Epigenetic reprogramming via EZH1/2 inhibition enhances T cell-mediated immunotherapies against multiple myeloma

Abstract Introduction: Although BCMA-targeted CAR-T therapies and CD3×BCMA bispecifics (e.g., teclistamab) have shown clinical promise, most r/r MM patients eventually relapse due to tumor-intrinsic resistance and T-cell dysfunction. Emerging evidence implicates epigenetic regulators promote immune evasion, tumor cell stemness, and therapeutic resistance in MM. We previously demonstrated that the catalytic subunits of the polycomb repressive complex 2, EZH1 and EZH2, contribute to immune escape and resistance to immunotherapies in germinal center–derived B-cell lymphomas, thus influencing the efficacy of anti-CD19 CAR-T therapy. In MM, EZH2 is frequently overexpressed in cytogenetically high-risk disease and at relapse. We, therefore, hypothesized that inhibition of EZH1 and EZH2 could enhance T cell–based immunotherapy for MM by both directly impairing tumor growth and survival, and by boosting T cell–mediated cytotoxicity.Methods and Results: We first demonstrated a strong tumor-intrinsic anti-clonogenic effect of the EZH2 inhibitor (tazemetostat) and the dual EZH1/2 inhibitor (valemetostat) on the MM cell line RPMI-8226. Colony formation assays revealed a significant, progressive reduction in clonogenic growth following treatment with tazemetostat and valemetostat, with the most pronounced effect observed with dual inhibition (day 10, number of colonies, RPMI-8226: DMSO vs EZH1/2i, 622.5 vs 292.5; p=0.0046). We next employed clinically relevant anti-BCMA CAR-T constructs and performed long-term killing assays combining MM cell lines with EZH1/2 inhibitors, with or without CAR-T cells. Notably, EZH1/2 inhibitors significantly enhanced cytotoxicity against MM cell lines (e.g., RPMI-8226; day 5; CAR-T-BCMA+EZH1/2 inhibitor vs CAR-T-BCMA+DMSO, p<0.001). Similarly, in assays using the FDA-approved anti-CD3/anti-BCMA bispecific antibody teclistamab and human T cells, we observed an additive tumor-killing effect (48 hrs; teclistamab+T cells+EZH1/2 inhibitor vs teclistamab+T cells+DMSO, p<0.01). Importantly, EZH1/2 inhibition did not impair T-cell viability or effector function. ELISA of co-culture supernatants showed dose-dependent increases in IL-2 and TNF following teclistamab dose escalation, with no significant impact from the inclusion of EZH1/2 inhibitor, confirming intact T-cell cytokine production (24 hrs; teclistamab 1 nM; IL-2 [pg/mL]: DMSO vs EZH1/2 inhibitor, 29 vs 23.7, p=ns; TNF: 503 vs 528, p=ns). Finally, we developed an intraosseous orthotopic xenograft model of MM using RPMI-8226 cells. Three days after tumor implantation, mice were administered valemetostat (100 mg/kg, orally, once daily for five weeks) or a vehicle control. One week after the RPMI-8226 injection, the mice were randomized to receive CART-BCMA therapy (0.8 × 10⁶ cells per mouse, intravenously). In this model, continuous valemetostat treatment enhanced the tumor-suppressive effect of CART-BCMA, supported T cell expansion in peripheral blood, and was not associated with any notable toxicities such as weight loss or GVHD. Of note, valemetostat/CART-BCMA combination doubled complete response rate (80% vs 40%) and showed increased serum levels of IFN-γ, a key cytotoxic cytokine, indicating heightened T cell activation and cytolytic function. Mechanistically, EZH1/2 inhibition did not alter BCMA expression at either the RNA or protein level, indicating that target antigen expression was preserved. However, RNA-seq analysis of RPMI-8226 and MM1.S cells treated with EZH1/2 inhibitors revealed upregulation of genes involved in cell adhesion (e.g., LAMA1, LAMA2, VCAN, PECAM1), immunogenic signaling (e.g., CXCL9, CXCL10, CXCL14, CD70, TNFSF9), and differentiation (SDC2), along with downregulation of cell cycle–associated genes. These results suggest that EZH1/2 inhibitors reprogram MM cells to a more immunogenic and less proliferative state, enhancing their susceptibility to immune-mediated attack. Conclusions: These findings support a dual mechanism of action EZH1/2 inhibitors directly suppress MM clonogenicity and immune evasion, while concurrently enhancing the function and cytotoxicity of T cell–based immunotherapies. By preserving T-cell activity and reprogramming tumor cells, EZH1/2 inhibition represents a promising strategy to improve the durability and depth of response to both CAR-T and bispecific antibody therapies in MM. These data support the development of clinical trials combining EZH1/2 inhibitors with CART and bispecific antibodies in r/r MM.