORC2 phosphorylates Akt on S473 in vitro which facilitates T308 phospho-
rylation [15]. Thus, mTORC2 can function as the elusive PDK-2 which phos-
phorylates Akt-1 on S473 in response to growth factor stimulation [40]. Akt
and mTOR are linked to each other via positive and negative regulatory circuits,
which restrain their simultaneous hyperactivation through mechanisms involving
p70S6K and PI3K [15, 31]. Assuming that equilibrium exists between these two
complexes, when the mTORC1 complex is formed, it could antagonize the forma-
tion of the mTORC2 complex and reduce Akt activity. Thus, at least in principle,
inhibition of the mTORC1 complex could result in Akt hyperactivation. This is
one problem associated with therapeutic approaches using rapamycin or modified
rapamycins (rapalogs) that block some actions of mTOR but not all.
mTOR is a 289 kDa S/T kinase. mTOR was the first identified member of
the phosphatidylinositol 3-kinase-related kinase (PIKK) family [15]. mTOR has
been referred to as the gatekeeper of autophagy [41]. mTOR regulates transla-
tion in response to nutrients and growth factors by phosphorylating components
of the protein synthesis machinery, including p70S6K and eukaryotic initiation
factor (eIF)-4E binding protein-1 (4EBP-1), the latter resulting in the release of
the eukaryotic initiation factor-4E (eIF-4E) allowing eIF-4E to participate in the
assembly of a translational initiation complex [2]. p70S6K phosphorylates the 40S

rpS6, leading to translation of “weak” mRNAs. Integration of a variety of signals
(mitogens, growth factors, hormones) by mTOR assures cell cycle entry only if
nutrients and energy are sufficient for cell duplication [15].
Unphosphorylated 4E-BP1 interacts with the cap-binding protein eIF4E and
prevents the formation of the 4F translational initiation complex (eIF4F) by com-
peting for the binding of eukaryotic initiation factor 4G (eIF4G) to eIF4E. 4E-BP1
phosphorylation by mTORC1 results in the release of the eIF4E, which then asso-
ciates with eIF4G to stimulate translation initiation [15].
eIF4E is a key component for translation of 5′ capped mRNAs, which include
transcripts mainly encoding for proliferation and survival promoting proteins, such
as c-Myc, cyclin D1, cyclin-dependent kinase-2 (CDK-2), signal activator and
transducer of transcription-3 (STAT-3), ornithine decarboxylase, surviving, B-cell
lymphoma 2 (Bcl-2), Bcl-xL, myeloid cell leukemia-1 (Mcl-1) [2].
13.4 Overview of Pathway Inhibitors
Sites of intervention with signal transduction inhibitors in the Ras/Raf/MEK/

ERK are presented in Fig. 13.1. Some of the inhibitors are currently being used
to treat patients with specific cancers, and others have been or are being evaluated
in numerous clinical trials with many different types of cancer patients. Effective
inhibitors, specific for many of the key components of the Ras/Raf/MEK/ERK and
Ras/PI3K/PTEN/mTOR pathways, have been developed [1, 15]. In many cases,
these inhibitors have been examined in clinical trials. Furthermore, inhibitors that
target the mutant protein more than the wild-type (WT) protein of various genes
(e.g., BRAF and PIK3CA) either have been or are being characterized.
13.4.1 Raf Inhibitors
Raf inhibitors have been developed, and some are being used for therapy while
others are being evaluated in clinical trials. Raf inhibitors have, in general, exhib-
ited greater response rates in clinical trials than MEK inhibitors which may be
related to the broader therapeutic index of Raf inhibitors that suppress ERK activ-
ity in a mutant-allele-specific fashion as opposed to MEK inhibitors which sup-
press MEK activity in tumor and normal cells [42].
13.4.1.1 Sorafenib
Sorafenib (Bayer) was initially thought to specifically inhibit Raf but has been
subsequently shown to have multiple targets (e.g., VEGF-R, Flt-3, PDGF-R) [43].
However, that does not preclude its usefulness in cancer therapy. Sorafenib is
approved for the treatment for certain cancers (e.g., renal cell Carcinoma (RCC)
and patients with unresectable HCC and was further evaluated in the Sorafenib
Hepatocellular carcinoma Assessment Randomized Protocol (SHARP) trial, which
demonstrated that the drug was effective in prolonging median survival and time
to progression in patients with advanced HCC [44, 45]. Sorafenib is generally well
tolerated in HCC patients with a manageable adverse events profile [44]. While
sorafenib is not considered effective for the treatment for most melanomas with
BRAF V600E mutations, it may be effective in the treatment for a minority of mela-
nomas with G469E and D594G mutations which express constitutive ERK1/2 but
low levels of MEK. These melanomas are sensitive to sorafenib, potentially because
they signal through Raf-1. Raf-1 also exerts anti-apoptotic effects at the mitochon-
drion in association with Bcl-2 family members [46].
13.4.1.2 Vemurafenib
Vemurafenib (Zelboraf, PLX-4032, Plexxikon/Roche) is a B-Raf inhibitor that

has and is being evaluated in many clinical trials [47–49]. Vemurafenib has been
approved by the US Food and Drug Administration (FDA) for the treatment of
patients with unresectable or metastatic melanoma carrying the BRAF(V600E)
mutation. For vemurafenib to be clinically effective, it needs to suppress down-
stream ERK activation essentially completely [47].
13.4.1.3 Dabrafenib
Dabrafenib (GSK2118436) is an ATP-competitive inhibitor of mutant B-Raf, WT

B-Raf, and WT Raf-1 developed by GlaxoSmithKlein (GSK) [50]. Dabrafenib is
in clinical trials [51, 52].
13.4.1.4 CCT239065
CCT239065 is a mutant B-Raf inhibitor developed at the Institute of Cancer

Research in London, UK. It inhibits mutant BRAF V600E signaling and prolifera-
tion more than those cells containing WT BRAF [53]. Its effects are more selective
for cells containing mutant BRAF than WT BRAF. CCT239065 is well tolerated
in mice and had good oral bioavailability. It suppressed tumors containing BRAF-
mutant gene but not WT BRAF tumors in mice tumor xenograft studies.
13.4.1.5 GDC-0879
GDC-0879 is a BRAF-mutant allele-selective inhibitor developed by Genentech

[54]. The efficacy GDC-0879 is related to the BRAF V600E mutational status in
the cancer cells and inhibition of downstream MEK and ERK activity.
13.4.1.6 AZ628
AZ628 is a selective Raf inhibitor developed by Astra Zeneca. It has been shown
that when BRAF-mutant melanoma cells, which are normally very sensitive to
AZ628, are grown for prolonged periods of time, they become resistant to AZ628
by upregulating the expression of Raf-1 [55].
13.4.1.7 XL281
XL281 is an oral active wild-type and mutant RAF kinase-selective inhibitor

developed by Exelixis and Bristol-Myers Squibb. It has been examined in clinical
trials primarily with patients having BRAF mutations (colorectal cancers (CRC),
melanoma, papillary thyroid cancers (PTC), and NSCLC) [56].
13.4.1.8 PLX5568
PLX5568 is a selective Raf kinase inhibitor developed by Plexxicon. It is being

examined for the treatment for polycystic kidney disease (PKD). In the kidney,
Raf-1 is localized to the tubular cells where it is linked with many physiologically
important functions. PLX5568 suppressed cyst enlargement in a rat model of PKD
but did not improve kidney function as fibrosis was not suppressed [57].
13.4.1.9 Raf-265
Raf-265 is an ATP-competitive pan-Raf inhibitor developed by Novartis.

Treatment for bronchus carcinoid NCI-H727 and CM-insulinoma cells with Raf-
265 enhanced sensitivity to TRAIL-induced apoptosis. These cells were normally
resistant to PI3K/mTOR inhibitors when combined with TRAIL. Raf-265 was
shown to decrease Bcl-2 levels which correlated with their sensitivity to TRAIL-
mediated apoptosis. This approach may be effective in the therapy of neuroendo-
crine tumors [58].
13.4.1.10 Regorafenib
Regorafenib (BAY 73-4506) is an oral multi-kinase inhibitor of angiogenic,

stromal, and oncogenic RTKs developed by Bayer. Regorafenib inhibits RTKs
such as VEGF-R2, VEGF-R1/3, PDGF-Rβ, fibroblast growth factor receptor-1
as well as mutant Kit, RET, and B-Raf. The effects of regorafenib on tumor
growth have been evaluated in human xenograft models in mice, and tumor
shrinkages were observed in breast MDA-MB-231 and renal 786-O carcinoma
models [59].
13.4.2 MEK Inhibitors
Most MEK inhibitors differ from most other kinase inhibitors as they do not
compete with ATP binding (non-ATP competitive), which confers a high specific-
ity [60–62]. Most MEK inhibitors are specific and do not inhibit many different
protein kinases [62] although as will be discussed below, certain MEK inhibitors
are more specific than others.
Molecular modeling studies indicate that many MEK bind to an allosteric bind-
ing site on MEK1/MEK2. The binding sites on MEK1/MEK2 are relatively unique
to these kinases and may explain the high specificity of MEK inhibitors. This
binding may lock MEK1/2 in an inactivate conformation that enables binding of
ATP and substrate, but prevents the molecular interactions required for catalysis
and access to the ERK activation loop [61].
A distinct advantage of inhibiting MEK is that it can be targeted without
knowledge of the precise genetic mutation that results in its aberrant activation.
This is not true with targeting Raf as certain Raf inhibitors will activate Raf and
also certain B-Raf-specific inhibitors will not be effective in the presence of RAS
mutations.
An advantage of targeting MEK is that the Ras/Raf/MEK/ERK pathway is a
convergence point where a number of upstream signaling pathways can be blocked
with the inhibition of MEK. For example, MEK inhibitors, such as Selumetinib
(AZD6244), are also being investigated for the treatment for pancreatic cancers,
breast cancers, and other cancers such as hematopoietic malignancies, including
multiple myeloma [1, 63].
13.4.2.1 Selumetinib
Selumetinib inhibits MEK1 in vitro with an IC50 value of 14.1 ± 0.79 nM [64–67].

It is specific for MEK1 as it did not appear to inhibit any of the approximately
40 other kinases in the panel tested. Selumetinib is not competitive with ATP.
Selumetinib inhibited downstream ERK1/ERK2 activation in in vitro cell line
assays with stimulated and unstimulated cells and also inhibited activation in tumor
transplant models. Selumetinib did not prevent the activation of the related ERK5
that occurs with some older MEK1 inhibitors, which are not being pursued in clini-
cal trials. Inhibition of ERK1/2 suppresses their ability to phosphorylate and modu-
late the activity of Raf-1, B-Raf, and MEK1 but not MEK2 as MEK2 lacks the
ERK1/ERK2 phosphorylation site. In essence, by inhibiting ERK1/2 the negative
loop of Raf-1, B-Raf, and MEK phosphorylation is suppressed, and hence, there
will be an accumulation of activated Raf-1, B-Raf, and MEK [67]. This biochem-
ical feedback loop may provide a rationale for combining Raf and MEK inhibi-
tors in certain therapeutic situations. Selumetinib has also been shown to suppress
cetuximab-resistant CRCs which had KRAS mutations both in vitro and in vivo
models [68].
13.4.2.2 PD-0325901
The PD-0325901 MEK inhibitor is an orally active, potent, specific, non-ATP-

competitive inhibitor of MEK. PD-0325901 demonstrated improved pharma-
cological and pharmaceutical properties compared with PD-184352, including a
greater potency for inhibition of MEK and higher bioavailability and increased
metabolic stability. PD-0325901 has a Ki value of 1 nM against MEK1 and MEK2
in in vitro kinase assays. PD-0325901 inhibits the growth of cell lines that prolif-
erate in response to elevated signaling of the Raf/MEK/ERK pathways [62]. PD-
0325901 has undergone phase I clinical trials [62, 69–71]. Although the initial trial
results were not encouraging, it was determined that some tumors which prolifer-
ate in response to the Raf/MEK/ERK pathways may be sensitive to PD0325901
[72]. Although the clinical trials with PD-0325901 were initially suspended, there
are now some clinical trials with PD-0325901 in combination with other pathway
inhibitors.
13.4.2.3 Refametinib
Refametinib (RDEA119) is a more recently described MEK inhibitor developed

by Ardea Biosciences [73]. It is a highly selective MEK inhibitor that displays a
>100-fold selectivity in kinase inhibition in a panel of 205 kinases. In contrast,
in the same kinase specificity analysis, other recently developed MEK inhibitors
(e.g., PD0325901) also inhibited the Src and RON kinases.
13.4.2.4 Trametinib
Trametinib (GSK1120212) is an allosteric MEK inhibitor developed by GSK. It

has been shown to be effective when combined with dabrafenib in certain dab-
rafenib-resistant BRAF V600 melanoma lines that also had mutations at NRAS
or MEK1 [52]. The combination of trametinib and the PI3K/mTOR dual inhibi-
tor GSK2126458 also enhanced cell growth inhibition in these B-Raf inhibitor-
resistant BRAF-mutant melanoma lines.
13.4.2.5 GDC-0973
GDC-0973 (XL518) is a potent and selective MEK inhibitor developed by

Genentech [74]. The effects of combining GDC-0973 and the PI3K inhibitor
GDC-0941 on the proliferation of BRAF and KRAS mutant cancer cells indicated
the combination efficacy both in vitro and in vivo.
13.4.2.6 AS703026
AS703026 (MSC1936369B) is a MEK inhibitor developed by EMD Serono.

