Reprogramming T Cell EZH2 to Counteract CAR T Cell Dysfunction during Tumor Control

T cell dysfunction, including memory loss and exhaustion, is a major limitation of CAR T cell therapy efficacy. Manipulating transcription factor (TF) activity ameliorates their exhaustion and memory loss during tumor control suggests tumor exposure may cause dysregulation of transcriptional programs in CAR T cells. EZH2, which catalyzes trimethylation of H3 at lysine 27 (H3K27me3) to orchestrate expression of multiple gene programs, plays a central role in the regulation of T cell immune response in mice, such as the reaction to tumor, infection and alloantigens. However, whether EZH2 is required for human CAR T cells to destroy tumor and whether tumor targets T cell EZH2 to induce CAR T cell dysfunction have never been previously defined. Here we demonstrate that EZH2 is a master regulator of CAR T cell responses critical for tumor control. Prolonged tumor exposure disrupts EZH2 function and reprogramming EZH2 in CAR T cells renders them enhanced capacity to resist tumor-induced dysfunction and memory loss. We observed that CRIPSR-Cas9 mediated EZH2 ablation in CD19-directed human CAR T cells resulted in significantly impaired expansion of CAR T cells in human xenograft Raji leukemia-bearing NSG mice, decreased CD45RA^-CD62L⁺ central memory (T_CM) phenotype cells, and increased short-lived CD45RA⁺CD62L^- T_EMRA cells and PD-1⁺TIM3⁺ terminal exhausted (T_EX) cells, and ultimately the failure to eliminate leukemia. EZH2 regulated genes that are critical for sustaining cell proliferation (CDK1, CCNB1, CCNB2, TOP2A) but limiting effector T cell differentiation (PRDM1, T-BET), T cell interactions with extracellular matrix (ITGA3, ITGB3, ITGA5) and T cell expression of genes encoding protein kinases (SRC and FAK). Furthermore, EZH2 inhibited constitutive activation of AKT in CAR T cells under either homeostatic or antigen-activation conditions, which drives T cell exhaustion and memory loss. Pharmacological inhibition of PI3K, SRC and FAK resulted in significant reduction of AKT activity in EZH2-sufficient CAR T cells, but to a much less extent in EZH2-ablated CAR T cells. Thus, EZH2 is important for repressing activation of gene programs that drive effector differentiation, exhaustion and memory loss, acting as a master regulator for CAR T cells to eliminate tumor. To further address whether T cell EZH2 is a central target for tumor to induce CAR T cell dysfunction, we used a ‘stress test’ model in which infusion of low doses of CAR T cells was insufficient to eliminate leukemia, thereby creating leukemia persistence in NSG mice. We found that persistent leukemia caused significantly decreased cellular levels of EZH2 protein and H3K27me3 in CAR T cells, which was associated with reduction of T_CM phenotype cells and increases of PD-1⁺TIM3⁺ T_EX-like T cells. Leukemia persistence triggered EZH2 protein degradation in CAR T cells via an antigen activation-dependent mechanism. We finally examined whether ectopic expression of which form of EZH2 in CAR T cells may enable them resistance to leukemia-induced exhaustion and memory loss. Adoptive transfer experiments showed that all NSG mice receiving CAR T cells with enforced expression of wild-type EZH2 (EZH2^WT), AKT phosphorylation-resistant EZH2 (EZH2^S21A) or naturally occurring gain-of-function EZH2 mutant (EZH2^Y646N) died from leukemia. In contrast, CD19-CAR T cells transduced with EZH2^S21A/Y646N dual mutant acquired greater capacity than vector control, EZH2^WT, EZH2^S21A or EZH2^Y646N to eliminate leukemia in NSG mice, with 42.9% (6/14) of them surviving over 70 days after transfer. Reprogramming CD19-CAR T cells with EZH2^S21A/Y646N skewed them differentiation away from terminal T_EX cells towards T_CM-phenotype cells, inhibited expression of gene programs that regulate cytokine production, cytokine receptor signaling and cell-cell adhesion, and decreased AKT activity. Collectively, our findings provide novel epigenetic insights into the pathophysiology of T cell tumor immunity and form the conceptual basis to enhance CAR T cell therapy efficacy, thereby addressing a major barrier to the progression of CAR T cell therapy.