Gefitinib Reverses PD-L1–Mediated Immunosuppression Induced by Long-term Glutamine Blockade in Bladder Cancer

Abstract Glutamine is a major energy source for tumor cells, and blocking glutamine metabolism is being investigated as a promising strategy for cancer therapy. However, the antitumor effect of glutamine blockade in bladder cancer remains unclear, necessitating further investigation. In this study, we demonstrated that glutamine metabolism was involved in the malignant progression of bladder cancer. Treatment with the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) inhibited the growth of bladder cancer cells in vitro in several ways. In addition, we observed inhibition of tumor growth in bladder cancer–bearing mice by using JHU083, a prodrug that was designed to prevent DON-induced toxicity. However, the antitumor immune effect of T cells changed from activation to inhibition as the administrated time extended. We found that both in vitro treatment with DON and in vivo prolonged administration of JHU083 led to the upregulation of PD-L1 in bladder cancer cells. Mechanistically, glutamine blockade upregulated PD-L1 expression in bladder cancer cells by accumulating reactive oxygen species, subsequently activating the EGFR/ERK/C-Jun signaling pathway. Combination treatment of JHU083 and gefitinib reversed the upregulation of PD-L1 in bladder cancer cells induced by prolonged glutamine blockade, resulting in the alleviation of T-cell immunosuppression and a significant improvement in therapeutic outcome. These preclinical findings show promise for glutamine metabolism targeting as a viable therapeutic strategy for bladder cancer, with the potential for further enhancement through combined treatment with gefitinib.