Functional studies of the neoplastic B cells In chronic lymphocytic leukemia (Doctoral thesis)

Χαρτοματσίδου, Ελισάβετ

1.The histone methyltransferase EZH2 as a novel prosurvival factor in clinically aggressive chronic lymphocytic leukemiaThe histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 overexpression has been reported in many types of cancer and associated with poor prognosis. Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world, characterized by the progressive accumulation of small, mature B lymphocytes in blood, bone marrow and lymphatic tissues. CLL is a very heterogeneous malignancy at both clinical and biological levels. In our previous study we demonstrated that EZH2 is highly expressed in a subgroup of patients (subset #1), characterized by a stereotyped B cell receptor (BcR) with unmutated immunoglobulin heavy variable genes (U-CLL) and an aggressive disease course.Here we investigated in a larger cohort of patients the expression and functionality of EZH2 in CLL. Aggressive cases with unmutated IGHV genes (U-CLL) displayed significantly higher EZH2 expression compared to indolent CLL cases with mutated IGHV genes (M-CLL), both in mRNA and protein levels; furthermore, in U-CLL EZH2 expression was upregulated with disease progression. EZH2high cases displayed high H3K27me3 levels and increased viability suggesting that EZH2 is functional and likely confers a survival advantage to CLL cells. This argument was further supported by siRNA-mediated downmodulation of EZH2 which resulted in increased apoptosis and reduced H3K27me3 levels in EZH2high cases. Treatment of primary CLL cells with EZH2 inhibitors, namely EPZ6438, GSK343 and GSK126 induced downregulation of H3K27me3 levels leading to increased cell apoptosis.In conclusion, EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival. Pharmacologic inhibition of EZH2 catalytic activity promotes apoptosis, highlighting EZH2 as a novel potential therapeutic target for specific subgroups of patients with CLL. 2.Inhibition of EZH2 and immune signaling exerts synergistic anti-tumor effects in ChronicLymphocytic LeukemiaMultiple lines of evidence highlight the role of external drive in CLL pathogenesis and suggest that microenvironmental stimuli received from the CLL cells through their immune receptors (e.g. BcR, TLRs, CD40, cytokine receptors and complement receptors) play an important role in the survival and proliferation of CLL cells in vivo. Here, we explored if EZH2 expression and function could be affected by microenvironmental triggering and whether synergism may exist between EZH2 and signaling inhibitors. The study group included cases responsive to BcR stimulation. Ex vivo stimulation of primary CLL cells from these cases through the BcR, CD40 and, especially, TLR9 led to EZH2 upregulation, accompanied by elevated H3K27me3 levels and significant increase in cell viability, suggesting that EZH2 expression likely contributes to the anti-apoptotic phenotype induced by microenvironmental signals. Ex vivo exposure to Ibrutinib exerted a significantly higher pro-apoptotic effect in CLL cells from EZH2high versus EZH2low cases, further highlighting links between microenvironmental triggering and EZH2 expression and functionality. Next, we investigated whether signaling inhibition might also modulate EZH2 expression and functionality in vivo by longitudinal profiling of CLL patients under Ibrutinib treatment. EZH2 protein levels and H3K27me3 declined at +1 month under treatment. However, at later time points they returned to levels similar to the pre-treatment sample, suggesting that EZH2 expression and functionality may characterize persisting cells, retained after prolonged treatment. Ex vivo treatment of primary CLL cells and splenic B cells from Tcl1 leukemic mice (the most common murine model in CLL) by combined EZH2 and signaling inhibitors (Ibrutinib, Idelasisib) was more effective in promoting apoptosis than single inhibition. Finally, ex vivo exposure to EZH2 inhibitorsinduced apoptosis in primary CLL cells from cases with suboptimal response to signaling inhibitors, further highlighting that targeting both processes might represent a novel therapeutic strategy for CLL.
Alternative title / Subtitle: intrinsic and extrinsic mechanisms
ενδογενείς και εξωγενείς μηχανισμοί
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Subject classification: Lymphocytic leukemia
Keywords: ΕΖΗ2 Ιnhibitors,Ibrutinib,Αναστολείς μεθυλοτρανσφεράσης ΕΖΗ2,Αναστολείς σηματοδότησης
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