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Background EZH2, a histone methyltransferase and core component of PRC2, is frequently overexpressed in tumors and contributes to immune evasion. EZH2 inhibition has been shown to reprogram cancer cells toward a more immunogenic state, enhancing CAR-T cell activation, expansion, and tumor infiltration in preclinical models. These findings have led to growing interest in combining EZH2 inhibitors with adoptive cell therapies. Tazemetostat, a potent EZH2 inhibitor, is FDA-approved for epithelioid sarcoma and follicular lymphoma and is under investigation in multiple solid and liquid tumors. However, EZH2 also plays a critical role in preserving T-cell epigenetic stability and function. Here, we investigate this therapeutic tradeoff and show that while EZH2 inhibition may enhance tumor immunogenicity, it impairs long-term CAR-T cell persistence and efficacy.Methods Human CD8 + T cells were activated with CD3/CD28/CD2 and transduced on day 0 with a lentiviral vector encoding a CD19-specific CAR. On day 5, CAR-T cells were co-cultured with nuclear RFP-labeled NALM6 (CD19+) target cells in the presence or absence of 0.5 µM tazemetostat (EZH2i). Tumor cell burden 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. In parallel, CD8+ T cells were cultured with or without 1 µM EZH2i for 7 days. Histone H3K27me3 levels were measured by Western blot. Expression of PD-1, CTLA-4, and LAG-3 was assessed by immunoblotting, and polyfunctionality (IL-2+/IFN-γ+/GZMB+) was measured via flow cytometry.Results EZH2i treatment did not impair initial CAR-T cytotoxicity against CD19 + NALM6 cells. However, prolonged co-culture revealed a significant decline in tumor control beyond day 4. EZH2i-treated CAR-T cells sustained tumor clearance for an average of 5.7 killing rounds (48 hours/round) compared to 14.0 rounds in untreated controls (p=0.0002, n=3). Western blot analysis confirmed loss of the repressive histone mark H3K27me3 following 7-day culture with EZH2i (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-γ+/GZMB+) CD8+ T cells and a shift toward an exhausted phenotype upon EZH2i including transcriptional elevation of Tox2.Conclusions We demonstrate that while EZH2 inhibition does not impair the short-term function of CD19-specific 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.