Research
A variety of human malignancies show loss of a critical region in chromosomal band 13q14.3 that harbors a tumor suppressor mechanism. In more than 50% of patients suffering from B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemia of the western world, this region is deleted. Despite extensive efforts from a number of labs, the molecular nature of this mechanism has proven elusive. The region contains long non-coding RNA genes, micro-RNA genes and multicistronic transcripts. We have recently been able to show a complex epigenetic regulatory mechanism in this region which involves DNA-methylation, asynchronous replication timing and monoallelic expression. This epigenetic mechanism can explain some of the genetic peculiarities observed in patients. What we are currently doing is to elucidate the intricacies of the regulatory mechanism characterize the molecular function of the candidate tumor suppressor genes in the critical region. With the insights gained, we hope to impact not only on the diagnosis of B-CLL patients but also open new avenues for treatment strategies.
How does the microenvironment support leukemic cells?
Malignant cells of patients suffering from B-cell chronic lymphocytic leukemia (B-CLL) accumulate in the lymphatic organs and peripheral blood and finally result in the failure of the immune system. However, if the malignant cells are cultured in vitro, they rapidly undergo apoptosis unless they are supported by non-malignant stroma cells. This suggests an intimate interaction of the leukemia cells with their microenvironmant and an essential dependance on survival signals from the supporting cells. In a systematic approach, we want to characterize the delicate interplay of these anti-apoptotic signals which have been discovered to date. Even though each of these signals prolongs the life of CLL cells in vitro, none of them can alone substitute the anti-apoptotic effect that the stromal cells have on the leukemic cells in vitro. If we are able to describe this crucial balance of supporting signals and identify its central nodes, these could be directly targeted in the patients using low but effective doses of compounds, some of which are already clinically established.
The microenvironment as a therapeutic target in chronic lymphocytic leukemia
Figure 1. Schematic representation of a CLL cell with established and experimental drug targets, as well as a classification of respective drugs (approved and experimental). Names of drugs with approval for use in CLL are given in red; drugs approved for use in other indications are shown in blue; drugs in various stages of clinical development are shown in black.
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Malignant cells of patients suffering from B-cell chronic lymphocytic leukemia (B-CLL) accumulate in the lymphatic organs and peripheral blood and finally result in the failure of the immune system. However, if the malignant cells are cultured in vitro, they rapidly undergo apoptosis unless they are supported by non-malignant stroma cells. This suggests an intimate interaction of the leukemia cells with their microenvironmant and an essential dependence on survival signals from the supporting cells. In a systematic approach, we want to characterize the delicate interplay of these anti-apoptotic signals which have been discovered to date. Even though each of these signals prolongs the life of CLL cells in vitro, none of them can alone substitute the anti-apoptotic effect that the stromal cells have on the leukemic cells in vitro. If we are able to describe this crucial balance of supporting signals and identify its central nodes, these could be directly targeted in the patients using low but effective doses of compounds, some of which are already clinically established. From databases we identified compounds that mimic the transriptomic signature of CLL cells upon loss of microenvironmental support. Intriguingly, these substances act via disruption of hypoxia-inducible pathways which are the most important support in the leukemic niche, and we can show that they are therapeutically active in a leukemia mouse model.
Ubiquitination and NOTCH1 signaling in leukemia
Figure 2: Proposed model of NICD degradation in CLL patients with and without FBXW7 mutations. NICD is targeted by wt FBXW7 (top), whereas mutations in the FBXW7 substrate binding domain G423V and W425C result in insufficient recognition of NICD (bottom, triangles). In contrast, mutations A503V as well as α-specific mutations T15VR and V154I do not impact on NICD binding (bottom, circle).
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Tumor-associated mutations are diverse and occur for individual genes only relatively rarely. However, genes are components of signalling pathways, so that mutations in different genes can affect the same signalling pathway. We are able to illustrate this for the NOTCH1 signalling pathway in chronic lymphocytic leukemia (CLL), which can be affected by mutations in NOTCH1 itself as well as in the FBXW7 gene.
The regulated degradation of proteins is important for cell health. FBXW7 plays an important role by recognising proteins such as NOTCH1 and "labelling" them for degradation. In chronic lymphocytic leukemia (CLL), FBXW7 is mutated in 2-6% of patients, but the consequence of the mutation in CLL was not known. CLL is the most common form of leukaemia in about 5000 people in Germany every year. Numerous therapies make life with CLL easier, but do not cure the disease. Patients with therapy resistance have a significantly reduced prognosis and for a small number of patients CLL can transform into an aggressive lymphoma due to the accumulation of unfavourable mutations.
In order to better understand the role of the FBXW7 mutation in CLL, we have characterized the FBXW7 mutations and were able to show that FBXW7 mutations in CLL lead to a growth advantage for the leukemia cells by the indirect upregulation of the NOTCH1 signaling pathway.
Mutations can rarely be correlated with a response of patients to therapies. However, it is known that CLL patients with NOTCH1 mutations do not respond to therapy with anti-CD20 antibodies. Our finding that mutations in FBXW7 lead to upregulation of the NOTCH1 pathway similar to patients with NOTCH1 mutations means that patients with FBXW7 mutations are unlikely to respond to antibody therapy. These mutations can therefore also be used to predict response, but this needs to be further investigated.
Epigenetic aberrations explain leukemogenesis and help prognostication of patients
Figure 3. Instead of the conventional �watch-and-wait� treatment, the novel concept is the identification and integration deregulated epigenetic markers for a prognostic score and patient stratification
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Currently, a paradigm shift takes place in cancer therapy: Global DNA damaging chemotherapies are becoming replaced by personalized treatments with defined molecular therapeutic goals. This general development specifically applies to chronic lymphocytic leukemia (CLL) where novel therapies target microenvironmental interaction and B-cell receptor (BCR) signaling. In our previous work we have identified genome-wide changes of epigenetic networks in CLL, which allow for novel patient stratification schemes beyond conventional markers. We now want to take advantage of this unique opportunity and available retro- and prospective clinical trials. We take a mechanistic approach to the identification of novel epigenetic biomarkers by understanding how BCR signaling in CLL patient cells impacts on the deregulated transcriptional and epigenetic landscape and the effectivity of new therapeutic inhibitors of BCR signaling depending on the patient-specific epigenetic landscape. On this basis, we further develop the application of epigenetic biomarkers for testing in clinical trials: (i) to overcome chemoresistant disease, where we aim to understand the molecular mode of action via its imprint on the epigenetic networks in CLL cells and (ii) where we will exploit the opportunity to identify epigenetic signatures that predict response to first-line treatment in asymptomatic patients.