The PKMYT1-ALDOA axis drives chemoresistance in cholangiocarcinoma by enhancing glycolysis and MRE11 lactylation.

Cholangiocarcinoma (CCA) is a highly aggressive biliary malignancy with limited responses to existing treatments. Gemcitabine plus cisplatin (GC) remains the standard first-line chemotherapy for advanced disease, but its clinical efficacy is suboptimal, and drug resistance frequently emerges in patients. Through high-throughput synergistic drug screening, we identified PKMYT1 as a key synthetic lethal target regulating the chemosensitivity of cholangiocarcinoma cells. Functional validation confirmed that the PKMYT1 small-molecule inhibitor RP6306 exerts robust synthetic lethal effects when used in combination with gemcitabine or cisplatin. Data from colony formation assays and a series of cellular functional experiments showed that genetic or pharmacological inhibition of PKMYT1 substantially compromises DNA damage repair (DDR) and induces massive DNA double-strand breaks. Further mechanistic investigations revealed that PKMYT1 binds to Aldolase A (encoded by the ALDOA gene) and enhances its enzymatic activity, thereby upregulating CCA glycolysis levels and inducing lactate accumulation, which ultimately elevates the lactylation level of MRE11, accelerates DDR and consequently drives chemoresistance in CCA. Both cell-derived and patient-derived xenograft models verified that combined treatment with RP6306 and GC markedly inhibits tumor growth in vivo. Taken together, this work reveals that the PKMYT1-ALDOA-mediated protein lactylation pathway acts as a critical regulator of chemoresistance in CCA. Targeting PKMYT1 could reverse chemotherapy resistance and enhance therapeutic outcomes for advanced CCA, offering a promising combination strategy for clinical application.