AS703026 suppressed cetuximab-resistant CRCs which had KRAS mutations both
in vitro and in vivo models [68]. AS703026 inhibited growth and survival of mul-
tiple myeloma (MM) cells and cytokine-induced differentiation more potently
than selumetinib, and importantly, AS703026 was cytotoxic, where as most MEK
inhibitors are cytostatic [75]. AS703026 sensitized MM cells to a variety of con-
ventional (dexamethasone, melphalan) and novel (lenalidomide, perifosine, bort-
ezomib, rapamycin) drugs used to treat MM.
13.4.2.7 RO4987655
RO4987655 (CH4987655) is an allosteric, orally available MEK inhibitor devel-

oped by Roche/Chiron. It has been tested in humans and determined to inhibit
active ERK levels. At the levels of RO4987655 administered, it was determined to
be safe in healthy volunteers [76].
13.4.2.8 TAK-733
TAK-733 is a potent and selective, allosteric MEK inhibitor developed by Takeda

San Diego [77]. TAK-733 is being investigated in clinical trials.
13.4.2.9 MEK162
MEK162 (ARRY-162) is a MEK inhibitor developed by Novartis. It is in clinical

trials.
13.4.2.10 SL327
SL337 is a MEK inhibitor that has been used in many neurological and drug
addiction studies [78].
13.4.2.11 Other MEK Inhibitors
Other MEK inhibitors are being developed. RG422 is one such inhibitor.
13.4.3 Combining Raf and MEK Inhibitors
The possibility of treating certain patients with Raf and MEK inhibitors is a con-
cept which is gaining more acceptance as it may be a therapeutic possibility to
overcome resistance [42]. Raf inhibitors induce Raf activity in cells with WT RAF
if Ras is active [79]. The addition of a MEK inhibitor would suppress the acti-
vation of MEK and ERK in the normal cells of the cancer patient. Thus, B-Raf
would be suppressed by the B-Raf-selective inhibitor in the cancer patient, while
the consequences of Raf activation in the normal cells would be suppressed by the
MEK inhibitor. These concepts are being examined in clinical trials.
13.4.4 Combining MEK and Bcl-2 Inhibitors
The effects of combining MEK and Bcl-2/Bcl-XL inhibitors have been examined
in preclinical studies with AML cell lines and patient samples [80]. The Bcl-2
inhibitor, ABT-737, was observed to induce ERK activation and Mcl-1 expres-
sion. However, when the ABT-737 inhibitor was combined with the MEK inhibi-
tor PD0325901, a synergistic response was observed in terms of the induction of
cell death both on AML cell lines and on primary tumor cells with the properties
of leukemia stem cells. Furthermore, these studies were also extended into tumor
transplant models with the MOLT-13 cell line, and synergy between ABT-737 and
PD0325901 were also observed in vivo.
13.4.5 ERK Inhibitors
There are at least two ERK molecules regulated by the Raf/MEK/ERK cascade,
ERK1 and ERK2. Little is known about the differential in vivo targets of ERK1
and ERK2. The development of specific ERK1 and ERK2 inhibitors is ongoing
and may be useful in the treatment for certain diseases such as those leukemias
where elevated ERK activation is associated with a poor prognosis (e.g., AML,
ALL) [81]. ERK inhibitors have been described [82].
13.4.5.1 AEZS-131
AEZS-131 has been reported on the Internet to be a highly selective ERK 1/2
inhibitor developed by Aeterna Zentaris and has been examined on human breast
cancer cells.
13.4.5.2 Pyrimidylpyrrole ERK Inhibitors
A novel series of pyrimidylpyrrole ERK inhibitors has been developed at Vertex

Pharmaceuticals [82]. A lead compound, ERKi, has been evaluated for its abil-
ity to overcome MEK inhibitor resistance [83]. These studies performed in breast
and CRCs demonstrated that dual inhibition of MEK and ERK by small molecule
inhibitors was synergistic. Furthermore, inhibition of both MEK and ERK acted
to suppress the emergence of resistance and overcome the acquired resistance to
MEK inhibitors in these breast and CRC cell line models.
13.4.5.3 SCH772984
SCH772984 is reported to be an ERK inhibitor.
13.4.6 PI3K/Akt/mTOR Inhibitors
Numerous PI3K, Akt, mTOR, and dual PI3K/mTOR inhibitors have been devel-
oped and evaluated. The PI3K and mTOR inhibitors have been used in basic sci-
ence studies for years and have provided much information about the role of the
PI3K/Akt/mTOR pathway in many biologic and diseases processes. We will focus
on the newer inhibitors of this pathway and how they are now being used in clini-
cal trials.
13.4.6.1 PX-866
The modified wortmannin PX-866 has been evaluated as a PI3K inhibitor [84]. It
is being evaluated in phase II clinical trials for patients with advanced metastatic
prostate cancer by Oncothyreon.
13.4.6.2 GDC-0941
GDC-0941 is a PI3K inhibitor developed by Genentech. GDC-0941 inhibited

the metastatic characteristics of thyroid carcinomas by targeting both PI3K and
hypoxia-inducible factor 1α (HIF-1α) pathways [85]. GDC-0941 synergized with
the MEK inhibitor UO126 in inhibiting the growth of NSCLC [86]. It is being
evaluated in a clinical trial for advanced cancers or metastatic breast cancers
which are resistant to aromatase inhibitor therapy.
13.4.6.3 IC87114
IC87114 is a selective p110δ PI3K inhibitor. It decreased cell proliferation and

survival in AML cells and increased sensitivity to etoposide [87–90].
13.4.6.4 CAL-101
CAL-101(GS-1101) is a derivative of IC87114 [91–93]. CAL-101 is an oral p110δ

PI3K inhibitor developed by Calistoga Pharmaceuticals and Gilead Sciences.
CAL-101 is currently undergoing clinical evaluation in patients with various

hematopoietic malignancies including relapsed or refractory indolent B-cell NHL,
mantle cell lymphoma or CLL. An additional clinical trial will examine the effects
of combining CAL-101 with chemotherapeutic drugs and the αCD20 monoclonal
Ab (MoAb).
13.4.6.5 XL-147 (SAR245408)
XL-147 (SAR245408) is a PI3K inhibitor developed by Exelixis/Sanofi-Aventis

[94]. It is in clinical trials, either as a single agent or in combination with erlotinib,
hormonal therapy, chemotherapy, or MoAb therapy for various cancers including
lymphoma, breast, endometrial, glioblastoma, astrocytoma, or other solid cancers.
13.4.6.6 Novartis PI3K Inhibitors
NVP-BKM120 is an orally available pan-class I PI3-kinase inhibitor developed by

Novartis [95]. It is in many clinical trials, either as a single agent or in combi-
nation with other drugs or signal transduction inhibitors [96]. NVP-BKM120 is
clinical trial with patients having advanced cancers such as CRC, NSCLC, breast,
prostate, endometrial, squamous cell carcinoma of the head and neck, GIST, RCC,
melanoma, and advanced leukemias.
NVP-BYL719 (BYL719) is a PI3Kα-selective inhibitor developed by Novartis.
It is in clinical trials for patients with advanced solid tumors, some containing
mutations at PIK3CA. It is also being examined in a clinical trial in combination
with the MEK-162 inhibitor for patients with advanced CRC, esophageal, pancre-
atic, NSCLC, or other advanced solid tumors containing RAS or BRAF mutations.
13.4.7 Dual PI3K/mTOR Inhibitors
The catalytic sites of PI3K and mTOR share a high degree of sequence homol-
ogy. This feature has allowed the synthesis of ATP-competitive compounds that
target the catalytic site of both PI3K and mTOR. Several dual PI3K/mTOR inhibi-
tors have also been developed. In preclinical settings, dual PI3K/mTOR inhibitors
displayed a much stronger cytotoxicity against leukemic cells than either PI3K
inhibitors or allosteric mTOR inhibitors, such as rapamycin and its derivatives
(rapalogs). In contrast to rapamycin/rapalogs, dual PI3K/mTOR inhibitors tar-
geted both mTOR complex 1 and mTOR complex 2 and inhibited the rapamycin-
resistant phosphorylation of eIF4B-1 and inhibited protein translation of many
gene products associated with oncogenesis (enhanced proliferation) in leukemic
cells. The dual inhibitors strongly reduced the proliferation rate and induced an
important apoptotic response [16].
The kinase selectivity profile of the dual PI3K/mTOR modulators is consist-

ent with the high sequence homology and identity in the ATP-catalytic cleft
of these kinases. Dual PI3K/mTOR inhibitors have demonstrated significant,
concentration-dependent cell proliferation inhibition and induction of apopto-

sis in a broad panel of tumor cell lines, including those harboring PI3K p110α
(PIK3CA) activating mutations [97].
Moreover, the in vitro activity of these ATP-competitive PI3K/mTOR modula-
tors has translated well in in vivo models of human cancer xenografted in mice.
They were well tolerated and achieved disease stasis or even tumor regression
when administered orally [98]. In spite of their high lipophilicity and limited water
solubility, the pharmacological, biologic, and preclinical safety profiles of these
dual PI3K/mTOR inhibitors supported their clinical development.
There may be some benefits to treating patients with an inhibitor that can tar-
get both PI3K and mTOR as opposed to treating patients with two inhibitors, that
is, one targeting PI3K and another specifically mTOR. An obvious benefit could
be lowered toxicities. Treatment with a single drug could have fewer side effects
than treatment with two separate drugs. The effects of detrimental Akt activation
by mTOR inhibition might be avoided upon treatment with a dual kinase inhibi-
tor. Furthermore, the negative side effects of mTOR inhibition on the activation
of the Raf/MEK/ERK pathway might be eliminated with the PI3K inhibitor activ-
ity in the dual inhibitor. There remains, however, considerable uncertainty about
potential toxicity of compounds that inhibit both PI3K and mTOR enzymes whose
activities are fundamental to a broad range of physiological processes. Although
it should be pointed out that there are some clinical trials in progress to determine
whether it is beneficial to treat cancer patients with a PI3K/mTOR dual inhibitor
and an mTORC1 blocker such as NVP-BEZ235 and RAD001, preclinical studies
have documented the benefits of combining RAD001 with NVP-BEZ235 [99].
13.4.7.1 PI-103
PI-103 was the first reported ATP-competitive kinase inhibitor of mTOR which
also blocked the enzymatic activity of PI3K p110 isoforms. It was developed at
UCSF in 2006. PI-103 exhibits good selectivity over the rest of the human kinome
in terms of non-selective inhibition of other kinases [100, 101]. PI-103 is a pan-
class I PI3K inhibitor with IC50 values in the 2 nm (p110α PI3K) to 15 nm range
(p110γ PI3K) PI-103 inhibits both mTORC1 (IC50 = 0.02 μm) and mTORC2
(IC50 = 0.083 μm).
13.4.7.2 Novartis Dual PI3K/mTOR Inhibitors
NVP-BEZ235 is a dual PI3 K/mTOR inhibitor developed by Novartis. Importantly

and in contrast to rapamycin, NVP-BEZ235 inhibited the rapamycin-resistant
phosphorylation of 4E-BP1, causing a marked inhibition of protein translation
in AML cells. This resulted in the reduced levels of the expression of c-Myc,
cyclin D1, and Bcl-xL known to be regulated at the translation initiation level
[102]. NVP-BEZ235 suppressed proliferation and induced an important apoptotic
response in AML cells without affecting healthy CD34+ cell survival. Importantly,
it suppressed the clonogenic activity of leukemic, but not healthy, CD34+ cells
[103]. NVP-BEZ235 targeted the side population (SP) of both T-ALL cell lines
and patient lymphoblasts, which might correspond to Leukemia-Initiating Cells
(LIC), and synergized with several chemotherapeutic agents (cyclophosphamide,
cytarabine, dexamethasone) currently used for treating T-ALL patients [104].
Also, NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells
by co-culturing with MS-5 stromal cells, which mimic the bone marrow micro-
environment [105]. In this study, NVP-BEZ235 was cytotoxic to T-ALL patient
lymphoblasts displaying pathway activation, where the drug dephosphorylated
4E-BP1, in contrast to the results with obtained rapamycin. Taken together, these
findings indicated that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR
network with NVP-BEZ235, either alone or in combination with chemothera-
peutic drugs, may be an effective therapy for of those T-ALLs that have aberrant
upregulation of this signaling pathway.
NVP-BEZ235 has been evaluated also in a mouse model consisting of
BA/F3 cells overexpressing either WT BCR-ABL or its imatinib-resistant BCR-
ABL mutants (E255K and T315I) [106]. NVP-BEZ235 inhibited proliferation of
both cytokine-independent WT BCR-ABL and mutant BCR-ABL (E255K and
T315I) overexpressing cells, whereas parental cytokine-dependent Ba/F3 cells
were much less sensitive. The drug also induced apoptosis and inhibited both
mTORC1 and mTORC2 signaling. Remarkably, the drug displayed cytotoxic
activity in vivo against leukemic cells expressing the E255K and T315I BCRABL
mutant forms. However, in this experimental model, NVP-BEZ235 induced an
overactivation of MEK/ERK signaling, most likely due to the well-known com-
pensatory feedback mechanism that involves p70S6K [39]. NVP-BEZ235 has
been intensively investigated and is in clinical trials for patients with advanced
cancers [107]. In some trials, NVP-BEZ235 is being evaluated in combination
with either paclitaxel or trastuzumab (herceptin). NVP-BTG226 is a recently
developed PI3K/mTOR inhibitor [15].
13.4.7.3 Pfizer Dual PI3K/mTOR Inhibitors
PKI-587, also known as PF-05212384, inhibited class I PI3Ks, PI3Kα mutants,

and mTOR. PKI-587-suppressed proliferation of approximately 50 diverse human
tumor cell lines at IC50 values less than 100 nmol/L. PKI-587-induced apopto-
sis in cell lines with elevated PI3K/Akt/mTOR signaling. PKI-587 inhibited the
tumor growth in various models including breast (MDA-MB-361, BT474), colon
(HCT116), lung (H1975), and glioma (U87MG). The efficacy of PKI-587 was
enhanced when administered in combination with the MEK inhibitor, PD0325901,
the topoisomerase I inhibitor, irinotecan, or the HER2 inhibitor, neratinib [108].
PF-04691502 is an ATP-competitive PI3K/Akt inhibitor which suppresses the

activation of Akt. PF-04691502 suppressed the transformation of avian cells in
response to either WT or mutant PIK3CA. PF-04691502 inhibited tumor growth in
various xenograft models including U87 (PTEN null), SKOV3 (PIK3CA mutation)
and gefitinib (EGFR inhibitor) and erlotinib-resistant NSCLC [109]. Both PKI-
587 and PF-04691502 are in clinical trials to treat endometrial cancers.
PKI-402 is a selective, reversible, ATP-competitive, PI3K and mTOR inhibitor. It
suppress PI3Ks, PI3Kα mutant, and mTOR equally. PKI-402 inhibited the growth of
many human tumor cell lines including breast, glioma, pancreatic, and NSCLC [110].
13.4.7.4 XL765
XL765 (SAR25409) is a dual PI3K/mTOR inhibitor developed by Exelixis/Sanofi-

Aventis. XL765 has been investigated in brain and pancreatic cancer models either
as a single agent or in combination with temozolomide [111] or the autophagy
inhibitor chloroquine [112]. XL765 downregulated the phosphorylation of Akt
induced by PI3K/mTORC2 and reduced brain tumor growth [111]. Combining
XL765 with chloroquine suppressed autophagy and induced apoptotic cell death in
pancreatic tumor models [112]. Clinical trials are being performed with XL765 in
combination with temozolomide to treat patients with glioblastoma or in combina-
tion with erlotinib to treat NSCLC patients.
13.4.7.5 Genentech Dual PI3K/mTOR Inhibitors
GNE-477 is a dual PI3K/mTOR inhibitor developed by Genentech [113]. GDC-0980

is similar to GNE-477 and has been shown to have high activity in cancer models
driven by PI3K pathway activation [114]. GDC-0980 is in a clinical trial for patients
with advanced cancers or metastatic breast cancers which are resistant to aromatase
inhibitor therapy.
13.4.7.6 GSK Dual PI3K/mTOR Inhibitors
GSK2126458 is a dual PI3K/mTOR inhibitor developed by GSK [52]. It is in at least

two clinical trials with advanced cancer patients. In one trial, it is being combined with
the MEK inhibitor GSK1120212. GSK1059615 is a dual PI3K/mTOR inhibitor devel-
oped by GSK. It was in a clinical trial with patients with solid tumors, metastatic breast
cancer, endometrial cancers, and lymphomas which was terminated.
13.4.7.7 WJD008
WJD008 (Chinese Academy of Sciences, Shanghai) is a dual PI3K/mTOR [115].

