An integrative epigenomic and single-cell framework identifies a prognostic DNA methylation risk score and ZNF638 silencing in urothelial carcinoma

BACKGROUND: Urothelial carcinoma (UC) exhibits profound molecular heterogeneity, yet how epigenetic dysregulation shapes tumor identity, the immune microenvironment, and clinical risk remains incompletely defined. METHODS: We integrated genome-wide DNA methylation (EM-seq, n = 72; WGBS, n = 9) with scRNA-seq (n = 9) to delineate epigenetically driven subtypes. A DNA Methylation Risk Score (DMRS) was constructed from 441 prognostic methylation haplotype blocks and validated across four independent cohorts (n = 574). Functional studies, including ZNF638 overexpression, proliferation assays, migration and invasion assays, RNA-seq, and CUT&Tag in UC cell lines, were performed to dissect downstream mechanisms of the key effector ZNF638. RESULTS: T-cell exhaustion. Methyl-low tumors exhibited less aggressive features. The DMRS robustly stratified patient prognosis across all cohorts, outperforming conventional clinicopathological variables. Promoter hypermethylation was associated with reduced ZNF638 expression, and functional assays supported a tumor-suppressive role for ZNF638. Low ZNF638 expression was associated with poor survival and reduced immunotherapy benefit. Integrative RNA-seq and CUT&Tag analyses supported a model in which ZNF638 binds regulatory regions of antiviral innate immunity genes (RIG-I, ZC3HAV1, PMAIP1, IFIT2, N4BP1) and may promote their transcriptional activation, thereby potentiating type I interferon signaling. Epigenetic silencing of ZNF638 may attenuate this antiviral program and contribute to immune suppression and tumor progression. CONCLUSIONS: DNA methylation is associated with UC heterogeneity by linking tumor cell identity, immune microenvironment remodeling, and clinical outcome. The DMRS shows potential utility for risk stratification, and ZNF638 emerges as a candidate epigenetically regulated immune-associated factor whose therapeutic relevance warrants prospective and functional validation, advancing precision medicine in UC. TRIAL REGISTRATION: The research was retrospectively registered (Date of Registration 2022-10-01 Registration number: ChiCTR2200064248). Ethics approval and informed consent were obtained (Peking University First Hospital, 2022SR338).