High DNA methylation age deceleration defines an aggressive phenotype with immunoexclusion environments in endometrial carcinoma.

Like telomere shortening, global DNA hypomethylation occurs progressively with cellular divisions or aging and functions as a mitotic clock to restrain malignant transformation/progression. Several DNA-methylation (DNAm) age clocks have been established to precisely predict chronological age using normal tissues, but show DNAm age drift in tumors, which suggests disruption of this mitotic clock during carcinogenesis. Little is known about DNAm age alterations and biological/clinical implications in endometrial cancer (EC). Here we address these issues by analyzing TCGA and GSE67116 cohorts of ECs. Horvath clock analysis of these tumors unexpectedly revealed that almost 90% of them exhibited DNAm age deceleration (DNAmad) compared to patient chronological age. Combined with an additional clock named Phenoage, we identified a subset of tumors (82/429) with high DNAmad (hDNAmad+) as assessed by both clocks. Clinically, hDNAmad+ tumors were associated with advanced diseases and shorter patient survival, compared to hDNAmad- ones. Genetically, hDNAmad+ tumors were characterized by higher copy number alterations (CNAs) whereas lower tumor mutation burden. Functionally, hDNAmad+ tumors were enriched with cell cycle and DNA mismatch repair pathways. Increased PIK3CA alterations and downregulation of SCGB2A1, the inhibitor of PI3K kinase, in hDNAmad+ tumors, might promote tumor growth/proliferation and stemness. In addition, the inactivation of aging drivers/tumor suppressors (TP53, RB1, and CDKN2A) while enhanced telomere maintenance occurred more frequently in hDNAmad+ tumors, which supports sustained tumor growth. Prominently, hDNAmad+ tumors were featured with immunoexclusion microenvironments, accompanied by significantly higher levels of VTCN1 expression while lower PD-L1 and CTLA4 expression, which indicates their poor response to immune checkpoint inhibitor (ICI)-based immunotherapy. We further showed significantly higher levels of DNMT3A and 3B expression in hDNAmad+ than in hDNAmad- tumors. Thus, the tumor suppressive function of aging-like DNA hypomethylation is severely impaired in hDNAmad+ tumors, likely due to enhanced expression of DNMT3A/3B and dysregulated aging regulators. Our findings not only enrich biological knowledge of EC pathogenesis but also help improve EC risk stratification and precision ICI immunotherapy.