WJD008 inhibited the increased activity of the PI3K pathway normally induced
by PIK3CA H1047R and suppressed proliferation and colony formation of

transformed RK3E cells containing PIK3CA H1047R.
13.4.8 PDK Inhibitors
Some compounds have been reported to be PDK inhibitors, including the osteo-
arthritis drug celecoxib [116], the modified celecoxib, OSU-03012 [76, 117], and
2-O-BN-InsP(5) [118]. Celecoxib (Celebrex, Pfizer) obviously has other targets
than PDK, such as cyclooxygenase-2 (Cox-2). Celecoxib is used to treat CRC
patients to reduce the number of polyps in the colon. OSU-03012 is reported
not to inhibit Cox-2 [117]. 2-O-BN-InsP(5) is based on the structure of inositol
1,3,4,5,6-pentakisphosphate, it may inhibit both PDK and mTOR [118].
13.4.9 Akt Inhibitors
Many attempts to develop Akt inhibitors have been performed over the years. In
many of the earlier attempts, the various Akt inhibitors either lacked specificity or
had deleterious side effects. Part of the deleterious side effects is probably related
to the numerous critical functions that Akt plays in normal physiology. Namely,
some Akt inhibitors will alter the downstream effects of insulin on Glut-4 translo-
cation and glucose transport.
13.4.9.1 Triciribine
Triciribine (API-2) is an Akt inhibitor that has been used in many studies: at least
92 are listed on PubMed. Triciribine suppressed the phosphorylation of all three
Akt isoforms in vitro and the growth of tumor cells overexpressing Akt in mouse
xenograft models [119]. The mechanism(s) by which triciribine inhibits Akt activ-
ity are not clear. The drug has been evaluated in a phase I clinical trial in patients
with advanced hematologic malignancies, including refractory/relapsed AML. In
this trial, triciribine was administered on a weekly schedule. The drug was well
tolerated, with preliminary evidence of pharmacodynamic activity as measured by
decreased levels of activated Akt in primary blast cells [120]. Triciribine has also
been examined in clinical trial with Akt+ metastatic cancers.
13.4.9.2 MK-2206
MK-2206 (Merck) is an allosteric Akt inhibitor which inhibits both T308 and
S473 phosphorylation. It also inhibits the downstream effects of insulin on Glut-4
translocation and glucose transport [121]. MK-2206 decreased T-acute lympho-

cytic leukemia (T-ALL) cell viability by the cells in the G0/G1 phase of the cell
cycle and inducing apoptosis. MK-2206 also induced autophagy in the T-ALL
cells. MK-2206 induced a concentration-dependent dephosphorylation of Akt
and its downstream targets, GSK-3α/β and FOXO3A. MK-2206 also was cyto-
toxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset
(CD34+/CD4−/CD7−) which is enriched in LICs. [122]. MK-2206 is in at least 43
clinical trials either as a single agent or in combination with other small molecule
inhibitors or chemotherapeutic drugs with diverse types of cancer patients.
13.4.9.3 GSK Akt Inhibitors
GSK690693 is a pan-Akt inhibitor developed by GSK. GSK690693 is an ATP-

competitive inhibitor effective at the low nanomolar range. Daily administration of
GSK690693 resulted in significant anti-tumor activity in mice bearing various human
tumor models including SKOV-3 ovarian, LNCaP prostate and BT474 and HCC-1954
breast carcinoma. The authors also noted that GSK690693 resulted in acute and tran-
sient increases in blood glucose level [123]. The effects of GSK690693 were also exam-
ined 112 cell lines representing different hematologic neoplasia. Over 50 % of the cell
lines were sensitive to the Akt inhibitor with an EC50 of less than 1 μm. ALL, non-
Hodgkin lymphomas, and Burkitt lymphomas exhibited 89, 73, and 67 % sensitivity to
GSK690693, respectively. Importantly, GSK690693 did not inhibit the proliferation of
normal human CD4+ peripheral T lymphocytes as well as mouse thymocytes.
GSK2141795 is a GSK Akt inhibitor under development. It is reported by GSK
to be an oral, pan-Akt inhibitor which shows activity in various cancer models,
including blood cancer and solid tumor models. In addition, it is reported by GSK
to delay tumor growth in solid tumor mouse xenograft models. It has been investi-
gated further in clinical trials.
13.4.9.4 KP372-1
KP372-1 inhibits PDK1, Akt, and Fms-like tyrosine kinase 3 (Flt-3) signaling and
induces mitochondrial dysfunction and apoptosis in AML cells but not normal
hematopoietic progenitor cells [124]. It also suppressed colony formation of pri-
mary AML patient sample cells but not normal hematopoietic progenitor cells. It
has also been investigated in other cancer types, including squamous cell carcino-
mas of the head and neck, thyroid cancers, and glioblastomas.
13.4.9.5 Enzasturin
Enzasturin (LY317615) is a protein kinase C-β (PKC-β) and Akt inhibitor devel-
oped by Lilly. It has been investigated in clinical trials either by itself or in
combination with other agents in various types of cancer patients including brain
[125] and NSC [126], CRC [127] as well as other cancer types. It is reported to be
in approximately 48 clinical trials on the ClinicalTrials.gov website.
13.4.9.6 Perifosine
Perifosine (KRX-0401, Keryx/AOI Pharmaceuticals, Inc., and licensed to AEterna

Zentaris) is an alkylphospholipid that can inhibit Akt [128]. The effects of perifosine
have been examined on many different tumor types. Perifosine induces caspase-depend-
ent apoptosis and downregulates P-glycoprotein expression in multi-drug-resistant
T-ALL cells by a JNK-dependent mechanism [104]. Perifosine is or has been in at
least 43 clinical trials to treat various cancer patients, with either blood cancers or solid
tumors, either by itself, or in combination with other agents. It has advanced to phase III
clinical trials for CRC and MM. In the USA, it has orphan drug status for the treatment
for MM and neuroblastoma.
13.4.9.7 Erucylphosphocholine and Erucylphosphohomocholine
Erucylphosphocholine (ErPC) and Erucylphosphohomocholine (ErPC3) have

been shown to inhibit Akt and induce apoptosis in malignant glioma cell lines
which are normally resistant to the induction of apoptosis. They are structurally
related to perifosine [129]. ErPC enhanced radiation-induced cell death and clo-
nogenicity [130]. These effects on the induction of apoptosis were correlated with
increased Bim levels and decreased Bad and Foxo-3 phosphorylation, potentially
consequences of decreased Akt activity. ErPC3 is the first intravenously applicable
alkylphosphocholine. ErPC3 was cytotoxic to AML cells through JNK2- and PP2-
dependent mechanisms [131].
13.4.9.8 PBI-05204
PBI-05204 (oleandrin) is an Akt inhibitor. PBI-05024 is a botanical drug candidate

derived from Nerium oleander and developed by Phoenix Biotechnology. It also
has other targets including FGF-2, NF-κB, and p70S6K. PBI-05204 is in clinical
trials for cancer patients with advanced solid tumors [132]. Interestingly, PBI-
05204 also provides significant neuroprotection to tissues damaged by glucose and
oxygen deprivation which occurs in ischemic stroke [133].
13.4.9.9 RX-0201
RX-0201 (Akt1AO, Rexahn Pharmaceuticals, Inc.) is an Akt-1 antisense oligo-

nucleotide molecule. RX-0201 downregulated Akt-1 expression at nanomolar
concentrations in multiple types of human cancer cells. RX-0201 also inhibited

tumor growth in mice xenografted with U251 human glioblastoma and MIA
human pancreatic cancer cells [134]. RX-021 is in a clinical trial in combination
with gemcitabine for patients with metastatic pancreatic cancer [135].
13.4.9.10 XL-418
XL-418 is reported to be a dual Akt/p70S6K inhibitor by developed by

Exelixis/GSK. It was in clinical trials for patients with advanced cancer; however,
those trials were suspended.
13.4.10 mTORC1 Inhibitors
Rapamycin (Rapamune, Pfizer) was approved by the FDA in 1999 to prevent

transplant rejection in organ transplant patients. Rapamycin/rapalogs act as allos-
teric mTORC1 inhibitors and do not directly affect the mTOR catalytic site [15].
They associate with the FK506-binding protein 12 (FKBP-12) and by so doing,
they induce disassembly of mTORC1, resulting in the repression of its a ctivity
[136, 137]. The rapalogs have been examined in clinical trials of various can-
cers including brain, breast, HCC, leukemia, lymphoma, MM, NSCLC, pancre-
atic, prostate, and RCC [138, 139]. The rapalogs Torisel (Pfizer) and Afinitor
(Novartis) were approved in 2007 and 2009 (respectively) to treat RCC patients
[140]. In 2008, Torisel was approved to treat Mantle cell lymphoma patients. In
2010, Afinitor was approved to treat subependymal giant cell astrocytoma (SEGA)
tumors in tuberous sclerosis (TS) patients. In 2011, Afinitor was approved to treat
patients with pancreatic neuroendocrine tumors [141]. Ridaforolimus (also known
as AP23573 and MK-8669; formerly known as deforolimus) is a rapalog devel-
oped by ARIAD and Merck. Ridaforolimus has been evaluated in clinical trials
with patients having metastatic soft-tissue or bone sarcomas where it displays
promising results in terms of the risk of progression or death [142]. Recently, the
ability of rapamycin and rapalog to treat various viral infections including AIDS
has been considered [143, 144]. Clearly, rapamycin has proven to be a very useful
drug.
13.4.11 mTOR Inhibitors
Small molecules designed for inhibiting the catalytic site of mTOR have shown
promising effects on the suppression of signaling downstream of mTOR. mTOR
kinase inhibitor has been developed which directly inhibits mTORC1 and
mTORC2. The mTOR kinase inhibitors have advantages over rapamycin and the
rapalogs as mTOR inhibitors will inhibit both mTORC1 and mTORC2, while
rapamycin and the rapalogs only inhibit mTORC1. Also, the mTOR kinases inhib-
itors do not induce the feedback pathways which result in Akt activation. In vitro
studies with purified mTOR and PI3K proteins have demonstrated that the mTOR
inhibitors selectively bind mTOR more than PI3K.
13.4.11.1 OSI-027
OSI-027 is a pan-TOR inhibitor developed by OSI Pharmaceuticals/Astellas

Pharma Inc. OSI-027 has been shown to be effective in inducing apoptosis in dif-
ferent types of cancer, including breast and leukemias [145, 146]. OSI-027 has
been shown to inhibit the growth of imatinib-resistant CML cells which contain
the BCR-ABL T315I mutation that are resistant to all BCR-ABL inhibitors [147].
OSI-027 has been evaluated in clinical trials with patients with advanced solid
tumors and lymphoma [148].
13.4.11.2 Intelllikine mTOR Inhibitors
PP-242 is a potent inhibitor of both mTORC1 and mTORC2. INK-128 is a deriva-

tive of PP-242 which has shown anti-tumoral effects on multiple cancer types
including RCC, MM, NHL, and prostate [149, 150]. INK-128 is in phase I clinical
trials for patients with relapsed or refractory multiple myeloma or Waldenstrom’s
macroglobulinemia or patients with solid malignancies.
13.4.11.3 AstraZenica mTOR Inhibitors
AZD8055 and AZD2014 are pan-mTOR inhibitors with potent anti-tumor activity
[151]. They are being evaluated in clinical trials patients with gliomas who have not
responded to standard glioma therapies as well as patients with other types of cancer.
13.4.11.4 Palomid 529
Palomid 529 (Paloma Pharmaceuticals) is a pan-mTOR inhibitor which has potent

anti-tumor affects and reduces tumor angiogenesis and vascular permeability
[152]. Palomid 529 is undergoing phase I clinical trials for patients with macular
degeneration.
13.4.11.5 Pfizer mTOR Inhibitors
WAY600, WYE353, WYE687, and WYE132 were developed by Wyeth (Pfizer).

These inhibitors were derived from WAY001 which was more specific for PI3Kα
than either mTORC1 or mTORC2. These inhibitors were modified which resulted
in WYE132 (WYE125132)/WYE132 has 5000-fold greater selectivity for mTOR
over PI3K. It caused tumor regression in breast, glioma, lung, renal tumors [153].
13.4.11.6 Other mTOR Inhibitors
Many other TOR inhibitors have been described which include Ku0063794
(KuDOS Pharmaceuticals) [154] and OXA-01 (OSI Pharmaceuticals) [155]. Torin2
has been developed by optimizing from Torin1 [156]. TORKiCC223 is a pan-TOR
inhibitor developed by Celgene. Other companies are developing mTOR inhibitors;
clearly, this is a very competitive but important research and clinical area.
13.5 Increasing the Effectiveness of Targeting the Raf/

MEK/ERK and PI3K/PTEN/Akt/mTOR Pathways
by Simultaneous Treatment with Two Pathway
Inhibitors
In the following section, we discuss the potential of combining inhibitors that tar-
get two pathways to more effectively limit cancer growth. Treatment for inducible
murine lung cancers containing KRAS and PIK3CA mutations with PI3K/mTOR
(NVP-BEZ235) and MEK (selumetinib) inhibitors led to an enhanced response
[157]. Synergistic responses between sorafenib and mTOR inhibitors were
observed in xenograft studies with a highly metastatic human HCC tumor [158].
Some recent studies in thyroid cancer have documented the benefit of combining
Raf and PI3K/mTOR inhibitors [159].
Intermittent dosing of MEK and PI3K inhibitors has been observed to suppress
the growth of tumor xenografts in mice [74]. This study demonstrated that con-
tinuous administration of MEK and PI3K inhibitors is not required to suppress
xenograft growth. These important results were obtained by performing washout
studies in vitro and alternate dosing schedules in mice with MEK and PI3K inhibi-
tors with cancer cells having mutations at BRAF and KRAS.
The combined effects of inhibiting MEK with PD-0329501 and
mTOR with rapamycin or its analog, the rapalog AP-23573 (ARIAD
Pharmaceuticals/Merck) were examined in human NSCLC cell lines, as
well as in animal models of human lung cancer. PD-0325901 and rapamy-
cin demonstrated synergistic inhibition of proliferation and protein translation.
Suppression of both MEK and mTOR inhibited ribosomal biogenesis and was
associated with a block in the initiation phase of translation [160]. The pan-
TOR inhibitor AZD-8055 has been examined as a single agent and in combina-
tion with the MEK inhibitor selumetinib in a NSCLC xenograft and increased
cell death and tumor regression [151, 161]. These preclinical results support the
suppression of both the MEK and mTOR pathways in lung cancer therapy and
indicate that both pathways converge to regulate the initiation of protein transla-
tion. ERK phosphorylates Mnk1/2 and p90Rsk, which regulate the activity of the
eukaryotic translation initiation factor eIF4E. The phosphorylation of 4EBP1 is
altered in cells containing BRAF mutations. It should also be pointed out that
4EBP1 is also regulated by Akt, mTOR, and p70S6K. This may result in the effi-
cient translation of certain mRNAs in BRAF-mutant cells. This could explain
how co-inhibition of MEK and PI3K/Akt/mTOR synergizes to inhibit protein
translation and growth in certain lung cancer cells.
13.6 Clinical Trials Based on Inhibiting Both the Raf/MEK/

ERK and PI3K/PTEN/Akt/mTOR Pathways
Combinations of Raf and PI3K/Akt/mTOR or MEK and PI3 K/Akt/mTOR

inhibitors are in clinical trials. The results of a phase 1 clinical trial on patients
with advanced solid tumors indicate that the combined dosing appears to be well
tolerated, at least as well as single agent dosing. Some anti-tumor effects were
observed, and dose-escalation trials were performed [162]. Clinical trial com-
bining MEK and Akt inhibitors (GSK1120212 and GSK2141795, respectively)
is in progress. A clinical trial for patients with advanced cancers combining the
PI3K/mTOR inhibitors (PF-04691502 and PF-05212384) with the MEK inhibi-
tor (PD-0325901) or irinotecan is in progress. The study will include patients
with metastatic CRC patients who have received previous therapy for their dis-
ease and whose cancers have a mutant KRAS gene. The dual PI3 K/mTOR inhib-
itor NVP-BEZ235 is in a combination clinical trial with RAD001 (everolimus)
in patients with advanced solid cancers. A phase 1 clinical trial is in progress
combining the MEK1/2 inhibitor MEK162 and the PI3K/mTOR dual inhibitor
NVP-BEZ235. This combination will be evaluated in various cancer patients,
for example, NSCLC with mutations at EGFR who have progressed after treat-
ment with EGFR inhibitors, triple-negative breast, CRC, melanoma, and pan-
creatic cancers. In addition, patients with other advanced solid tumors with
KRAS, NRAS, and/or BRAF mutations will be included in the study. A trial is
underway testing the effects of combining two experimental drugs, SD703026
(MSC1936369B) (a MEK inhibitor) and XL755 SAR245409 (a PI3K/mTOR
inhibitor) for the treatment for locally advanced or metastatic solid tumors.
Patients with breast, NSCLC, melanoma, and colorectal cancers will be treated
with this inhibitor combination. A clinical trial is examining the effects of com-
bining MK-2206 (an Akt inhibitor) with selumetinib (a MEK inhibitor) in cancer
patients with advanced solid tumors. A combination clinical trial combining the
MEK inhibitor selumetinib and the Akt inhibitor MK-2206 in patients with stage
III or stage IV melanoma that previous failed after treatment with vemurafenib
or dabrafenib is in progress.
13.7 Trials Based on Combining Raf/MEK,

