Background Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve complete and durable responses. Identifying new strategies to enhance the cancer killing capacity of these cells is therefore crucial in the effort to improve therapies. One avenue for CTL manipulation is through altering cellular metabolic programs. Prior studies have shown pan-inhibition of glutamine metabolism (glutaminolysis) using the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) enhances CTL function. However, lack of specificity and toxicity associated with DON demonstrate the need for a more directed glutaminolysis targeting approach.
Methods To investigate the role of individual enzymes targeted by DON on anti-tumor CTLs, I utilized a glutaminolysis focused pooled CRISPR library. I developed a model in which CRISPR-edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts.
Results While deletion of multiple DON targets decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – the enzyme responsible for catalyzing the conversion of glutamate to glutamine – conferred a fitness advantage to CTLs. Upon inhibition of GS, cells may increase cellular levels of glutamate and subsequent downstream metabolites. Mitochondrial stress test data suggests this results in a decrease in glycolysis and concurrent increase in mitochondrial respiration capacity. Consistent with this finding, GS knockout CTLs contain more mitochondria with increased mitochondrial polarization by flow cytometric analysis. Importantly, increased mitochondrial respiration is associated with long-lived memory subsets of T cells, which are crucial for durable responses in patients receiving cell-based immunotherapies. Lastly, in vitro data demonstrate increased inflammatory cytokine production of CTLs upon CRISPR-mediated genetic knockout or pharmacologic inhibition of GS.
Conclusions Together, these results identify glutamine synthetase as a potential metabolic target to enhance anti-tumor immunity of CTLs, offering a new strategy to enhance immunotherapy.
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