RNAseq of activated CD8+ T cells with and without Glutamine Synthetase

Cellular immunotherapy has revolutionized the treatment of hematologic malignancies yet has limited success in the solid tumor microenvironment (TME). While insufficient nutrients can lead to T cell metabolic stress in the TME, the glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) can paradoxically enhance anti-tumor immunity. Because DON inhibits a range of glutamine metabolizing enzymes, including some that are essential and may contribute to dose-limiting toxicities, mechanisms underlying DON-induced anti-tumor activity have remained unclear. Here we aimed to identify specific DON targets responsible for increased T cell anti-tumor activity and test if more selective inhibition of glutamine metabolism could replicate the effects of DON with reduced toxicity. CRISPR screening in the TME of DON-relevant glutamine metabolizing enzymes identified some targets that were essential in tumor infiltrating CD8 T cells (TILs), but that TILs lacking the DON target glutamine synthetase (GS) were enriched. Upon adoptive T cell transfers, GS-deficient CD8+ T cells displayed improved survival, a more stem-like phenotype, and greater anti-tumor function. GS converts glutamate to glutamine and GS-deficient cells exhibited increased intracellular glutamate and reduced glutathione levels, which correlated with enhanced mitochondrial respiration and resistance to reactive oxygen species (ROS). Pharmacological inhibition of GS reduced tumor burden in multiple orthotopic murine tumor models in a manner dependent on adaptive immunity. Our findings establish GS as a key metabolic regulator of CD8+ T cells in the TME. By preserving intracellular glutamate, GS inhibition reprograms T cells for improved survival and function, offering a promising therapeutic strategy to enhance immune-based cancer treatments.