Combining PRC2 inhibitors with metronomic chemotherapy: A promising therapeutic strategy for diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma (NHL). Most attempts to combine standard chemo-immunotherapy with targeted drugs have failed due to limited synergy or excessive toxicity. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) acting as a transcriptional repressor by methylating histone H3 at lysine 27 (H3K27me3). Activating mutations in EZH2 in NHL correlate with poor survival. We aimed at using metronomic chemotherapy (mCHEMO)-the frequent, regular administration of low-dose cytotoxic drugs that maintain prolonged and active drug-plasma levels-in combination with new epigenetic therapies as a promising, more effective and less toxic approach for DLBCL treatment. In vitro proliferation assays were performed on both EZH2 Y641F mutant (SU-DHL10) and EZH2 wild-type (OCI-LY3, Toledo) DLBCL cell lines exposed to thrice-weekly vinorelbine (mVNR) and daily PRC2 inhibitors (PRC2i: tazemetostat, valemetostat, ORIC-944), alone and in concomitant combination, for 144h. Synergism was measured by the Combination Index method and the Loewe additivity model. PRC2i target engagement was assessed by H3K27me3 western blot. The modulation of gene expression in DLBCL cells treated with 144h-daily valemetostat was evaluated by RNA sequencing. The 144h exposure of daily PRC2i and mVNR inhibited the DLBCL cell viability in a concentration-dependent manner. Among the PRC2i, valemetostat exhibited the greatest cytotoxic effect on SU-DHL10, OCI-LY3 and Toledo cells (IC 50 s: 3.09nM, 371nM, and 303nM, respectively). ORIC-944 also effectively reduced DLBCL cell growth (IC 50 s: 8.7nM, 721nM, and 1,388nM, respectively). Tazemetostat significantly inhibited DLBCL cell viability, with higher IC 50 values (1,389nM, 3,481nM, and 8,597nM, respectively). mVNR substantially arrested the proliferation of all cell lines (IC 50 s: 508.9pM, 62.9pM, and 675.6pM, respectively). The strongest synergistic effect was observed for the mVNR+valemetostat combination, while mVNR+ORIC-944 showed synergism only for high percentage of affected DLBCL cell fractions. PRC2i markedly reduced H3K27me3 levels in DLBCL cells. Among the differentially expressed genes, we observed more up-regulated than down-regulated transcripts upon PRC2 inhibition, and an increased expression of key p53-dependent pro-apoptotic genes. These findings offer a solid rationale for combining PRC2i and mCHEMO as an encouraging therapeutic approach for DLBCL, particularly in elderly or fragile patients, due to their low toxicity profiles. Overall, these results warrant additional in vitro investigations to elucidate the mechanisms underlying the reported effects and in vivo experiments in DLBCL models to validate our new therapeutic strategy, which holds potential for rapid translation into future clinical trials.