This study investigates the mechanism by which tumor-repopulating cells (TRCs) evade ferroptosis to resist chemotherapy and radiotherapy. Using a soft fibrin gel culture system, the researchers found that TRCs, which have stem-cell-like characteristics, are resistant to ferroptosis, a form of iron-dependent lipid peroxidation-mediated cell death. Mechanistically, they identified that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4, a key enzyme in phospholipid remodeling, leading to increased ferroptosis resistance. TRCs downregulate PCK2 expression, reducing its activity and maintaining a structural ferroptosis-resistant state. Notably, higher levels of PCK2 and pACSL4(T679) were correlated with better response to chemotherapy and radiotherapy in nasopharyngeal carcinoma (NPC) cohorts, suggesting clinical relevance. The study also revealed that STAT3 activation in TRCs promotes PCK2 degradation, further enhancing ferroptosis resistance. In vivo experiments validated the role of the PCK2-pACSL4(T679) axis in ferroptosis sensitivity, and clinical data confirmed the correlation between PCK2 and pACSL4(T679) levels with treatment response and survival in NPC patients. This work highlights the importance of PCK2 in mediating ferroptosis resistance in TRCs and provides insights into the non-canonical functions of metabolic enzymes in cancer therapy resistance.This study investigates the mechanism by which tumor-repopulating cells (TRCs) evade ferroptosis to resist chemotherapy and radiotherapy. Using a soft fibrin gel culture system, the researchers found that TRCs, which have stem-cell-like characteristics, are resistant to ferroptosis, a form of iron-dependent lipid peroxidation-mediated cell death. Mechanistically, they identified that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4, a key enzyme in phospholipid remodeling, leading to increased ferroptosis resistance. TRCs downregulate PCK2 expression, reducing its activity and maintaining a structural ferroptosis-resistant state. Notably, higher levels of PCK2 and pACSL4(T679) were correlated with better response to chemotherapy and radiotherapy in nasopharyngeal carcinoma (NPC) cohorts, suggesting clinical relevance. The study also revealed that STAT3 activation in TRCs promotes PCK2 degradation, further enhancing ferroptosis resistance. In vivo experiments validated the role of the PCK2-pACSL4(T679) axis in ferroptosis sensitivity, and clinical data confirmed the correlation between PCK2 and pACSL4(T679) levels with treatment response and survival in NPC patients. This work highlights the importance of PCK2 in mediating ferroptosis resistance in TRCs and provides insights into the non-canonical functions of metabolic enzymes in cancer therapy resistance.