Generating EZH2-inhibitor resistant CAR-T cells to sustain function for combination therapy

Background: EZH2, a histone methyltransferase and core component of the PRC2 complex, is often overexpressed in tumors and contributes to immune evasion. EZH2 inhibition has been shown to reprogram cancer cells toward a more immunogenic state and enhance lymphocyte infiltration into tumors in preclinical models. These findings have led to growing interest in combining EZH2 inhibitors with adoptive cell therapies. EPZ-6438 (Tazemetostat), a potent EZH2 inhibitor (EZH2i), is FDA-approved for epithelioid sarcoma and follicular lymphoma and is under investigation in multiple solid and liquid tumors. However, EZH2 is also critical for preserving T cell epigenetic stability and function. Here, we investigate this therapeutic tradeoff and show that while EZH2i may enhance tumor immunogenicity it impairs long-term CAR-T cell persistence. Methods: Human CD8⁺/CD3⁺ T cells were activated with CD3/CD28/CD2 and transduced on day 0 with a CD19-specific CAR lentiviral vector. On day 3, CAR-T cells were edited with CRISPR/Cas9 to introduce EZH2i-resistant mutations into the endogenous EZH2 gene. On day 9, CAR-T cells were co-cultured with nuclear RFP-labeled CD19⁺ target cells with or without 0, 0.5, 1, 2 μM EZH2i. Tumor cell count was measured every 4 hours using automated imaging, and fresh tumor cells and drug were added every 48 hours until loss of T cell tumor control. This was repeated in the OT1 T cell murine system with RFP-labeled SIINFEKL + B16F10 melanoma cells. In parallel, CD8⁺/CD3⁺ T cells cultured with or without chronic exposure to 1 μM EZH2i were used for phenotype and functional analysis. Histone H3K27me3 levels and PD-1, CTLA-4, and LAG-3 expression were measured by immunoblot. Polyfunctionality (IL-2⁺/IFN-gamma⁺/GZMB⁺) was measured via flow cytometry. Results: EZH2i treatment did not impair initial CAR-T cytotoxicity against tumor cells. However, prolonged co-culture revealed a significant decline in tumor control past the fourth round of co-culturing. EZH2i-treated WT CAR-T cells sustained tumor clearance for an average of 5.7 killing rounds compared to 14.0 rounds in untreated controls (p=0.0002, n=3). Immunoblots confirmed loss of the repressive histone mark H3K27me3 following chronic EZH2i exposure (p<0.0001, n=8), alongside elevated expression of exhaustion markers: PD-1 (1.6-fold, p=0.0502, n=8), CTLA-4 (7.4-fold, p<0.0001, n=6), and LAG-3 (53.3-fold, p=0.0198, n=6). Flow cytometry further revealed a reduced frequency of polyfunctional (IL-2⁺/IFN-gamma⁺/GZMB⁺) CD8⁺ T cells and a shift toward an exhausted phenotype. Conclusions: We demonstrate that while EZH2i does not impair the short-term function of CAR-T cells, prolonged exposure significantly reduces persistence and anti-tumor efficacy. To overcome this limitation, we are developing strategies to engineer CAR-T cells resistant to EZH2i, enabling future combination therapies that harness both EZH2 inhibition and adoptive T-cell therapy.