Integrative multi-omics analysis identifies an enhancer-driven ELF3-CLDN4 regulatory axis that shapes an immunosuppressive microenvironment and predicts prognosis in high-grade serous ovarian cancer.

OBJECTIVES: High-grade serous ovarian cancer (HGSOC) is marked by genomic instability, immune suppression, and poor outcome, yet enhancer-gene networks coordinating malignant epithelial programs and CD4⁺ T-cell states remain unclear. We aimed to identify enhancer-associated regulatory circuits linking tumor plasticity, CD4⁺ T-cell polarization, and prognosis in HGSOC. METHODS: We integrated H3K27ac ChIP-seq, Hi-C, single-cell RNA-seq, spatial transcriptomics, TCGA-OV data, CD4⁺ T-cell eQTLs, and pharmacogenomic annotations to reconstruct cell-type-specific enhancer-gene regulatory networks. Motif enrichment, SCENIC, Louvain clustering, CellChat, pseudotime analysis, spatial deconvolution, GSVA, and survival modeling were applied. Seven-color multiplex immunofluorescence was performed in 90 primary HGSOC and 10 non-malignant ovarian tissues. RESULTS: HGSOC showed redistribution of H3K27ac signals toward distal regulatory regions linked to immune and tumor-cell programs. Single-cell profiling identified an aneuploid malignant epithelial compartment and a Treg-skewed CD4⁺ T-cell landscape. Integrated network analysis highlighted ELF3, FLI1, KLF4, and ZNF250 as shared hub transcription factors. Among these, an ELF3-associated enhancer-CLDN4 axis was supported by chromatin contacts and CD4⁺ T-cell eQTL evidence. eGRN-active CD4⁺ Th17 cells showed altered communication patterns, aberrant pseudotime positioning, and preferential peritumoral localization, particularly in JAK1-high spatial samples, where they co-localized with Tregs and macrophages. Multiplex immunofluorescence confirmed elevated ELF3 and CLDN4 expression in HGSOC, positive associations with Treg accumulation, inverse associations with Th17 infiltration, and correlations with poor overall survival. ELF3-high and CLDN4-high status remained independent adverse prognostic factors. CONCLUSIONS: An enhancer-associated ELF3-CLDN4 axis links tumor-cell regulatory plasticity with CD4⁺ T-cell immunosuppression and adverse prognosis in HGSOC.