Lactate Enhances Non-Homologous End Joining Repair and Chemoresistance Through Facilitating XRCC4–LIG4 Complex Assembly in Ovarian Cancer

Background: Non-homologous end joining (NHEJ) is a crucial pathway for repairing DNA double-strand breaks and a key contributor to chemoresistance in cancer. The assembly of the DNA Ligase IV (LIG4)–XRCC4 complex is essential for NHEJ fidelity, however, the regulatory mechanisms governing this complex in cancer remain poorly understood. This study aims to investigate whether and how lactate, a key metabolic byproduct of the Warburg effect, regulates the XRCC4–LIG4 complex and influences chemoresistance. Methods: The functional role of lactate in NHEJ was assessed using DNA repair reporter assays in ovarian cancer cells. Protein–protein interactions were examined through co-immunoprecipitation and pull-down assays. The molecular mechanism of lactate’s action was delineated using a combination of site-directed mutagenesis, in vitro binding assays, and molecular docking. Finally, the physiological relevance of lactate-mediated NHEJ was validated in a preclinical ovarian cancer mouse model treated with cisplatin. Results: We demonstrated that lactate enhances NHEJ repair efficiency and confers resistance to DNA-damaging chemotherapeutics. Mechanistically, lactate directly binds to XRCC4 at key residues, including Y66, E55, and S110, thereby strengthening the XRCC4–LIG4 association. This interaction is independent of protein lactylation. In vivo studies confirmed that lactate-driven NHEJ promotes chemoresistance in ovarian cancer. Conclusions: Our findings reveal lactate as a novel metabolic regulator of the NHEJ pathway by directly allosterically modulating the XRCC4–LIG4 complex. This work establishes a direct molecular link between the Warburg effect and DNA repair-driven chemoresistance, offering new insights into potential therapeutic strategies for ovarian cancer.