PI3K/Akt/mTOR Inhibitors with Chemotherapy
or MoAbs
Treatment of mice xenografted with vemurafenib-resistant BRAF-mutant CRCs

with various combinations of vermurafenib and chemotherapeutic drugs (capecit-
abine, irinotecan), MoAbs [bevacizumab (avastin, targets VEGFα), cetuximab
(erbitux, targets EGFR)], the Akt inhibitor MK-2206, or the EGFR inhibitor erlo-
tinib, increased survival [163]. Combination of the Akt inhibitor MK-2206 and
either EGFR/HER2-targeted therapy [erlotinib or lapatinib (tykerb, a dual EGFR
and HER2 inhibitor from GSK)] or chemotherpapeutic drugs doxorubicin, camp-
tothechin, gemcitabine, 5-flurouracil, docetaxel or carboplatin resulted in synergis-
tic responses in lung (NCI-H460) and ovarian (A2780) cancer cell lines. In some
cases, the timing of drug addition was determined to be important as MK-2206
suppressed the Akt activation induced by carboplatin and gemcitabine [164].
The effects of combining the dual PI3K/mTOR inhibitor NVP-BEZ235 and vari-
ous chemotherapeutic drugs as well as other targeted therapies are being exam-
ined (doxorubicin, melphalan, vincristine, bortezomib) [165, 166]. The anti-tumor
effects of WYE132 (a mTOR inhibitor) could be enhanced upon combination with
bevacizumab in lung and breast xenograft models [153]. A clinical trial with INK-
128 in combination with paclitaxel, either in the absence or in the presence of tras-
tuzumab, is in progress in patients with advanced solid malignancies.
Clinical trials are ongoing based on combining the dual PI3K/mTOR inhibi-
tor NVP-BEZ235 with PI3K and MEK inhibitors (BKM120, MEK162) and
chemotherapeutic drugs (paclitaxel, trastuzumab) to treat advanced solid cancers
and metastatic breast cancers which are difficult to treat (see below). BKM120
is a pan-PI3 K inhibitor. It is being included in some clinical studies since NVP-
BEZ235 does not inhibit PI3Kβ [167]. Furthermore, NVP-BEZ235 is not effective
in suppressing the growth of tumors which have the KRAS G12D mutation [157].
Thus, to achieve effective suppression of cancer growth in some situations, it may
be important to combine PI3K/mTOR inhibitors with pan-PI3K inhibitors.
Palomid 529, a pan-mTOR inhibitor, is in some circumstances is effective as a
single agent. However, when Palomid 529 was combined with either cisplatin or
docetaxel, it had a better effect on hormone-refractory prostate cancers [168]. It
also improved the effects of radiotherapy on prostate cancer cells [169].
The effects of inhibiting Akt in combination with other pathways, inhibitors,
and chemotherapy are being evaluated in numerous phase I clinical trials. These
trials highlight the importance of targeting multiple molecules to suppress the
growth of cancer which are resistant to most therapies. Combination clinical tri-
als with the Akt inhibitor MK-2206 and the dual EGFR/HER2 inhibitor lapatinib
are in progress with patients having advanced or metastatic solid tumors or breast
cancer patients. The effects of combining MK-2206 and erlotinib, docetaxel, or
carboplatin + paclitaxel are being examined in clinical trials in certain patients
with advanced cancers. Clinical trials with NSCLC patients are underway to
examine the effects of combining MK-2206 with gefitinib (iressa, EGFR inhibitor
AstraZenica). Clinical trials with postmenopausal metastatic breast cancer patients
are in progress to examine the effects of combining anastrozole, letrozole, exemes-
tane (aromatase-inhibitors), or fulvestrant (an estrogen receptor antagonist).
Clinical trials are also underway examining the effects of combining MK-2206
with bendamustin (nitrogen mustard alkylating agent) and Rituximab (chimeric
monoclonal antibody targeting CD20 from IDEC Pharmaceuticals/Genentech)
on CLL cancer patients who have relapsed or on cancer patients with small
lymphocytic lymphoma. Clinical trials combining MK-2206 and various other
drugs including dalotuzumab (a MoAb which targets IGF-1R from Merck) and
MK-0752 (a Y-secretase inhibitor which inhibits the NOTCH pathway from
Merck) are in progress. The effects of MK-8669 (ridaforolimus an mTORC1
inhibitor from Merck) and dalotuzumab are being examined in patients with
advanced cancers. Clinical trials combining MK-2206 and paclitaxel in cancer
patients with locally advanced, metastatic solid tumors, or metastatic breast can-
cers are in progress. The above mentioned clinical trials document the importance
of targeting Akt and other signaling molecules as well as critical targets involved
in cellular division. Furthermore, the clinical trials document how basic research
on these pathways is being translated into clinical therapy for cancer and other
types of patients.
13.8 Enhancing the Effectiveness of Raf/MEK

and PI3K/mTOR Inhibitors with Radiotherapy
Radiotherapy is a common therapeutic approach for treatment for many diverse

cancers. A side effect of radiotherapy in some cells is induction of the Ras/Raf/
MEK/ERK cascade [2]. Various signal transduction inhibitors have been evaluated
as radiosensitizers. The effects of pre-treatment for lung, pancreatic, and prostate
cancer cells with selumetinib were evaluated in vitro using human cell lines and
in vivo employing xenografts [170]. The MEK inhibitor treatment radiosensitized
the various cancer cell lines in vitro and in vivo. The MEK inhibitor treatment was
correlated with decreased Chk1 phosphorylation 1–2 h after radiation. The authors
noticed the effects of the MEK inhibitor on the G2 checkpoint activation after irra-
diation, as the MEK inhibitor suppressed G2 checkpoint activation. Since ERK1/
ERK2 activity is necessary for the carcinoma cells to arrest at the G2 checkpoint,
suppression of phosphorylated Chk1 was speculated to lead to the abrogated G2
checkpoint, increased mitotic catastrophe, and impaired activation of cell cycle
checkpoints. Mitotic catastrophe was increased in cells receiving both the MEK
inhibitor and radiation when compared to the solo-treated cells. It was also pos-
tulated in this study that the MEK inhibitor suppressed the autocrine cascade in
DU145 prostate cancer cells that normally resulted from EGF secretion and EGFR
activation. Suppression of this autocrine cascade by the MEK inhibitor may have
served as a radiosensitizer. The other two cancer cell lines examined in this study
(A549 and MiaPaCa2) had KRAS mutations and both were radiosensitized by the
MEK inhibitor. Although these studies document the ability of a MEK inhibitor to
radiosensitize certain cells, clearly other cancer cell lines without activating muta-
tions in the Ras/Raf/MEK/ERK pathway or autocrine growth stimulation should
be examined for radiosensitization by the MEK inhibitor as the KRAS mutation
may also activate the PI3K pathway which could lead to therapy resistance.
PI3K/Akt/mTOR inhibitors will sensitize the tumor vasculature to radiation
both in vitro in cell lines and in vivo in xenografts [171, 172]. mTOR and radia-
tion play critical roles in the regulation of autophagy [173, 174]. When mTOR is
blocked by rapamycin, there is an increase in autophagy. This is important as apop-
totic cell death is a minor component to cell death in many solid tumors. These
studies document the potential beneficial use of combining mTOR inhibitors and
radiation to improve the induction of autophagy in the treatment for solid tumors.
13.9 Conclusions
Inhibitors to the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways.

Initially, MEK inhibitors were demonstrated to have the most specificity. However,
these inhibitors may have limited effectiveness in treating human cancers, unless
the particular cancer proliferates directly in response to the Raf/MEK/ERK path-
way. Moreover, MEK inhibitors are often cytostatic as opposed to cytotoxic; thus,
their ability to function as effective anticancer agents in a monotherapeutic setting
is limited, and they may be more effective when combined with other small mol-
ecule inhibitors, MoAbs, chemo- or radiotherapy. Raf inhibitors have also been
developed, and some are being used to treat various cancer patients (e.g., sorafenib
and vemurafenib). This particular Raf inhibitor also inhibits other receptors and
kinases which may be required for the growth of the particular cancer. This pro-
miscuous nature of sorafenib has contributed to the effectiveness of this particular
Raf inhibitor for certain cancers. Raf inhibitors such as vemurafenib, dabrafenib,
and GDC-0879 are promising for the treatment for melanoma, CRC, thyroid as
well as other solid cancers, and leukemias/lymphomas/myelomas which have
mutations at BRAF V600E. However, problems have been identified with certain
Raf inhibitors as they will be ineffective if Ras is mutated/amplified, if an exon of
BRAF is deleted, if BRAF is amplified, if there are mutations at MEK1 and various
other genetic mechanisms that result in resistance [175]. Combination therapies
with either a traditional drug/physical treatment or another inhibitor that targets a
specific molecule in either the same or a different signal transduction pathway are
also key approaches for improving the effectiveness and usefulness of MEK and
Raf inhibitors.
Modified rapamycins (rapalogs) are being used to treat various cancer patients
(e.g., patients with RCC and some other cancers). While rapalogs are effective
and their toxicity profiles are well known, one inherent property is that they are
not very cytotoxic when it comes to killing tumor cells. This inherent property of
rapamycins may also contribute to their low toxicity in humans.
Mutations at many of the upstream receptor genes or RAS can result in abnor-
mal Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathway activation. Hence,
targeting these cascade components with small molecule inhibitors may inhibit
cell growth. The usefulness of these inhibitors may depend on the mechanism of
transformation of the particular cancer. If the tumor exhibits a dependency on the
Ras/Raf/MEK/ERK pathway, then it may be sensitive to Raf and MEK inhibitors.
In contrast, tumors that do not display enhanced expression of the Ras/Raf/MEK/
ERK pathway may not be sensitive to either Raf or MEK inhibitors, but if the Ras/
PI3K/Akt/mTOR pathway is activated, these cancers may be sensitive to specific
inhibitors that target this pathway. Finally, it is likely that many of the inhibitors
that we have discussed in this review will be more effective in inhibiting tumor
growth in combination with MoAb, cytotoxic chemotherapeutic drugs, or radia-
tion. This is documented by the large number of clinical trials combining signal
transduction inhibitors with these various therapeutic agents.
Some scientists and clinicians have considered that the simultaneous targeting
of Raf and MEK by individual inhibitors may be more effective in cancer therapy
than just targeting Raf or MEK by themselves. This is based in part on the fact
that there are intricate feedback loops from ERK which can inhibit Raf and MEK.
For example, when MEK1 is targeted, ERK1,2 is inhibited, and the negative feed-
back loop on MEK is broken and activated MEK accumulates. However, if Raf
is also inhibited, it may be possible to completely shut down the pathway. This is
a rationale for treatment with both MEK and Raf inhibitors. Likewise, targeting
both PI3K and mTOR may be more effective than targeting either PI3K or mTOR
by themselves. If it is a single inhibitor which targets both molecules, such as the
new PI3K and mTOR dual inhibitors, this becomes a realistic therapeutic option.
Although it should be pointed out that some studies are examining the effects of
combining rapamycins and PI3K/mTOR inhibitors. Finally, an emerging concept
is the dual targeting of two different signal transduction pathways, Raf/MEK/ERK
and PI3K/PTEN/Akt/mTOR, for example. This has been explored in some preclin-
ical models as discussed in the text. The rationale for targeting of both pathways
may be dependent on the presence of mutations in either/or both pathways or in
upstream Ras in the particular cancer which can activate both pathways.
It is not always clear why a particular combination of a signal transduction
inhibitor and chemotherapeutic drug works in one tumor type but not at all in a
different tumor type. This has also been experienced with the development of indi-
vidual chemotherapeutic drugs, some work in some cells but not others. This may
result from many different complex interacting events. Some of these events could
include percentage of cells in different phases of the cell cycle, persistence of can-
cer stem cells, presence of multiple mutated activated oncogenes, or repressed
tumor suppressor genes, epigenetic modifications and many other factors. Finally,
chemotherapeutic drug therapy and other types of therapy (radiotherapy, antibody
therapy) may induce certain signaling pathways (e.g., the reactive oxygen spe-
cies generated by chemotherapy and radiotherapy induce the Ras/Raf/MEK/ERK
pathway). The induction of these signaling pathways may counteract some of the
effects of the signal transduction inhibitors. These effects could indicate that the
timing of each therapy will be important for effective treatments.
In summary, targeted therapy has advanced from basic research studies to the
treatment of cancer patients in less than 25 years. We have learned a lot regard-
ing how specific inhibitors exert their effects and how the Ras/Raf/MEK/ERK and
PI3K/Akt/mTOR pathways function; we still need to discover more about resist-
ance mechanisms and how we can overcome therapeutic resistance and improve
the effectiveness of targeted therapy.
Acknowledgments MC and GM were supported in part by grants from the Italian

“Ministero dell’Istruzione, dell’Università e della Ricerca (Ministry for Education,
Universities and Research)—MIUR” PRIN 2008 and FIRB-MERIT n. RBNE08YYBM. MC
was also supported in part by a grant to the CNR from the Italian Ministry of Economy
and Finance for the Project FaReBio di Qualità. ML was supported in part by a grant from
the Italian Ministry of Health, Ricerca Finalizzata Stemness 2008 entitled “Molecular
Determinants of Stemness and Mesenchymal Phenotype in Breast Cancer”. AMM was
supported in part by grants from Fondazione del Monte di Bologna e Ravenna, MinSan
2008 “Molecular therapy in pediatric sarcomas and leukemias against IGF-IR system: new
drugs, best drug–drug interactions, mechanisms of resistance and indicators of efficacy”,
MIUR PRIN 2008 (2008THTNLC), and MIUR FIRB 2010 (RBAP10447J-003) and 2011
(RBAP11ZJFA_001). MM was supported in part from the Italian Association for Cancer
Research (AIRC), the Cariplo Foundation, and the Italian Ministry of Health. AT was
supported in part by grants from the Italian “Ministero dell’Istruzione, dell’Università e
della Ricerca (Ministry for Education, University and Research) —MIUR—PRIN 2008 and
grant from “Sapienza”, University of Rome 2009–11.
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Chapter 14
Activation of Immune-Mediated Tumor Cell
Death by Chemotherapy
Melanie J. McCoy, Anna K. Nowak and Richard A. Lake
Abstract Like pathogens, tumor cells express a range of antigens that can be

recognized by the immune system and are therefore susceptible to immune-mediated
death. It is widely recognized that the immune system plays a significant role in pre-
venting cancer development through the elimination of malignant and pre-malignant
cells, and in controlling tumor growth, once this protective mechanism has failed.
The capacity of the immune system to contribute to the success of anti-cancer ther-
apy, however, is a more recent concept. Originally assumed to have a detrimental
effect on anti-tumor immunity due to its indiscriminate targeting of proliferating
cells, including lymphocytes, it has now emerged that chemotherapy can enhance
anti-tumor immune responses through altering the level and context of antigen pres-
entation to immune effectors and through altering the immunological milieu, creat-
ing a favorable environment for the generation of anti-tumor immunity. Activation
of immune-mediated tumor cell death by chemotherapy opens the door to the pos-
sibility of novel treatment strategies combining standard chemotherapy with immu-
notherapy agents aimed at enhancing such responses. Preclinical studies and early
phase trials of combination chemoimmunotherapy have produced promising results.
However, it is becoming clear that synergy is dependent not only on the drugs
selected, but also on the intrinsic properties of the host and the tumor. Development
of such combination regimens will therefore require careful design and an individu-
alized approach.
M. J. McCoy (*) · A. K. Nowak · R. A. Lake

