BCL-2 disruption during CAR T cell production augments potency through non-apoptotic adaptive changes

BH3 mimetics were initially designed to induce cancer cell death through targeting key anti-apoptotic members within the BCL-2 family of proteins. Venetoclax, the only FDA-approved BCL-2-specific BH3 mimetic, has demonstrated robust clinical efficacy in killing acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells attributed to its direct apoptotic tumor effect. However, little is known regarding the direct effect of venetoclax on tumor infiltrating T cells. Recent studies have reported apoptosis-independent immunomodulatory activities of venetoclax that still are not fully understood. Given the vital role of BCL-2 family proteins in the dynamic processes of T cell activation, differentiation to effector or memory phenotype and contraction, it becomes imperative to dissect the direct effect of BH3 mimetics on T cell functions. We previously demonstrated that murine T cells expanded for 5 days in vitro in the presence of venetoclax acquire adaptive-like reprograming leading to cell death resistance. Further, long-term in vivo treatment with venetoclax results in transcriptional alterations of signaling pathways involved in T cell activation and exhaustion resistance. Herein, we investigate whether this venetoclax-induced T cell reprograming can be employed to improve the efficacy of adoptive T cell therapy. Using murine and human CD19-targeted chimeric antigen receptor (CAR) T cell therapy (CD19CART) as a model, we show that venetoclax treatment during expansion of CD19CARTs prepared from T cells isolated from healthy donors and patients exhibit enhanced antitumor efficacy in vitro and in vivo using pre-clinical xenograft models of lymphoma and leukemia. Mechanistically, bulk and single-cell RNA sequencing analyses reveal that venetoclax treatment during CART manufacturing transcriptionally reprograms CART cells at key T cell signaling (JAK/STAT5, PI3K/AKT, NFKB) and metabolic (PGC1α, OXPHOS, glycolysis) pathways to enhance CAR T cell fitness and durability, mechanisms dependent upon BCL-2 affinity to venetoclax. Our study highlights a possible novel therapeutic approach to induce adaptive anti-apoptotic reprograming to enhance the efficacy of CAR T cell therapy and supports the continued examination of BH3 mimetics as immune modulators.