Lactylation represents a newly discovered post-translational modification triggered by lactate that influences tumor metabolic processes. Its importance and the specific proteins it modifies in bladder cancer (BLCA) are still largely unexplored. Immunohistochemical staining was applied to patient-derived tissues. Comprehensive lactylation proteomics was performed using LC–MS/MS. Functional experiments were conducted in cell cultures and animal models. CRISPR/Cas9 gene editing, plasmid-based overexpression, and drug treatments were utilized to investigate the impact of P300-catalyzed lactylation of HNRNPA1 at lysine 350 on the aggressive behavior of BLCA cells. Metabolomics analysis and measurements of glycolytic rates helped evaluate the metabolic effects. Gene expression profiling and splicing pattern assessments provided further molecular validation. Potential HNRNPA1 inhibitors were screened using molecular docking approaches. Overall, protein lactylation was notably higher in BLCA samples than in normal tissues and was strongly associated with unfavorable clinical outcomes. Among the targets, HNRNPA1 stood out as a major lactylated protein. Enhanced glycolysis led to lactate accumulation, which, in turn, drove P300 to add a lactyl group to HNRNPA1 at K350. This change favored the splicing of PKM pre-mRNA into the PKM2 variant, thereby increasing glycolytic metabolism and promoting tumor expansion. Suppressing glycolysis pharmacologically or reducing LDHA expression reduced HNRNPA1 lactylation, decreased PKM2 production, and hindered BLCA cells’ proliferation, migration, and invasion. Metabolic profiling confirmed that HNRNPA1-K350 lactylation intensified aerobic glycolysis within these cancer cells. Through molecular docking, a candidate small-molecule inhibitor was found that binds HNRNPA1, reduces PKM2 levels, and limits cell growth. The research identifies a novel lactate-dependent pathway that connects alternative splicing regulation to metabolic shifts in BLCA via HNRNPA1 lactylation. Lactylation at K350 of HNRNPA1 is an important driver of glycolysis-fueled cancer progression. These insights support the development of therapies targeting HNRNPA1 for the treatment of BLCA.
