Effect of loss of KDM6A-mediated genomic instability and metabolic reprogramming on response to therapeutic perturbations in bladder cancer.

816 Background: Mutations in genes encoding critical epigenetic regulators are frequently observed in advanced bladder cancer; however, the mechanisms by which these alterations shape therapeutic responses remain incompletely understood. Through retrospective analyses of multiple bladder cancer patient cohorts, we identified that loss-of-function mutations in KDM6A , a histone demethylase, are found in approximately 26% of patients with advanced bladder cancer. Notably, inactivating mutations in KDM6A are associated with reduced overall survival following cisplatin-based chemotherapy whereas they correlate with improved outcomes after anti–PD-1/anti–PD-L1 therapy. Methods: To investigate the divergent therapeutic outcomes, we generated KDM6A -deficient murine and human bladder cancer cell lines using CRISPR-Cas9 and evaluated their phenotypes both in vitro and in syngeneic mouse models of bladder cancer. Whole-genome sequencing, ChIP-sequencing, and RNA-sequencing were performed to define the genomic and epigenomic alterations associated with KDM6A loss. In addition, mass cytometry (CyTOF) and metabolic profiling were conducted to characterize Kdm6a -dependent metabolic reprogramming and tumor microenvironment remodeling. Results: Whole-genome sequencing revealed increased generation of extrachromosomal circular DNA (eccDNA) encompassing gene loci linked to chemoresistance in KDM6A -deficient human bladder cancer cells, establishing a mechanistic connection between KDM6A loss and cisplatin resistance. In parallel, KDM6A loss impaired DNA repair pathways and reprogrammed tumor metabolism, reducing glycolysis and lactate production. This metabolic shift diminished histone lactylation (H3K9la, H3K18la) in regulatory T cells (Tregs), suppressing key immunoregulatory genes ( Foxp3, Tgfβ, Pdcd1 ) and limiting the expansion of PD-1 hi Tregs. Functionally, this enhanced the cytotoxic T cell-to-Treg ratio and improved response to anti–PD-1 therapy in Kdm6a-deficient tumor-bearing mice. Conclusions: Our findings establish KDM6A as a key epigenetic regulator that links eccDNA biogenesis, maintenance of genomic stability, and remodeling of the tumor immune microenvironment thus providing a unifying mechanistic basis for its role as a predictive biomarker to guide precision therapy in advanced bladder cancer.