School of Medicine and Pharmacology, The University of Western Australia,
Crawley, Western Australia
M. J. McCoy
St John of God Health Care, Subiaco, Western Australia
A. K. Nowak · R. A. Lake
National Centre for Asbestos Related Diseases, Perth, Australia
A. K. Nowak
Department of Medical Oncology, Sir Charles Gairdner Hospital, Nedlands, Western Australia
374 M. J. McCoy et al.
14.1 Introduction
The primary aim of chemotherapy is to eradicate tumor cells through direct

cytotoxicity. However, chemotherapy drugs also impact on the immune system,
and this impact may not be entirely negative as has been widely thought. Instead,
evidence is mounting for an immune-mediated component to chemotherapy-
induced tumor cell death. In this chapter, we will discuss the role of the immune
system in controlling tumor development, the clinical significance of generat-
ing anti-tumor immunity, the mechanisms and characteristics of chemotherapy-
induced activation of immune-mediated tumor cell death, and the implications for
the development of future anti-cancer treatment strategies.
14.2 Cancer and the Immune System
14.2.1 The Three Es of Tumor Development: Elimination,

Equilibrium and Escape
The concept of cancer immunosurveillance, whereby the immune system recognizes

and destroys malignant and premalignant cells to prevent the development of cancer,
was first introduced by Paul Ehrlich in the early twentieth century and has remained
a contentious issue since formally presented to the scientific community by Burnet
over 50 years ago [1]. The theory brought into question the established and easily
understood notion of tolerance to self and protection from non-self. While still not
universally accepted, there is now overwhelming evidence in favor of a complex
and ongoing relationship between the immune system and a developing tumor. This
relationship can be divided into three distinct stages: elimination, equilibrium, and
escape [2] (Fig. 14.1). During the elimination phase, the immune system remains in
control, identifying and destroying all malignant cells, that is, there is effective can-
cer immunosurveillance. If, however, tumor cell variants evolve that are less immu-
nogenic, or active suppression of immune effectors occurs, tumor development
enters the equilibrium phase, where the immune system struggles to maintain the
balance of power. The final phase, escape, occurs when the host anti-tumor immune
response is no longer sufficient and the tumor expands uncontrollably.
14.2.2 Generating an Anti-Tumor Immune Response:

Cross-Presentation of Tumor Antigens
Tumors bear a range of antigens that can be recognized by autologous T cells.

Some are tumor or tumor type-specific, some are shared by several different can-
cers, others are also expressed on normal tissue but display increased or altered
14 Activation of Immune-Mediated Tumor Cell Death by Chemotherapy 375
Elimination: Malignant and pre-malignant cells are recognized

and destroyed by immune effectors
Equilibrium: Balance between the proliferation and immune-

mediated destruction of malignant cells
Escape: Malignant cells overcome immune control and a

tumor develops
Fig. 14.1 Cancer immunosurveillance—the three Es of tumor development
expression on malignant cells [3]. Induction of tumor-specific CD8+ cytotoxic

T lymphocytes (CTL) through the cross-presentation of tumor antigens by den-
dritic cells (DC) is thought to be the primary mechanism by which anti-tumor
immune responses are generated [4–6]. Cross-presentation refers to the pro-
cess whereby exogenous antigen is processed and presented to CD8+ T cells in
association with major histocompatibility complex (MHC) class I, that is, exog-
enous antigen crosses into the MHC class I pathway of professional antigen-pre-
senting cells (APC), which include DC, B cells, and macrophages. MHC class
I molecules, constitutively expressed by all healthy cells except red blood cells,
classically present endogenous peptides derived from proteins inside the cell
to CD8+ T cells. This is known as direct presentation and enables the detection
and elimination of cells synthesizing foreign proteins, such as those infected by
viruses [7]. Exogenous antigens, on the other hand, are usually processed and
presented to CD4+ T cells in association with MHC class II, expressed only by
APC (indirect presentation) [8]. The phenomenon of cross-presentation was first
observed by Bevan and colleagues in the 1970s using mouse strains differing in
minor histocompatibility antigens [9]. Immunization of one mouse strain with
lymphoid cells from the other resulted in the priming of CD8+ T cells restricted
by host MHC class I, which were able to lyse the transferred cells, thus demon-
strating that CTL could be generated in the absence of direct antigen presentation.
Precisely, how exogenous antigen enters the MHC class I presentation pathway
is not completely understood. Currently, the most widely accepted mechanism is
that following internalization into phagosomes, exogenous proteins escape into
the cytosol where they enter the direct presentation pathway, undergoing proteo-
somal degradation into oligopeptides and transportation to the endoplasmic reticu-
lum (ER) via the transporter associated with antigen processing (TAP) for MHC
class I loading (reviewed in [10]). Depending on the context of tumor antigen
cross-presentation, CD8+ T cells are either stimulated to differentiate into CTL,
which migrate to the tumor site and kill tumor cells displaying the relevant anti-
gen via perforin/granzyme-induced apoptosis or ligation of death receptors (cross-
priming), or are tolerized and subsequently deleted (cross-tolerization) [11].
14.2.3 CD8+ T Cells: Key Players in Anti-Tumor Immunity
There are several lines of evidence that suggests CD8+ T cells are critical to the
development of anti-tumor immunity. Depletion of CD8+ T cells prevents the
rejection of chemically or UV-induced tumors, or transplanted tumors formed
in immuno-compromised hosts, all of which are highly immunogenic in normal
mice [12–14]. Mice deficient in the cytolytic molecule perforin are not only more
susceptible to chemically induced tumor formation [15], but also develop spon-
taneous lymphoma in later life [13]. While perforin is required for both CTL and
natural killer (NK) cell cytotoxicity, Smyth et al., demonstrated that CTL were
essential for protection from lymphoma, since tumors that developed in perforin
knockout mice were rejected when transplanted into wild-type mice depleted of
NK cells, but not in those depleted of CD8+ T cells. The efficacy of anti-tumor
immunotherapy has also been shown to be dependent on the presence of CD8+
T cells in a variety of murine models [16–19]. In humans, tumor infiltration by
CD8+ T cells has been associated with improved prognosis in many different can-
cers, as will be discussed in more detail later in this chapter.
The role of CD4+ T cells in generating an anti-tumor immune response is not
so well understood. Traditionally, activated CD4+ T cells have been divided into
two major subsets according to their cytokine secretion profile. T helper 1 (Th1)
cells secrete cytokines usually associated with inflammation including interleu-
kin-2 (IL-2), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and
interleukin-12 (IL-12) and classically promote cell-mediated immunity against
intracellular bacteria and viruses. T helper 2 (Th2) cells generally promote
humoral immunity against extracellular pathogens and secrete IL-10, IL-4, and
transforming growth factor beta (TGFβ) [20, 21] More recently, a third subset of
T helper cells, characterized by secretion of the pro-inflammatory cytokines IL-17
and IL-22, has been defined. Termed Th17 cells, this subset protects against a vari-
ety of pathogens including intracellular and extracellular bacteria [22]. However,
with CD4+ T cells often showing non-classical cytokine expression profiles and
increasing evidence that the tissue in which an immune response occurs, as well
as the pathogen invading it, plays a major role in fine-tuning the class of response
[23], the division of activated CD4+ T cells into distinct helper subsets is perhaps
less meaningful than once thought.
As their name suggests, CD4+ helper T cells support CD8+ T-cell-driven
responses, not only through IL-2 production, which is required for T-cell expan-
sion, but also through the licensing of APC via CD40–CD40L interactions
[24–27]. Interaction of the co-stimulatory molecule CD40 (expressed on APC)
with its ligand CD40L (expressed on CD4+ T cells and mast cells) results in the
production of IL-12 by APC and their upregulation of the co-stimulatory mol-

ecules CD80 and CD86, both of which are required for CD8+ T-cell priming.
Optimal CD8+ T-cell cross-priming is dependent on the presence of CD4+ T cells
in several different animal models [27–29]. CD4+ T cells can also kill tumor cells
directly [30] and indirectly through the activation of eosinophils and macrophages,
which can then lyse tumor cells via superoxide and nitric oxide production [31].
Yet CD4+ T-cell depletion has frequently been found to have no effect on the abil-
ity of mice to reject tumors [14, 16, 18] and has even been shown to enhance the
efficacy of anti-tumor immunotherapy [32]. This paradox may be explained by the
presence of regulatory T cells (Treg), another subset of CD4+ T cells that nega-
tively regulate immune responses. Treg are vital for the maintenance of self-toler-
ance and prevention of immune-mediated pathology following infection [33, 34].
However, Treg also suppress anti-tumor immunity, with their depletion leading to
immune-mediated tumor regression in many animal models (reviewed in [35]).
The role of Th17 cells in anti-tumor immunity is not fully understood, but appears
complex since their signature cytokine IL-17 can promote CD8+ T-cell-driven
anti-tumor immune responses but can also aid angiogenesis and metastasis, pro-
moting tumor growth [36].
There is to date no evidence that B cells are required for the development of an
anti-tumor immune response. In fact, B cells may inhibit the activation and expan-
sion of tumor-specific CTL. Antigen presentation by B cells has been found to
result in the tolerization and subsequent Fas-mediated deletion of CD8+ T cells
[37], while B-cell-deficient mice have been shown to be more resistant to tumor
growth than their wild-type littermates [38]. Qin and colleagues hypothesized that
the negative effect of B cells may be due to competition for tumor-derived antigen,
which would otherwise be taken up by dendritic cells, thereby interfering with
tumor-specific T-cell priming.
14.2.4 Interplay Between Innate and Adaptive Immune

Effectors
T and B lymphocytes, the effectors of cell-mediated and humoral immunity,

respectively, together form the adaptive arm of the immune system and provide a
targeted response to specific antigens. When T and B cells encounter their cognate
antigen and receive appropriate co-stimulatory signals, they become activated and
undergo clonal expansion [39]. However, T- and B-cell clonal expansion and dif-
ferentiation into effector/helper and plasma cells respectively, takes several days.
Adaptive immune responses are therefore not immediate. In contrast, the innate
arm of the immune system, comprised of macrophages, monocytes, NK cells,
and mast cells, provides a rapid, non-specific, first-line defense against pathogens
(reviewed in [40]). It is of note, however, that the identification of cell subsets with
characteristics of both innate and adaptive immune cells, including NKT cells and
γδ T cells, suggests that such a clear division between the two arms of the immune
system may be overly simplistic [41].
Research into immunity against infection has shown that the generation of a
potent adaptive immune response is usually dependent upon the initial stimulation
of a strong innate immune response [42–44]. Consistent with this finding, it is now
becoming clear that the generation of an effective anti-tumor immune response
involves a complex interplay between the innate and adaptive arms of the immune
system. In the early stages of neoplasia, it seems that NK cells play a vital role
in the recognition of cancerous and precancerous cells. NK cells detect cell stress
markers through receptors such as NK group 2 member D (NKG2D), and reduced
MHC class I expression (lack of “self”), both of which are a common feature of
malignant cells. NK cells, like CTL, can exert direct anti-tumor activity through
secretion of perforin and granzymes, and via Fas and tumor necrosis factor-related
apoptosis inducing ligand (TRAIL)-mediated cell death [45–48]. IFNγ secre-
tion by NK cells stimulates DC maturation [49] and the induction of chemokines
including IFN-inducible protein-10 (IP-10), monokine induced by IFNγ (MIG)
and interferon-inducible T-cell α chemoattractant (I-TAC) [50, 51], which pro-
motes lymphocyte recruitment. Mature, activated DC are then able to migrate to
the draining lymph node, where they can cross-present antigen to tumor-specific
CD8+ T cells.
Type I IFN (IFNα/β) are known to play an important role in the initiation of an
innate immune response [52]. Injection of activated plasmacytoid DC (pDC) has
been shown to induce NK and CD8+ T-cell-dependent rejection of transplanted
tumors in a mouse melanoma model through secretion of type I IFN [53], illus-
trating the close collaboration between innate and adaptive immunity in generat-
ing an effective anti-tumor immune response. However, mice deficient in both an
adaptive immune system and type I and type II IFN signaling (RAG2−/− STAT−/−
double knockout mice) are more susceptible to spontaneous tumor development
than either RAG2−/− or STAT−/− single knockouts [54]. This suggests that while
both the innate and adaptive arms of the immune system are required for optimal
immunosurveillance, each plays a partially independent role.
14.2.5 Avoiding Immune-Mediated Destruction
There are therefore three vital sequential steps in the generation of an anti-tumor
immune response. Firstly, tumor cells must be recognized by innate and/or adap-
tive immune effectors. Secondly, recognition of tumor cells must lead to activa-
tion of an immune response rather than tolerance. Thirdly, immune effectors must
effectively kill the tumor cells. Interruption of any one of these three steps can lead
to immuno subversion and uncontrolled tumor growth, that is, transition from the
elimination phase to escape. Tumors employ many mechanisms to subvert anti-
tumor immunity (Table 14.1). One key means by which tumors avoid recognition
by the immune system is through loss or reduced expression of tumor antigens.
Table 14.1 Mechanisms used by tumors to subvert anti-tumor immunity

