External Stimuli Modulate EZH2 Expression in Chronic Lymphocytic Leukemia. Rationale and Evidence for Synergistic Anti-Tumor Effects of B Cell Signaling and EZH2 Inhibitors

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). We have previously reported that EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Moreover, we demonstrated that EZH2 pharmacological inhibition induced down-regulation of H3K27me3 levels leading to increased cell apoptosis. Here we sought to investigate if EZH2 expression could be affected by microenvironmental triggering, a key driver of CLL development and evolution.

The study group included 10 CLL cases (9 with unmutated and 1 with mutated IGHV genes) that had previously been screened and found responsive to cross-linking of the B cell receptor (BcR) with anti-IgM. CD19⁺ cells were negatively selected from peripheral blood and stimulated or not through the BcR for 14 hours. Using RQ-PCR we found that stimulation with anti-IgM showed opposing effects on EZH2 mRNA levels, since 5 cases upregulated while the remaining 5 downregulated EZH2. In contrast, stimulation with CpG or CpG/CD40L for 14 hours caused pronounced and consistent upregulation of EZH2 mRNA levels compared to unstimulated cells (FD=8.7, p=0.03 and FD=11.3, p=0.03, respectively; n=6). This effect was also obvious at the protein levels in cells treated with CpG, CD40L and CpG/CD40L for 24 hours, where EZH2 protein was significantly upregulated as revealed by Western blot analysis (FD=3.9, p=0.002; FD=1.7, p=0.005; and FD=2.9, p=0.01, respectively; n=6). In CpG-stimulated cultures, the induction of EZH2 was accompanied by a significant increase in cell viability (FD=2, p=0.002; n=6), supporting the conclusion that EZH2 expression likely contributes to the anti-apoptosis phenotype induced in CLL cells by microenvironmental signals. Prompted by these observations, we then investigated whether B cell signaling inhibition might also modulate EZH2 expression and H3K27me3 levels in vivo by longitudinal profiling of another group of 9 CLL patients that were under ibrutinib treatment. In patients at +1 month under ibrutinib, when, as shown by us and others, CLL cells become anergized through the BcR, we observed a significant decrease in EZH2 protein (FD=3, p=0.0007; n=9) as well a trend for reduced H3K27me3 levels (FD=1.4, p=0.07; n=6) compared to the pre-treatment samples, further highlighting the links between BcR signaling and EZH2 expression and functionality. Next, we assessed ex vivo the effects of ibrutinib on CLL cell viability in cases with differential EZH2 expression [EZH2_high (n=7) vs EZH2_low (n=6)] and found decreased viability in treated cells compared to control cells (DMSO-exposed) in EZH2^high cases (FD=1.9, p=0.0025), with no difference in EZH2^low cases.

Finally, we examined potential synergistic effects of B cell signaling inhibitors [ibrutinib (IB), idelalisib (IDE)] and EZH2 inhibitors (GSK343, GSK126) in primary CLL cells. To this end, CD19⁺ cells from 6 CLL cases were first pre-stimulated with CpG for 24 hours and then exposed to either single inhibitors (IB, IDE, GSK343, GSK126) or combinations of B cell signaling and EZH2 inhibitors (IB/GSK343, IDE/GSK343, IB/GSK126). Flow cytometry analysis after 3 days in culture revealed significantly lower CLL cell viability and H3K27me3 levels in CLL cells exposed to combined versus single treatment (FD≥ 1.5, p<0.05).

In conclusion, we show that EZH2 expression is affected by signals emanating from CLL microenvironment. Moreover, we provide for the first time evidence that the combination of BcR signaling inhibitors widely used in CLL therapy and EZH2 inhibitors currently tested in phase I/II clinical trials in lymphoid malignancies has synergistic effects, suggesting that targeting both processes might represent a rational and promising therapeutic strategy.