Precise metabolomics identifies glycolysis-related pyruvate kinase M activity as regulator of the S-phase-specific radiation response in triple-negative breast cancer cells.

Metabolic reprogramming is known to critically contribute to therapy response in cancer, including the resistance of triple-negative breast cancer (TNBC) patients to conventional radio/(chemo)therapy. Our study, therefore, characterized the metabolic response of TNBC cell models to irradiation using untargeted metabolomics. We identified radiation-induced metabolic changes by harnessing a metabolite-gene interaction network comprising 44 genes. Nine genes with significant radiosensitizing potential in TNBC cells were identified by RNAi-screening. Among the druggable genes, high expression of pyruvate kinase M (PKM) was associated with worse overall survival in the METABRIC-TNBC cohort. Pharmacological PKM inhibition led to radiosensitization in TNBC cell models, which was attributed to S-phase related metabolic changes. Selective disruption of the S-phase by PKM inhibition led to both reduced DNA synthesis and increased replication stress, as evidenced by increased DNA damage at active replication forks. This resulted in prolonged cell cycle arrest after irradiation. Metabolic profiling upon PKM depletion and irradiation revealed two positively PKM-associated gene clusters, predominantly involved in the glycolysis pathway. Each cluster was functionally distinct, one shared the PKM-associated effects in survival reduction, while the other correlated with its radiosensitization. High expression of the first cluster was significantly correlated with worse overall survival, whereas high expression of the second cluster, consisting of PKM, ENO1, GAPDH and GPI, predicted poor response to radiotherapy in the METABRIC-TNBC cohort. Thus, our study suggests that glycolytic-associated metabolic reprogramming upon irradiation plays a major role in radioresistance of TNBC. Analysis of our identified glycolysis-related gene cluster in patients could predict the response to radiotherapy, and targeting PKM and the related glycolytic pathway holds promise for radiosensitization in TNBC patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02803-5.