Abstract BCAT1, the enzyme responsible for the reversible transamination of leucine in the cytosol, has recently been implicated in the development and growth of various types of cancer. The limited expression of BCAT1 in adult tissues under physiological conditions suggests that the enzyme is a sensible target for drug development. To better understand the role of BCAT1 in cancer, we examined its gene expression at the single cell level (scRNAseq) in specimens obtained from lung, colorectal, breast, and ovarian cancer patients (n=36; >200,000 single cells). In those samples, Bcat1 gene expression was primarily associated with myeloid cells and fibroblasts with the exception of ovarian cancer in which robust Bcat1 presence was also seen in cancer cells. In tumor infiltrating lymphocytes (TILs), enrichment in Bcat1 gene expression was observed in exhausted CD4+ and CD8+ T cells compared to other T cell subtypes; this was independent of the cancer type examined. We speculated that inhibition of BCAT1 in TILs reverses exhaustion and has a therapeutic impact in cancer. In the syngeneic CT26 colon cancer model, combination treatment of a BCAT1 inhibitor (ERG245; ip administration of 5 mg/kg, given at days 7, 8 and 9 after tumor cell inoculation) and an anti-PD1 antibody (ab) (clone RMP1-14; ip administration given at days 7, 11, 14, and 18) led to eradication of established tumors. In the same model, limited treatment of large tumors (average size of ~600 mm3) with ERG245+anti-PD1 ab suppressed tumor growth and increased the frequency of CD8+ T cells expressing Granzyme B and IFNγ by ~50% compared to anti-PD1 monotherapy. Mechanistically, in vitro treatment of newly activated hCD8+ T cells with ERG245 impaired translocation of the lactate transporter MCT1 to the cell surface and increased the levels of intracellular lactate within 30 min of activation leading to upregulation of transcription factor ATF3. ERG245-driven metabolic reprogramming of CD8+ T cells towards an oxidative phenotype (increased OXPHOS) was also observed at 24h of treatment. Withdrawal of ERG245 restored intracellular lactate levels without reversing the metabolic reprogramming of the cells. We propose that these are elements of a mechanism that links temporal inhibition of BCAT1 to the rise of highly energetic CD8+ T cells with increased cytotoxicity and proliferative capacity in vitro and in vivo. Citation Format: Adonia E. Papathanassiu, Francesca Lodi, Hagar Elkafrawy, Michelangelo Certo, Hong Vu, Jeong Hun Ko, Jacques Behmoaras, Claudio Mauro, Diether Lambrechts. BCAT1 as a druggable target in immuno-oncology [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 3402.