Mechanism Stage affected Result
Loss/reduced expression of Tumor cell recognition Prevents cross-presentation of
tumor antigens [55–59] antigen to CD8+ T cells,
indirect presentation to
CD4+ T cells and direct rec-
ognition by CD8+ T cells.
Loss/altered expression of Tumor cell recognition Impedes direct recognition by
MHC class I [60, 61] CD8+ T cells.
Non-classical MHC expres- Tumor cell recognition/killing Protects from NK and CTL-
sion (e.g. HLA-G) usually mediated death.
restricted to immune-privi-
leged cells [173–175]
Reduced expression of NK cell Tumor cell recognition Inhibits NK-mediated killing.
ligands [176]
Induction of NKG2D down- Tumor cell recognition Inhibits NK and CTL-mediated
regulation [177] killing.
Secretion of IL-10 and TGF-β Immune effector activation Inhibits DC recruitment,
[62–64] prevents cross-priming of
CD8+ T cells, and promotes
Treg generation.
Expression of inhibitory Immune effector activation/ Impaired T cell proliferation
molecules including PDL-1 tumor cell killing and function.
[65, 66]
Expression of IDO [178] Immune effector activation Tryptophan-depletion, causing
T cells to undergo cell cycle
arrest.
Sterol synthesis [179] Immune effector activation Prevents CCR7 expression on
maturing DC inhibiting their
traffic to lymph node.
Mutations in death receptors Tumor cell killing Protects from NK and CTL-
including Fas and DR4/DR5 mediated death.
[68, 67]
Expression of Fas-L [69] Tumor cell killing Induces apoptotic cell death in
NK cells and activated T
cells.
CTL cytotoxic T lymphocyte, DR death receptor, HLA human leukocyte antigen, IDO indoleam-
ine 2,3-dioxygenase, PDL-1 programmed death ligand-1
This has been observed in human and mouse cell lines resistant to CTL lysis
in vitro [55, 56], in outgrowing tumors following immunotherapy in mouse mod-
els [57, 58] and in recurrent melanoma in patients treated with antigen-specific
immunotherapy [59]. Aberrant human leukocyte antigen (HLA) expression, which
impedes direct recognition by CD8+ T cells, has also been detected in a variety
of human cancers (reviewed in [60]), and human tumor cell lines lacking HLA
class I expression evade CTL-mediated cell death in vitro [61]. Both loss of tumor
antigen and HLA expression likely represent passive selection rather than active
adaptation, reflecting survival of the least immunogenic cells.
Secretion of soluble factors including IL-10 and TGFβ by tumors can skew the
immune system toward tolerance rather than active immunity. IL-10 can inhibit
DC recruitment [62], while TGFβ prevents the cross-priming of CD8+ T cells [63]
and promotes the generation of Treg [64]. Tumor cells can also express inhibitory
molecules on their surface, which negatively regulate T-cell function. For exam-
ple, a variety of human tumors express programed death ligand-1 (PDL-1), which
inhibits T-cell proliferation and cytokine production through interaction with its
receptor, programed death-1 (PD-1), expressed by activated T cells [65, 66].
Finally, tumor cell evolution can also select for cells less susceptible to CTL
and NK cell-mediated cell death. Mutations in death receptors including Fas and
TRAIL receptors, DR4 and DR5, have been observed in B-cell lymphoma [67]
and in metastatic breast cancer [68]. Interestingly, tumor cells have also been
found to express Fas ligand (Fas-L) [69], creating a role reversal situation in which
tumor cells can induce apoptotic cell death in activated T cells.
14.2.6 Antitumor Immunity: Does it Matter?
While original evidence for the existence of anti-tumor immunity came from ani-
mal models, this has now been corroborated by data demonstrating that anti-tumor
immune responses can be detected in cancer patients, even in the context of pro-
gressive disease, and that such responses have clinical significance.
Melanoma is considered one of the most immunogenic cancers with tumor-
associated antigen (TAA)-specific CD8+ T cells detectable in around 50 % of
patients [70]. Vitiligo-like hypopigmentation, thought to result from the destruc-
tion of normal melanocytes by autoantibodies and autoreactive T cells, is also
observed in melanoma patients and is associated with improved outcome [71].
While less frequently, circulating TAA-specific T cells have now been detected
in most cancer types [72–77]. Cancer patients are also commonly found to have
an increased proportion of peripheral Treg, consistent with the idea of an under-
lying anti-tumor immune response blocked by the concurrent expansion of Treg
[78–81].
Perhaps, the most convincing evidence for the clinical relevance of anti-tumor
immunity in patients is the strong prognostic significance of the balance of T-cell
subsets within the local tumor environment. Tumor infiltration by CD8+ T cells
has been associated with an improved prognosis in many cancer types including
ovarian [82], colorectal [83], esophageal [84], and lung [85]. Conversely, tumor-
infiltrating Treg predict poorer survival in most solid cancers [86–89], again
consistent with the suppressive role played by this T-cell subset. An exception,
however, appears to be colorectal cancer, where a higher density of tumor-infiltrat-
ing (Ti) Treg has been identified as a good prognostic factor in several independ-
ent studies [90–93]. One explanation for this seemingly paradoxical finding is that
expansion of Treg within the tumor occurs in response to the activation and expan-
sion of tumor-specific CD8+ T cells, thereby representing an indirect measure of
an anti-tumor immune response. Alternatively, it has been suggested that Treg may
limit the tumorigenic effects of Th17 cell-mediated inflammation [94]. In support
of this, Th17 cells are more frequently identified in colorectal tumors than other
cancer types, likely due to the abundance of pathogens present in the gut [95], and
a lower density of Ti-Th17 cells has recently been associated with improved dis-
ease-free survival [96]. Ti-Treg have also been associated with improved outcome
in nasopharyngeal and head and neck squamous cell carcinoma [97, 98], both of
which also develop in a non-sterile environment. It may be therefore that it is the
balance of T-cell subsets within a tumor, in relation to the stage of tumor develop-
ment and the environment in which this occurs, that is important. In any case, this
serves to highlight the complexity of the relationship between the immune system
and a developing tumor and the potential value of harnessing anti-tumor immunity
to improve the efficacy of cancer therapy.
14.3 Driving Anti-Tumor Immunity with Chemotherapy
14.3.1 Cytotoxic Chemotherapy: Mechanisms of Action
The major classes of cytotoxic drugs currently used for the treatment of cancer
include anti-metabolites, anti-folates, alkylating agents, topoisomerase inhibi-
tors, anthracyclines, vinca alkaloids, and taxanes (Table 14.2). While differing
in their precise mode of action, most interfere with DNA synthesis resulting
in apoptotic tumor cell death. The anti-metabolites gemcitabine and 5-fluoro-
uracil (5-FU), for example, are synthetic nucleoside analogues, which upon
intracellular conversion to their active forms, cause apoptosis by two different
mechanisms. They are incorporated into DNA (and RNA in the case of 5-FU),
disrupting DNA synthesis and/or RNA processing and function, and also inhibit
enzymes essential for DNA synthesis and repair [99, 100]. Alkylating agents,
including cyclophosphamide, and platinum-based drugs such as cisplatin and
oxaliplatin, cross-link DNA, inhibiting DNA synthesis and RNA transcription,
disrupting the cell cycle and activating several DNA damage-mediated signal
transduction pathways, leading to apoptosis [101, 102]. Anthracycline antibi-
otics, including doxorubicin, idarubicin and epirubicin also intercalate DNA/
RNA base pairs, but primarily cause apoptotic cell death through inhibition of
topoisomerase II activity, which is vital for DNA structuring and repair [103].
Vinca alkaloids (e.g., vinorelbine and vinblastine) and taxanes (paclitaxel and
docetaxel) differ in their mode of action in that they inhibit mitosis rather than
DNA synthesis through stabilizing or destabilizing microtubules and, thereby
preventing cell cycle progression [104]. However, like the other cytotoxics men-
tioned, this ultimately results in apoptosis. Most commonly used chemotherapy
drugs are therefore not specifically toxic to tumor cells, but target all dividing
cells, including lymphocytes. Consequently, chemotherapy has traditionally
Table 14.2 Major classes of cytotoxic drugs and mechanisms of action

Mechanism(s) of DNA damage
Class Examples / apoptosis induction
Anti-metabolites gemcitabine, 5-fluorouracil Incorporation into DNA/RNA
causing strand termination
and inhibition of enzymes
required for DNA synthesis/
repair including thymidylate
synthase, ribonucleoside
reductase and DNA poly-
merase [99, 100].
Anti-folates pemetrexed, methotrexate Inhibition of enzymes involved
in folate metabolism and
purine/pyrimidine synthesis
[180, 181].
Alkylating agents cyclophosphamide, ifospha- Addition of an alkyl group to
mide, dacarbazine DNA resulting in inter and
intra-strand cross-links
[101].
Platinum-based drugs cisplatin, carboplatin Binding of DNA bases resulting
oxaliplatin in inter and intra-strand
cross-links [102].
Topoisomerase inhibitors irinotecan, topotecan, Inhibition of topoisomerase I/II
etoposide preventing DNA structuring
and repair [182].
Anthracyclines doxorubicin, idarubicin Intercalation of base pairs and
epirubicin inhibition of topoisomerase
II [103].
Vinca alkaloids vinorelbine, vinblastine, Binding of tubulin preventing
vincristine microtubule formation and
cell cycle progression [104].
Taxanes docetaxel, paclitaxel Stabilization of microtubules
preventing their breakdown
and causing cell cycle arrest
[104].
been viewed as immunosuppressive, and combining chemotherapy with immu-

notherapy appeared counterintuitive. However, over the past decade, there has
been a paradigm shift, with the potential of chemotherapy to induce or enhance
anti-tumor immunity increasingly recognized.
It has now been demonstrated in a variety of animal models that optimal effi-
cacy of many cytotoxic drugs is dependent upon an intact immune system.
Oxaliplatin, for example, effectively controls tumor growth in immunocompe-
tent wild-type mice bearing EL4 thymomas, but is ineffective in mice athymic
nude mice lacking T cells, in CD8+ T-cell-depleted wild-type mice, or in mice
with a deficiency in the IFNγ signaling pathway [105]. Similarly, the efficacy
of doxorubicin against CT26 colon cancer and methylcholanthrene-induced sar-
coma is lost in the absence of IFNγ signaling [105]. Using a mouse model of
Fig. 14.2 Mechanisms by which chemotherapy can alter the level and/or context of antigen
presentation and the immunological milieu to promote anti-tumor immunity: 1 Physical destruc-
tion of the tumor-releasing antigen into the cross-presentation pathway. 2 Induction of “eat me”
and “danger” signals from dying tumor cells including CRT translocation and HMGB1/ATP
release. 3 Upregulation of tumor or cell-surface receptors including MHC class I and NKG2D. 4
Promotion of DC expansion and activation. 5 Homeostatic T-cell proliferation. 6 Selective Treg
depletion. 7 Selective B-cell depletion. ATP adenosine triphosphate, CRT calreticulin, CTL cyto-
toxic T lymphocyte, DC dendritic cell, HMGB1 high-mobility group box 1, MHC major histo-
compatibility complex, NK natural killer, Treg regulatory T cell
mesothelioma, we have found gemcitabine and the anti-folate pemetrexed to be

less effective in nude versus wild-type mice [106], and that the curative effect of
the DNA alkylating agent cyclophosphamide we observe in wild-type mice, is
CD8+ T-cell-dependent [107]. Much evidence has arisen to support the existence
of multiple mechanisms through which chemotherapy may induce or enhance
anti-tumor immune responses. These can be divided into those that alter the level
and/or context of antigen presentation through effects on tumor cells and APC,
and those that alter the immunological milieu, through effects on lymphocyte
populations, providing a favorable environment for the generation of anti-tumor
immunity (Fig. 14.2).
14.3.2 Altering the Level and Context of Antigen Presentation
Perhaps, the most obvious way by which chemotherapy could increase the level
of antigen presentation, is through physical destruction of the tumor, releas-
ing antigens from the dead and dying cells for entry into the cross-presentation
pathway. It is likely that the availability of some tumor antigens, normally pre-
sent at levels too low to induce an immune response, could be increased above
the required threshold in this process. In our murine model of mesothelioma,
using hemagglutinin (HA) antigen-transduced tumor cells (AB1-HA) injected
subcutaneously, gemcitabine significantly increases the level of antigen pres-
entation to tumor-specific CD8+ T cells for a given tumor size [108]. For
example, proliferation of adoptively transferred HA-specific CD8+ T cells in
a mouse bearing a 40 mm2 tumor treated with gemcitabine was almost dou-
ble the level observed in animals with an untreated tumor of equivalent size.
Vaccination with the HA antigen-bearing PR8 virus following gemcitabine
treatment resulted in a marked decrease in tumor growth and increased sur-
vival compared with vaccination without gemcitabine. It is also plausible that
so-called cryptic antigens, not usually visible to the immune system, could be
released following chemotherapy-induced cell death, along with many of the
~90 neoepitopes generated by point mutations that are harbored by the average
tumor [109]. In support of this, we found that animals cured of AB1-HA tumors
following combination chemo-immunotherapy with gemcitabine and the CD40
activating antibody FGK45 were resistant to rechallenge with both AB1-HA
and non-transfected AB1 tumor cells, demonstrating memory to tumor antigens
other than HA [110].
With regard to altering the context of antigen presentation, chemotherapy can
induce the expression or release of molecules associated with dangerous, and
therefore immunogenic, cell death [111, 112]. Using an assay in which tumor
cells are exposed to cytotoxic drugs in vitro, injected into immunocompetent
naive mice, and assessed for their capacity to elicit a protective immune response
against rechallenge with live tumor cells, Zitvogel, Kroemer, and colleagues
have demonstrated that oxaliplatin and the anthracyclines doxorubicin, idaru-
bicin, and mitoxantrone, induce immunogenic cell death in several tumor mod-
els [105, 113–115]. Characteristics of immunogenic versus non-immunogenic
tumor cell death in their system include cell-surface exposure of the calcium
ion-binding protein, calreticulin [115], secretion of the non-histone chromatin-
binding nuclear constituent, high-mobility group box 1 (HMGB1) [113], and
release of adenosine triphosphate (ATP) [105]. Calreticulin is usually seques-
tered within the endoplasmic reticulum. Its translocation to the cell surface
occurs before cells become apoptotic and is required for uptake of dying tumor
cells by DC [115]. Blockade of cell-surface calreticulin with specific antibodies,
or knockdown of its expression with siRNA, inhibited phagocytosis of anthra-
cycline-treated tumor cells, while addition of exogenous recombinant calreticu-
lin, enabled uptake of treated tumor cells transfected with siRNA [115]. Whereas
calreticulin exposure therefore provides an “eat me” signal for DC, binding of
HMGB1 to toll-like receptor 4 (TLR4) on DC, and subsequent signaling through
the MyD88 pathway is thought to represent a danger signal required to induce
antigen processing and presentation. Mice deficient in TLR4 or the MyD88
adapter molecule were unable to mount a tumor-specific immune response fol-
lowing vaccination with oxaliplatin or doxorubicin-treated tumor cells and were
not protected against re-challenge [113]. Although WT and TLR4−/− DC were

equally efficient in engulfing dying tumor cells, TLR4−/− DC were defective in
their capacity to process and present antigen in association with MHC class I
[113]. Binding of ATP released from oxaliplatin-treated tumor cells to the P2rX7
receptor on DC results in activation of the NLRP3 inflammasome and subse-
quent secretion of IL-1β, which is required for effective CD8+ T-cell priming
[105]. It is extremely likely, however, that other factors distinguishing immuno-
genic from non-immunogenic cell death are yet to be discovered.
Some drugs, including gemcitabine, cisplatin, and paclitaxel, have also been
shown to induce upregulation of tumor cell-surface receptors/ligands, result-
ing in increased antigen presentation and/or susceptibility to immune effector-
mediated cell death. Liu et al., found that gemcitabine induced upregulation of
HLA class I expression on human tumor cell lines [116], thereby increasing the
opportunity for direct recognition by CD8+ T cells, while Ramakrishnan et al,
demonstrated that increased sensitivity of murine and human tumor cells to CTL
lysis following cisplatin or paclitaxel exposure was mediated via the upregula-
tion of mannose receptors, increasing their permeability to granzyme B [117].
This allowed bystander killing of antigen-deficient tumor cells by CTL activated
through recognition of neighboring antigen-expressing tumor cells. Sensitization
of tumor cells to TRAIL-mediated cell death can also contribute to the anti-
tumor effects of cytotoxic drugs. In our murine mesothelioma model, the effi-
cacy of cyclophosphamide is reduced in TRAIL-deficient mice, while agonistic
anti-DR5 antibodies, which mimic TRAIL ligation, rescued the partial effect of
the drug observed in athymic nude mice [118]. Increased susceptibility of tumor
cells to NK cell-mediated death has also been observed following exposure to
cytotoxic drugs, mediated both by upregulation of NKG2D ligands [119] and
downregulation of c-type lectin receptor ligands [120], which leaves tumor cells
vulnerable to NK cell-mediated killing in accordance with the “missing self
hypothesis” [121].
Cytotoxic drugs may also enhance tumor antigen presentation through direct
effects on DC. In several mouse models, cyclophosphamide has been shown to
promote DC expansion from bone marrow progenitors [122–124], while exposure
of DC to non-toxic concentrations of paclitaxel in vitro can lead to upregulation of
co-stimulatory molecules and an increased capacity to present antigen [125, 126].
14.3.3 Altering the Immunological Milieu
Although seemingly paradoxical, chemotherapy-induced lymphodepletion is

one of the major mechanisms through which the immunological milieu can be
altered to favor the generation of anti-tumor immune responses. As the size and
composition of the T-cell repertoire is under constant regulation in the periph-
ery [127], lymphodepletion triggers a period of homeostatic T-cell prolifera-
tion, driven by IL-7 and IL-15 [128–130]. Being at least partially dependent
upon self-antigen-MHC interactions, homeostatic T-cell proliferation results in a

