Radiation-induced transient metabolic rewiring uncovers a glutamine transport therapeutic vulnerability in triple-negative breast cancer

Glutamine metabolism is a vulnerability in triple-negative breast cancer (TNBC), but is buffered by metabolic flexibility. We posited that ionizing radiation (IR) enforces a short-lived glutamine dependency that can be targeted. TNBC cell lines and in vivo models were subjected to IR with or without pharmacological inhibitors of glutamine metabolism. We quantified glutamine flux using stable isotope tracing, DNA repair kinetics, cell cycle progression, apoptosis, tumor growth/metastasis, and changes in the immune microenvironment. IR triggered a radiation-associated anabolic shift (RAAS) characterized by increased ASCT2-mediated glutamine uptake and transient upregulation of glutamine-driven de novo nucleotide synthesis. Isotope tracing revealed that exogenous glutamine supplies nitrogen for the post-IR synthesis of purine precursors, supporting nucleotide biosynthesis following radiation exposure. In vitro, inhibition of ASCT2 with V-9302 abolished RAAS, impaired double-strand break (DSB) DNA repair, caused cell cycle arrest, and increased apoptosis. Compared with other glutamine pathway inhibitors, ASCT2 blockade provided the most consistent radiosensitization across the TNBC lines. In vivo, low-dose V-9302 combined with fractionated radiotherapy slowed primary tumor growth and reduced lung metastasis without causing significant systemic toxicity. Combination therapy increased CD8 + T cell infiltration, pro-inflammatory macrophage polarization, and TNF-α production. IR temporarily induces glutamine dependence in TNBC cells. ASCT2 inhibition (V-9302) exploits this window to block glutamine-driven DNA repair and enhances local anti-tumor immune polarization, leading to radiosensitization and anti-metastatic effects. These results support further studies that combine inhibition of glutamine transport with radiotherapy for breast cancer. Ionizing radiation exposes transient metabolic vulnerability in triple-negative breast cancer by enforcing dependence on glutamine metabolism for stress adaptation and survival. Targeting this induced dependency is a mechanistically grounded therapeutic strategy resolving the existing crisis in glutamine therapy and overcoming the metabolic plasticity in aggressive breast cancer.