Abstract Direct competition between tumor-infiltrating lymphocytes and cancer cells for metabolic resources often renders T cells dysfunctional. It is becoming clearer that T cell metabolic changes can precede functional exhaustion. In the case of solid tumors, the driving force of this dysfunction is thought to be a result of inhibitory metabolism (e.g. glucose deprivation and mitochondrial dysfunction). The mechanisms connecting inhibitory metabolism and T cell metabolic exhaustion are largely unknown. Interestingly, T cells undergo a loss of the histone methyltransferase EZH2 (H3K27me3) during tumor infiltration. Methyltransferases, like EZH2, depend on mitochondria to supply S-Adenosyl-L-Methionine (SAM), the primary methyl donor. We sought to determine if there was a link between EZH2 and mitochondrial function. In this work, we utilized highly specific EZH2 inhibitors to model acute inhibition of EZH2 in CD8+ and CD4+ T cells. Taking an unbiased, multi-omics approach (proteomics, RNAseq, ChIPseq) we fully interrogate the downstream consequences of EZH2 inhibition in T cells. The loss of H3K27me3 does indeed lead to an induction of mitochondrial dysfunction, which in turn, drives a dependency on glycolytic metabolism and sensitivity to glucose withdrawal. The metabolic shift phenotype was confirmed using extracellular metabolic flux analysis. In T cells, we found the loss of H3K27me3 repression of the Cdkn2aArf locus to be a major contributor to mitochondrial dysfunction, independent of the canonical role to stabilize p53. Furthermore, we show Arf-/- mice are resistant to EZH2-inhibition induced mitochondrial dysfunction. Reprogramming tumor-specific T cells to exogenously express a gain-of-function EZH2 mutant (Y641F) resulted in an enhanced ability of T cells to inhibit solid tumor growth. This work demonstrates the potential for manipulation of EZH2 in cellular therapies for solid tumors with harsh metabolic conditions and sheds light on the dynamic interplay of epigenetics and metabolic sufficiency. Citation Format: Brian Koss, Bradley D. Shields, Erin M. Taylor, Aaron J. Storey, Stephanie D. Byrum, Allen J. Gies, Charity L. Washam, Samrat Roy Choudhury, Jeong Hyun Ahn, Hidetaka Uryu, Kimberly J. Krager, Tung-Chin Chiang, Samuel G. Mackintosh, Rick D. Edmondson, Nukhet Aykin-Burns, Gang Greg Wang, Alan J. Tackett. Epigenetic control of tumor-infiltrating lymphocyte metabolic-exhaustion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1029.