Asparaginase enhances CAR-T cell antitumor immunity by asparagine metabolic reprogramming and central memory induction in ALL.

High levels of asparagine synthetase (ASNS) in acute lymphoblastic leukemia (ALL) lead to immunotherapy resistance. Our study showed that ASNS overexpression (OE) in NALM6-GL cancer cells attenuated chimeric antigen receptor (CAR)-T cell-mediated cancer cell lysis. Asparaginase (ASPG) is an approved drug that breaks down circulating asparagine in leukemia cells, thereby depriving cancer cells of asparagine and inhibiting cancer growth. We proposed a hypothesis that ASPG-engineered CAR-T cells undergo phenotype switching to overcome immunotherapy resistance in ALL. Coculture killing assay showed ASPG-OE CAR-T cells exhibited increased killing efficacy against ASNS-OE cancer cells by enhancing the expression of granzyme B, interferon gamma, and tumor necrosis factor alpha, whereas ASPG-knockout (KO) CAR-T cells showed decreased cancer cell lysis efficiency. Phenotypic analysis revealed that ASPG-OE CAR-T cells exhibited distinct phenotypes, including increasing central memory T cells percentage, while decreasing effector memory T cells and effector memory cells that re-expressed CD45RA cells proportions. This distinct phenotype switch of ASPG-OE CAR-T cells toward central memory T cells exerted the increased killing efficacy against NALM6-GL cells even without ASNS-OE. The in vivo xenograft mouse model confirmed that ASPG-OE CAR-T cells exhibited superior anticancer activity against NALM6-GL cancer cells, while ASPG-KO CAR-T cells exhibited inferior anticancer activity. Taken together, ASPG orchestrates CAR-T cell distinct phenotype toward central memory T cells and reprogramming of asparagine metabolism for enhancing antitumor immunity.