freshly reconstituted T-cell pool biased toward self-antigen reactivity [131, 132].
Since most tumor antigens are self-antigens, lymphopenia in a tumor-bearing
host may result in increased anti-tumor immunity, as has been demonstrated in
sub-lethally irradiated mice, whereby homeostatic expansion of transferred autol-
ogous or syngenic T cells inhibited the growth of established tumors [133]. This
becomes even more important if the level of available antigen and the capacity
for presentation to effectors is simultaneously increased.
Another indirect way by which chemotherapy has been shown to enhance
anti-tumor immunity is through differential effects on lymphocyte subsets.
Despite also functioning as a potent immunosuppressor, Maguire and Ettore dis-
covered over 40 years ago that cyclophosphamide could, in fact, increase contact
sensitivity reactions in guinea pigs [134]. More than a decade later, this find-
ing was extended to humans, with antigen-specific delayed-type hypersensitivity
reactions shown to be augmented in a cohort of patients receiving cyclophospha-
mide treatment for metastatic melanoma or colorectal cancer [135], the authors
suggesting that this was due to inhibition of suppressor T-cell function, a distinct
Treg population not having been characterized at the time. More recently, it has
been demonstrated both in animal models and cancer patients that cyclophos-
phamide can be used to target Treg [136, 137]. Importantly, however, the effects
of cyclophosphamide are dose-dependent, with lower doses (50–100 mg/day)
causing selective Treg depletion and enhanced effector function, while higher
doses (200 mg/day) result in indiscriminate loss of all lymphocyte subsets ([137]
and our own unpublished data). This increased susceptibility of the Treg popu-
lation may not be limited to cyclophosphamide, with gemcitabine now shown
to cause transient selective Treg depletion in cancer patients receiving single
agent treatment [138] and remaining Treg in paclitaxel-treated mice found to
have reduced suppressive function [139]. Whether the effects of gemcitabine and
paclitaxel on immune cell populations are dose-dependent is not yet clear. Since
cytotoxic drugs target dividing cells, one explanation for this apparent selective
Treg depletion is their increased proliferative state, relative to other lymphocyte
subsets, which appears to be an intrinsic characteristic of this cell population
[138, 140, 141]. In support of this theory, those Treg most actively proliferating
are preferentially depleted by cyclophosphamide [107]. This may have impor-
tant implications for anti-tumor immunity as cycling Treg also express higher
levels of markers associated with maximal suppressive activity, including induc-
ible co-stimulatory molecule (ICOS) and tumor necrosis factor receptor type 2
(TNFR2) [107, 142–144]. Depletion of a maximally suppressive Treg population
by cytotoxic chemotherapy could favorably alter the context of antigen presen-
tation, removing the brakes on anti-tumor immunity. In the same vein, B cells
are more profoundly affected by some cytotoxics than T cells [145, 146]. With
evidence to date indicating that B cells have a negative, rather than a positive
impact on anti-tumor immunity, this again could potentially skew any ensuing
immune response in the right direction.
14.3.4 Immune-Mediated Tumor Cell Death Contributes

to Treatment Efficacy
Consistent with preclinical data demonstrating a requirement for an intact

immune system for optimal treatment efficacy, it is becoming increasingly clear
that anti-tumor effects of chemotherapy in cancer patients are not entirely due
to direct cytotoxicity; activation of anti-tumor immunity may also play a role.
In breast cancer, infiltration of CD3+ T cells and disappearance of Treg in sur-
gical specimens following adjuvant taxane/anthracycline-based chemotherapy
have both been associated with complete pathological response (i.e., no residual
tumor cells) [147, 148]. Similarly, the presence of Ti-T cells in samples taken
from patients undergoing surgical debulking for ovarian cancer, was associated
with complete response to adjuvant platinum-based chemotherapy [82], while in
rectal cancer, density of both CD8+ and CD4+ T cells in pre-treatment biopsy
samples was recently shown to predict complete response to 5-FU-based neoad-
juvant chemoradiotherapy [149].
Factors indicative of immunogenic tumor cell death following chemotherapy
in animal models have also been shown to influence clinical outcome in patients,
with loss of function mutations in both TLR4 and P2xr7 genes associated with
reduced disease-free survival following anthracycline-based chemotherapy in
breast cancer patients [105, 113], and calreticulin expression on tumor cells found
to correlate with the presence of tumor-infiltrating memory T cells and favorable
prognosis in colorectal cancer [150].
The immune system may even contribute significantly to the efficacy of tar-
geted therapies, a new class of anti-cancer drugs that interfere with specific mol-
ecules involved in tumor growth and progression. Examples of these include
the human epidermal growth factor 2 (HER-2) inhibitor, trastuzumab, used in
breast cancer; the vascular endothelial growth factor (VEGF) blocking anti-
body, bevacizumab used in lung, breast, colorectal, and renal cancer; the epi-
dermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, used
in lung and pancreatic cancer; and the multi tyrosine kinase inhibitor, imatinib,
used to treat gastrointestinal stromal tumors (GIST) and certain types of leu-
kemia. Approximately 80 % of human GISTs harbor an activating mutation in
the c-kit gene that encodes the receptor tyrosine kinase KIT [151]. Imatinib is
now the recommended first-line treatment for c-kit mutation positive GIST with
around 50 % of patients achieving at least a partial response [152]. While the
primary mechanism of action is thought to be a direct anti-proliferative effect
on tumor cells through inhibition of KIT signaling, imatinib can prevent the
in vivo growth of tumors refractive to the antiproliferative effects of imatinib
in vitro in several animal models through enhancement of DC-mediated NK cell
activation [153]. More recently, imatinib has been shown to induce CD8+ T-cell
activation and Treg apoptosis in a transgenic mouse model in which an activat-
ing c-kit gene mutation results in spontaneous development of GIST, through
reducing tumor cell expression of the immunosuppressive enzyme indoleamine

2,3-dioxygenase (IDO) [154]. The authors also demonstrated that imatinib sensi-
tivity in patients with GIST correlated with an increase in the intratumoral CD8+
T cell to Treg ratio and reduced IDO protein expression [154].
14.4 Cancer Therapy: Looking Forward
14.4.1 Combination Chemoimmunotherapy
Immunotherapies, when used as single agents, have proved largely disappoint-

ing to date. However, given that chemotherapy, at least in certain situations, can
enhance anti-tumor immunity, combining standard chemotherapy with immuno-
therapy treatment has the potential to increase the efficacy of anti-cancer therapy.
Synergy between chemotherapy and immunotherapy has been demonstrated in a
variety of animal models using several different strategies aimed at inducing or
enhancing an anti-tumor immune response. In murine mesothelioma, combina-
tion chemoimmunotherapy with gemcitabine and the αCD40-activating antibody
FGK45 can induce long-term cures and resistance to rechallenge in up to 80 % of
animals, a dramatic improvement over either treatment alone, which only delays
tumor growth [110]. Importantly, however, this superior effect is only observed
when FGK45 therapy follows gemcitabine treatment; reversing the order of
administration is actually less effective than chemotherapy alone. Combining gem-
citabine or pemetrexed with Treg depletion using the αCD25 antibody PC61 also
results in a synergistic treatment effect in this model [155].
Cisplatin, cyclophosphamide, and paclitaxel have also have also been shown
to synergize with immunotherapy in preclinical studies [139, 156–160]. Using
a model of human papilloma virus-associated malignancy, Tseng et al., demon-
strated that cisplatin followed by a tumor antigen-encoding DNA vaccine led to
reduced tumor growth and increased survival, associated with the development of
tumor-specific CTL [158]. Again, this synergistic effect was only observed when
chemotherapy preceded vaccination. Chemotherapy prior to vaccination was
also reported by Wada et al., to be the most effective treatment schedule, induc-
ing antigen-specific effector T-cell expansion and inhibiting tumor growth, using
a granulocyte–macrophage colony-stimulating factor (GMCSF)-secreting tumor
cell vaccine following cyclophosphamide treatment in an autochthonous prostate
cancer model [159]. However, other studies have found immunotherapy prior to
chemotherapy to be the best approach. For example, in a mouse lymphoma model,
Correale et al, found peptide vaccination administered 13 days before combination
chemotherapy gave the biggest synergistic effect [156]. Similarly, type I IFN gene
therapy was most effective when given prior to chemotherapy with cisplatin and
gemcitabine in murine mesothelioma and lung cancer models [157].
There have been few randomised clinical trials to date powered to evalu-
ate the efficacy of chemoimmunotherapy over chemotherapy alone. A recent
practice-changing study demonstrated that the anti-CTLA4 antibody ipilimumab

in combination with dacarbazine improved survival by around 2 months in patients
with metastatic melanoma, as compared to dacarbazine with placebo [161].
However, dacarbazine alone has extremely modest activity in melanoma, and ipili-
mumab has activity as a single agent [162]. While the combination appeared to
show a “tail on the curve” for long-term survival, it is unclear whether this repre-
sents a synergistic or merely additive response. Impressive results have been
reported in some early phase studies. For example, in a phase II trial of gemcitabine
plus FOLFOX (5-FU, oxaliplatin, and levofolinic acid) chemotherapy combined
with subcutaneous GMCSF and IL-2 injections for colorectal cancer, an objective
response rate of almost 70 % and an average time to progression of 12.5 months
were observed, significantly greater than previously reported for any other regimen
[163]. In lung cancer, a randomised phase II trial of the TLR9 agonist PF-3512676
in combination with first-line taxane plus platinum chemotherapy demonstrated a
higher objective response rate in patients who received combination therapy com-
pared to chemotherapy alone [164]. While too small, or not designed to determine
clinical benefit, several studies have demonstrated that combination chemoimmuno-
therapy is tolerable and results in enhanced anti-tumor immune responses [165–168].
Data from preclinical studies and early phase trials have therefore been encour-
aging. However, the success of chemoimmunotherapy will rely heavily on careful
drug selection and treatment scheduling. Chemotherapy drugs vary significantly
in their capacity to induce immunogenic tumor cell death [114] and to complicate
things further; this may be model-dependent [106, 169]. There are also arguments
to support both administration of immunotherapy prior to and following chemo-
therapy. Treg respond far more rapidly in the initial stages of an immune response
than naive conventional T cells [170], and Treg activation and proliferation have
been shown to precede and prevent the generation of tumor-specific effector T cells
in animal models [171]. Memory T cells, however, respond to antigen with similar
kinetics to Treg [170, 171] and are more resistant to cytotoxic chemotherapy than
other T-cell subsets [172]. Immunotherapy given prior to chemotherapy therefore
has the potential to generate tumor-specific memory T cells better placed to com-
pete with Treg in the context of increased antigen availability following chemo-
therapy-induced tumor cell death. Alternatively, immunotherapy administered
following chemotherapy, could capitalize on homeostatic T-cell proliferation fol-
lowing chemotherapy-induced lymphodepletion to skew newly generated T cells
toward anti-tumor activity and/or enhance T-cell priming and differentiation. As
discussed, evidence from animal models can be found to support either strategy.
14.4.2 Entering the Era of Personalized Medicine
While cytotoxic chemotherapy is still the mainstay of most cancer therapy, the
emphasis is now shifting from a one size fits all approach to more individualized
treatment strategies. Many factors contribute to chemotherapy efficacy and these
pertain as much to the tumor and to the host as to intrinsic properties of the drug.
Not least of these factors is the potential for development of an anti-tumor immune
response, and the capacity for chemotherapy to induce or enhance this. Patients
whose cancers have “druggable” targets may be treated with novel-targeted thera-
pies, and our understanding of the interaction between targeted therapies and the
immune response is even less mature. Nevertheless, many of these patients will
also have chemotherapy before or after treatment with a targeted agent.
Chemoimmunotherapy using carefully selected drug combinations rep-
resents a promising treatment option. However, it is likely that this strategy
will be most effective when used in an individualized setting. Adjunct immu-
notherapies may need to be tailored not only to the type of chemotherapy, but
also to the immunogenicity of the patient’s tumor (expression of tumor anti-
gens) and any defects in the patient’s immune system (e.g., TLR4/P2xr7 muta-
tions). Measurement of immunological parameters prior to initiating therapy
may prove a useful tool in predicting which patients may benefit from adjunct
immunotherapy and in selecting the type of immunotherapy to administer, for
example, tumor-specific CTL stimulating or Treg depleting. More work needs
to be done to determine how the precise balance of T-cell subsets within the
local tumor environment before and after chemotherapy affects response, and
whether assessment of peripheral blood T-cell subsets could potentially repre-
sent a less invasive surrogate measure, which could be more easily translated
into the clinic.
14.5 Conclusions
Although once a highly controversial topic, there is now overwhelming evi-

dence to support a role for the immune system in controlling cancer devel-
opment and influencing treatment response. It is also becoming increasingly
recognized that chemotherapy, rather than purely suppressing anti-tumor
immune responses due to lymphocyte toxicity, can actually enhance anti-tumor
immunity through a variety of mechanisms. This relatively recent discovery
has major implications for the development of cancer therapy. Harnessing
the capacity of certain cytotoxic drugs to synergize with the immune system
through combining chemotherapy with novel immunotherapy agents represents
an exciting new treatment strategy. Rather than using immunotherapy as an
alternative to chemotherapy, which has produced disappointing results to date,
in this context, we may instead see a real benefit. However, it is imperative
that combination chemoimmunotherapy regimens are carefully designed, not
just with regard to the potential for synergy between the selected drugs, but
also taking account the intrinsic properties of the tumor and the capacity of the
patient to generate an anti-tumor immune response. It is therefore likely that
the development of more effective treatment strategies for cancer patients will
increasingly require a more individualized approach.
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About the Editor
Dr. Daniel E. Johnson received his undergraduate training at North Park

University and his doctoral degree in molecular biology from Princeton
University. He was a postdoctoral fellow at the University of California, San
Francisco. In 1993, he joined the faculty at the University of Pittsburgh and the
University of Pittsburgh Cancer Institute, where he is currently a Professor in the
Departments of Medicine and Pharmacology and Chemical Biology. Dr. Johnson
has served as a standing member on study sections for the National Institutes of
Health and American Cancer Society and is a long-standing Section Editor for the
journal Leukemia. His research has focused on molecular mechanisms of apopto-
sis in leukemia and head and neck cancer, as well as the mechanisms of myeloid
differentiation. He has placed particular emphasis on the translation of findings
from his laboratory to the clinic and, together with physician scientist collabora-
tors, has helped to develop ongoing trials in both acute myeloid leukemia and head
and neck cancer.
Cell Death in Biology and Diseases, DOI: 10.1007/978-1-4614-5847-0,
Index
A Autophagy, 64, 87–89, 93–108, 162, 210, 212,

