Cisplatin-based first-line chemotherapy resistance and metastasis are distinctive features of advanced bladder cancer, and the underlying mechanisms are largely unveiled. Increasing studies revealed that lactylation, a novel lysine post-translational modication, links the bridge between metabolism and tumor progression. However, it has been rarely reported the role of lactylation in regulating drug resistance.

To map the global profiling of protein lysine lactylation in bladder cancer, combined lactylome and proteome analysis of surgically resected tumors and adjacent samples was conducted. Lactylated sites of ARHGDIB were validated through immunoprecipitation. In vivo and in vitro functional assays were performed to demonstrate the effects of ARHGDIB lactylation. Mass spectrometry and co-immunoprecipitation were employed to identify the lactylase. Relevant mechanisms were explored by western blotting, immunofluorescence, as well as rescue assays. Patient derived organoids were constructed to validate the drug sensitivity.

A total of 916 lactylated proteins and 1965 lactylated sites were identified. Lys47 (K47) and Lys50 (K50) was validated as the lactylated sites of ARHGDIB. Delactylation at K47 and K50 promoted metastasis of bladder cancer, and K50 delactylation also caused cisplatin resistance. Subsequently, we found that P300 and HDAC2 functioned as the lactyltransferase and delactylase of ARHGDIB respectively. Mechanically, ARHGDIB delactylation increased DNA damage repair through Rac1-MRN complex-ATM-CHK2 axis. Entinostat, a FDA-approved class I HDAC inhibitor, showed synergistic effects with cisplatin in vivo and in vitro. Finally, level of ARHGDIB-K50 lactylation was lower in cisplatin-resistant clinical samples and predicted better prognosis of bladder cancer.

Our data demonstrated the important role of ARHGDIB delactylation in metastasis and cisplatin resistance of bladder cancer. ARHGDIB lactylation level might be an ideal biomarker to guide cisplatin chemotherapy, and targeting lactylation dynamics with HDAC inhibitors presented a promising avenue for overcoming therapeutic resistance in advanced bladder cancer.