AMPK enhances CART function through increased oxidative capacity and improved control of cellular ROS 3375

Abstract Description Metabolic insufficiency limits Chimeric Antigen Receptor (CAR) T-cell therapy efficacy. The cellular energy sensor, AMP-activated protein kinase (AMPK), regulates energy homeostasis. Overexpressing the regulatory subunit (AMPKγ2) increases AMPK signaling and facilitates oxidative metabolism in human T cells. We hypothesized that AMPKγ2 overexpression would strengthen CART metabolic fitness and improve anti-leukemia function. Human T cells were co-transduced with a CD19 targeting CAR and either AMPKγ2 (AMPK-CART) or an empty vector. AMPK-CART cells displayed a 25% elevation in spare respiratory capacity and greater cytotoxicity against NALM6 leukemia cells in vitro. Consistent with these findings, transfer of AMPK-CARTs significantly improved survival in leukemia-bearing mice. Mechanistic studies comparing AMPKγ2-overexpressing (AMPKOE) to AMPKα1-knockout (AMPKKO) T cells via bulk RNA sequencing revealed that both groups increased the oxidative phosphorylation gene set, while only AMPKKO cells upregulated genes promoted by reactive oxygen species (ROS), suggesting AMPK-mediated regulation. Flow cytometry confirmed lower ROS levels, alongside increased mitochondrial mass, in AMPKOE cells. In summary, AMPKγ2 overexpression enhances CART oxidative capacity, anti-leukemia efficacy, and ROS control. Future studies will elucidate further AMPK’s role in ROS modulation and evaluate AMPKOE-CARTs in additional tumor models to advance therapeutic options against various cancer types. Funding Sources Supported by Curing Kids Cancer and Hyundai Hope on Wheels Scholar award. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)