A1/Bfl-1, 12, 232–234 213, 338, 350, 352, 360
ABT-263 (also called Navitoclax), 231, 232, AZ628, 341
237–240
ABT-737, 231–237, 240–242, 345
Acid sphingomyelinase (aSMase), 208, 211 B
Adaptive immunity, 377, 378 Bafilomycin A1, 94, 106
AEZS-131, 345 Base excision repair (BER), 106, 140,
Akt, 38, 39, 95, 99, 102, 128, 208, 209, 285, 142, 191
331, 332, 336, 337, 348, 350, 351, 353, Bax, 11–15, 97, 216, 232, 233, 237, 240, 258,
356–362 289, 314
AMP-activated protein kinase (AMPK), Bcl-2, 2, 36, 6, 11, 12, 19, 55, 62, 96–98, 208,
97–99, 338 209, 231–235, 237, 241, 258, 272, 289,
Antigen presentation, 306, 375, 377, 313, 314, 339, 345
383–386 Bcl-w, 11, 12, 232, 234, 237, 240
Anti-tumor immunity, 58, 373, 374, 376, 377, Bcl-XL, 11, 13, 15, 55, 56, 215, 231, 232, 237,
379–381, 386, 388, 390 240, 314, 339, 345, 349
Anti-tumor immune response, 373–378, BCR-ABL, 54, 56, 62, 63, 101, 187, 188, 194,
380, 381, 383, 385, 388–390See also 221, 340, 355
Anti-tumor immunityApaf-1, 3–5, 14, Beclin-1, 213, 232
18, 283 Bid, 5, 11, 14, 15, 211, 259, 282, 289, 314
APE1 (AP-endonuclease 1), 106, 142, 143, Bik, 11, 13, 289, 314
158, 159 Bim, 13, 62, 126, 232, 233, 289, 314, 353
Apoptosis, 1–5, 9, 11, 13–15, 61, 62, 70, Bortezomib, 62, 64, 100, 235, 236, 245, 288,
97–99, 104, 124, 127, 140, 157–159, 291, 304, 308, 310, 314–321, 344, 358
208, 211, 219, 241, 257, 260, 262, 264,
282, 313, 352, 353
Apoptosome, 5, 6, 259 C
AS703026, 344 CAL-101, 346, 347
Ataxia telangiectasia-mutated (ATM), 14, 97, Cancer-associated fibroblasts (CAFs), 55,
139, 140, 154, 159–161, 192, 262 57, 124
Ataxia telangiectasia-RAD3 related (ATR), Carfilzomib, 307, 309, 320, 321
14, 139–141, 146, 156, 159, 191 Caspase protease, 3, 7, 9, 232
ATG genes, 89 Caspase-3, 3, 6–10, 165, 216, 219, 260,
Autophagolysosome, 93, 95 267–269, 282, 283, 314
Autophagosome, 89, 90, 92–94, 97, 106 Caspase-7, 3, 6–9, 260, 267, 268
Cell Death in Biology and Diseases, DOI: 10.1007/978-1-4614-5847-0,
404 Index
C (cont.)
Caspase-8, 3, 5–7, 15–18, 62, 258–260, 262, E
267, 268, 282, 285–290 Enzastaurin, 352
Caspase-9, 3, 5–10, 62, 216, 259, 260, ERCC1, 144, 163
283, 289 ERK, 62, 65, 96–98, 126, 163, 193, 209, 214,
Caspase-10, 5–7, 16, 18, 258, 285 241, 332, 334, 335, 337–340, 344–346,
CCT239065, 340 359, 360
CD44, 68–70 ERK inhibitors, 97, 345, 346
CD95, 262, 268, 282–286 ER stress response, 88, 98
Ced-3, 2, 210 Erucylphosphocholine (ErPC), 353
Ced-4, 2, 3 Erucylphosphohomocholine (ErPC3), 353
Ced-9, 2 Extrinsic apoptosis pathway, 4, 5, 15, 17, 18,
Caenorhabditis elegans, 2, 3, 18, 103, 128 257, 264
Ceramide, 205–219
c-FLIP, 16, 18, 268, 285, 286, 289
Chaperones, 88, 182, 186, 188, 192, 314, 315 F
Chemoimmunotherapy, 373, 388–390 Fanconi pathway, 145
CHK1 (checkpoint kinase 1), 139, 141, 146, Fas, 3–5, 16, 17, 126, 220, 262, 282, 377–380
157, 159, 191 Fas-associated death domain protein (FADD),
CHK2 (checkpoint kinase 2), 139, 141, 156, 4, 5, 16–18, 258, 262, 267, 286
157, 192
Chloroquine (CQ), 94, 104–107, 350
Chronic myeloid leukemia (CML), 54, 55, G
127, 192, 220, 235 Galectin-1, 60, 61
cIAP1, 8–10, 256–258, 260–262, 264, 267, Galectin-3, 60–62
268, 270 Galectin-9, 60, 62
cIAP2, 8–10, 256, 258, 260–262, 264, 265, GDC-0879, 340, 360
267, 270 GDC-0941, 236, 343, 346
Co-chaperones, 183, 186, 193 GDC-0973, 343
Crabtree effect, 36, 37 Geldanamycin, 185, 195, 196
Cytochrome c, 3–6, 11, 13, 15, 18, 99, 210, Glutamine metabolism, 40, 41
216, 259, 283 Glycolysis, 35–39, 41, 99, 238
Cytotoxic T cells (CTL), 101, 375, 379, 383 GX15-070 (also called Obatoclax), 231–233,
236, 240–243
D
Dabrafenib, 340, 343, 357, 360 H
Death domain (DD), 4, 17, 258, 261, 262, HDAC inhibitors, 100, 101, 190, 289
282, 285 Heat Shock Proteins (HSPs), 106, 182
Death-effector domain (DED), 5, 18 Helper T cells, 376
Death-inducing signaling complex (DISC), 4, Histone methyl transferase, 45, 190, 191
5, 16–18, 258, 259, 282, 284–286, 288 Homologous recombination, 144, 145, 191
Delanzomib, 308 HSP90 (heat shock protein 90), 376
DNA damage response, 88, 98, 138, 139, Hypoxia, 63, 64, 99, 105, 189
144, 147 Hypoxia-inducible factor (HIF), 43, 63, 99,
DNA-dependent protein kinase (DNA-PK), 189, 346
144, 160
DNA glycosylases, 142, 164
DNA methyl transferase (DNMT), 190 I
DNA polymerases, 143, 158, 163, 164, 382 IC87114, 346
DNA repair, 14, 53, 63, 138–140, 143, Immune system, 2, 59, 88, 101, 102, 107,
146–148, 152, 155, 159, 161–163, 108, 282, 373, 374, 377, 378, 380–382,
166, 192 384, 387
Index 405
Immunoproteasome, 306, 310–312, 320 miR-17~92 cluster, 127

Immunosurveillance, 374, 375, 378 miR-106b~25 cluster, 127
Inhibitor of apoptosis (IAP), 8, 255–260, Mismatch repair, 140, 191
264–270, 283, 289 MGMT (06-methylguanine DNA methyl trans-
Innate immunity, 377, 378 ferase), 140–142, 149, 154–156, 165
Integrins, 59, 64–68, 70, 287 MK-2206, 351, 352, 357–359
Interleukin-1β converting enzyme (ICE), 2 MLN-9708, 308, 321
Intrinsic apoptosis pathway, 4–6, 9, 11, 13–15, mTOR, 39, 41, 95–97, 121, 180, 194, 332,
240, 283 335, 337–339, 346–350, 354, 356,
358, 360
Myc, 38, 41, 99, 104, 157, 238, 265, 314, 349
J Myeloid-derived suppressor cells (MDSCs),
JAK, 55–57, 62, 65, 188 58
K N
KP372-1, 252 NAD+ biosynthesis inhibitors, 162
Natural killer cells (NK cells), 376–379
Nuclear factor-κB (NF-κB), 102, 209, 215,
L 256, 259, 261, 262, 264, 265, 268, 270,
LC3-I, 92, 94 289, 313, 319, 337, 353
LC3-II, 92, 94 Neutral Sphingomyelinase (nSMase), 208,
Lin-4, 117, 128 211, 212, 217, 218
Non-homologous-end-joining (NHEJ), 144
NOXA, 11, 13–15, 62, 233, 237, 314
M Nucleotide excision repair (NER), 140, 191
3-methyladenine (3-MA), 105, 164
Marizomib, 307–309, 321
Mcl-1, 11–13, 15, 55, 62, 96, 231–234, 238, O
240, 314, 345 Oprozomib, 308, 309, 321
MDM2, 14, 189 OSI-027, 355
MEK, 65, 96, 97, 234, 332, 334, 335,
338–340, 342–346, 356–360
MEK inhibitors, 339, 342–344, 346, 358, P
360, 361 p53, 14, 15, 17, 62, 63, 98, 103, 105, 106,
MEK162, 344, 347, 357, 358 122–124, 127, 128, 139, 140, 156, 161,
Mesenchymal stem cells (MSCs), 55, 288 189, 190, 262, 285, 289, 314
MGMT (06-methylguanine DNA methyl trans- PARP [poly(ADP)ribose polymerase], 143,
ferase), 140–142, 149, 154–156, 165 152
microRNA (miRNA), 12 , 13, 41, 117, PBI-05204, 353
122, 123 PD-0325901, 343, 356, 357
miR-15a, 12, 122–124 Perifosine, 344, 353
miR-16-1, 12, 122–124 PI-103, 348
miR-21, 123, 125, 126, 128 PI3K, 39, 70, 89, 90, 95–97, 99, 102, 161,
miR-26a, 124 162, 193, 194, 213, 221, 236, 332,
miR-34, 124 335–339, 341, 343, 346–350, 355–362
miR-122, 120–122, 124 PLX5568, 341
miR-130/131, 123, 127 Proteasome, 162, 304, 306, 308, 310,
miR-143, 122, 125 312–315, 318, 320
miR-145, 125 PTEN, 95, 123, 127, 128, 154, 242, 332,
miR-155, 123, 128 335–337, 339, 350, 356, 357, 360, 361
miR-221, 126 PUMA, 11, 13–15, 98, 126, 127, 314
miR-222, 126 Purine and purine-like inhibitors, 196
406 Index
P (cont.)
PX-866, 346 Tumor necrosis factor (TNF), 3, 216, 282
Pyk2, 65, 70, 71 Tumor necrosis factor receptor--associated
death domain protein (TRADD), 17,
18, 261, 270
R Tumor necrosis factor receptor--associated
Raf-265, 341 factor -1/-2 (TRAF-1/-2), 17
Raf, 96, 163, 187, 188, 194, 208, 209, Tumor necrosis factor--related apoptosis-
332–335, 337–345, 356, 357, 359, 362 inducing ligand (TRAIL), 3, 16, 17, 97,
Raf inhibitors, 334, 339, 342, 345, 360, 361 162, 234, 236, 238, 242, 264, 267, 268,
Ras, 60, 65, 67, 95, 96, 99, 102, 125, 163, 208, 270, 282, 284–291
214, 332–339, 342, 345, 347, 359–362 TRAIL-R1, 16, 17, 282, 284, 285, 288–290
Receptor-interacting protein (RIP), 17, 313 TRAIL-R2, 16, 17, 282, 284, 285, 288, 289
Refametinib, 343 TRAIL-R3, 282, 284, 285
Regorafenib, 341 TRAIL-R4, 284, 285, 288
Regulatory T cells (Treg), 377 Trametinib, 343
Resorcinol class inhibitors, 197 Triciribine, 351
RO4987655, 344
RX-201, 353–354
U
Ubiquitin-proteasome pathway (UPP), 304
S Ubiquitin-proteasome system (UPS), 100, 304
SCH772984, 346 Unfolded protein response (UPR), 64, 314
Second mitochondria-derived activator of
caspase (SMAC), 9, 10, 18, 255, 256,
259, 260, 262–264, 266–269, 270 V
Selumetinib, 342, 344, 356, 357, 359 Vemurafenib, 340, 357, 358, 360
SL327, 344
SMAC mimetics, 256, 259, 266–268, 270
Sorafenib, 57, 124, 163, 234, 236, 241, 339, W
340, 356, 360 Warburg effect, 36, 42, 45
Sphingosine, 42, 205–208, 214, 216, 217, 220 WJD008, 350
Sphingosine kinase 1 (SK1), 207, 208, 214,
215, 217, 219, 221
Sphingosine kinase 2 (SK2), 207, 208 X
Sphingosine-1 phosphate (S1P), 205, 207 XIAP, 8–10, 256, 258–260, 262–267, 269, 270
STAT3, 55–57, 126 XIAP antagonists, 269
Steroid hormone receptors, 189 XL147, 347
XL281, 341
XL418, 354
T XL765, 350
TAK-733, 334
Tumor cell metabolism, 35

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Cell Death in Biology and Diseases: Series Editors

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What is the book about?

What is the book about?

What are some of the major topics discussed in the book?

What are some of the major topics discussed in the book?

Cell Death in Biology and Diseases

For further volumes:

Cell Death Signaling

ISBN 978-1-4614-5846-3 ISBN 978-1-4614-5847-0 (eBook)

Library of Congress Control Number: 2012952022

© Springer Science+Business Media New York 2013

Printed on acid-free paper

Humana Press is a brand of Springer

Cell death, or conversely cell survival, is a major biological phenomenon. Just

Xiao-Ming Yin MD, Ph.D.

Zheng Dong Ph.D.

Recent descriptions of aberrant microRNA expression in cancer cells and the

Daniel E. Johnson Ph.D.

1 Defective Apoptosis Signaling in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . 1

2 The Warburg Effect and Beyond: Metabolic Dependencies

3 Emerging Opportunities for Targeting the Tumor–Stroma

4 The Role of Autophagy in Drug Resistance and Potential

5 MicroRNAs in Cell Death and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . 117

6 Targeting DNA Repair Pathways for Cancer Therapy. . . . . . . . . . . . . 137

7 Molecular Chaperones and How Addiction Matters

8 Sphingolipid Metabolism and Signaling as a Target

9 Leading Small Molecule Inhibitors of Anti-Apoptotic

10 SMAC IAP Addiction in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

11 Harnessing Death Receptor Signaling for Cancer Treatment. . . . . . . . 281

12 Proteasome Inhibition as a Novel Strategy for Cancer Treatment. . . . 303

13 New Agents and Approaches for Targeting the

14 Activation of Immune-Mediated Tumor Cell Death

About the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

Cori Abikoff Division of Clinical Research, Fred Hutchinson Cancer Research

Richard A. Franklin Department of Microbiology and Immunology, Brody

Clorinda Massarino Department of Biomedical Sciences, University of Catania,

Tony Taldone Breast Cancer Service, Department of Medicine, Memorial Sloan-

Abstract Apoptosis is critically important during development, facilitating the

1.1 Characterization of a Human Disease and Biochemical

D. E. Johnson (ed.), Cell Death Signaling in Cancer Biology and Treatment, 1

biochemical changes, including externalization of plasma membrane phospholip-

Fig. 1.1 Activation of the Apoptosis Stimulus

Cleavage of caspase substrate proteins

Death ligand/Death receptor

“DISC” procasp-3 caspase-3

Fig. 1.2 The extrinsic and intrinsic apoptosis pathways

1.2 The Intrinsic and Extrinsic Apoptosis Pathways

Based on the seminal discoveries described above, two pathways of apoptosis

Fas (death receptor)

Death ligand/Death receptor

“DISC” procasp-3 caspase-3

Fig. 1.4 Bcl-2 inhibition of the intrinsic apoptosis pathway

migrate to the mitochondria and stimulate cytochrome c release and activation of

1.3 Defects in Caspase Signaling

1.3.1 Mutation or Dysregulated Expression of Caspase

Mandruzzato et al. [51] reported in 1997 a mutant form of caspase-8 expressed

1.3.2 Aberrant Expression of IAPs

Death ligand/Death receptor

procasp-8/-10 caspase-8/-10 XIAP

“DISC” procasp-3 caspase-3

1.1 Characterization of a Human Disease and Biochemical

1.2 The Intrinsic and Extrinsic Apoptosis Pathways

1.3 Defects in Caspase Signaling

1.3.1 Mutation or Dysregulated Expression of Caspase

1.3.2 Aberrant Expression of IAPs

1.4 Defects Affecting the Intrinsic Apoptosis Pathway

1.4.1 Overexpression of Anti-Apoptotic Bcl-2 Family

1.4.2 Mutation or Reduced Expression of Proapoptotic Bcl-2

1.5 Defects Affecting the Extrinsic Apoptosis Pathway

1.5.1 Loss or Mutation of Death Receptors

1.5.2 Regulation of Death Receptor Signaling by Decoy

1.5.4 Dysregulated c-FLIP Expression

2.1 Pre-Molecular Biology Research

2.2 Glycolytic Regulation in Cancer

2.3 Therapeutic Strategies Based on Glycolysis Inhibition

2.4 Alternative Mitochondrial Fuels

2.5 Screening for Additional Metabolic Dependencies

3.2 Cellular Components of the Tumor Microenvironment

3.2.1 Mesenchymal Stroma Cells

3.3 The Tumor Microenvironment Milieu and Drug

3.4 Hypoxia and Drug Resistance

3.5 Molecules Involved in the Interaction of the Tumor Cell

3.6 Signaling Pathways Activated Due to the Interaction