Abstract
Background: Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable success in hematologic malignancies but faces significant challenges in solid tumors due to the immunosuppressive tumor microenvironment (TME). Among these, hypoxia plays a vital role, yet the molecular mediators that link hypoxia to CAR-T dysfunction remain incompletely understood. Methods: Anti-mesothelin (MSLN) CAR-T cells were cultured under normoxic (21% O2) and hypoxic (1% O2) conditions for six days. We assessed cell expansion, phenotypes, cytotoxicity, and metabolic features. RNA sequencing was conducted to identify key gene expression changes induced by hypoxia. Findings were further validated using anti-CD70 CAR-T cells. Results: Hypoxia reduced CAR-T proliferation, increased apoptosis, lowered memory phenotypes, raised exhaustion, and weakened cytotoxicity in short-term and long-term assays. Transcriptomic and metabolic analyses showed metabolic reprogramming with increased glycolysis and reduced oxidative phosphorylation. Among the dysregulated genes, the serine/threonine-protein kinase PIM3 emerged as a previously underexplored mediator of hypoxia-driven dysfunction. Genetic or pharmacologic inhibition of PIM3 counteracted hypoxia-induced impairment, enhancing memory phenotypes of CAR-T cells, and improving their anti-tumor activity both in vitro and in vivo. Conclusions: This work identifies PIM3 as a previously underexplored target that links hypoxia to CAR-T cell dysfunction and demonstrates that PIM3 inhibition can reverse these effects. These findings provide a mechanistic rationale for incorporating PIM3 inhibition into CAR-T cell manufacturing or engineering to improve their therapeutic potential in hypoxic solid tumors.