A multi-omics integration identifies ENY2 hypomethylation as a driver of immunosuppression and progression in breast...

Aberrant DNA methylation contributes to breast cancer (BC) progression and immune microenvironment dysregulation, but its underlying epigenetic-immune crosstalk remains poorly defined. This study constructed a methylation-driven prognostic signature in BC using the SIMPO algorithm and Cox regression with TCGA cohort data, validated externally in the GSE76427 cohort. Bioinformatic analyses including GSEA, CIBERSORT and TIMER were applied to characterize the immune microenvironment. Functional assays were performed in BC cell lines and mouse models with ENY2 knockdown, overexpression and 5-Aza-dC treatment, combined with scRNA-seq, ATAC-seq, Western blot, flow cytometry and other molecular techniques. The established prognostic model stratified patients into high- and low-risk groups, with high-risk patients exhibiting poorer survival and an immunosuppressive phenotype. ENY2 was identified as a critical oncogene driven by promoter hypomethylation alongside genomic copy number gain, whose expression was inversely correlated with promoter methylation. ENY2 promoted cancer cell malignant progression. Mechanistically, ENY2 hypomethylation enhanced chromatin accessibility, facilitated the recruitment of the transcription factor E2F1, and increased active histone marks, thereby driving ENY2 transcription. Furthermore, ENY2 remodeled the immunosuppressive microenvironment not only by activating the TNF-NF-κB/PD-L1 axis, but also by elevating immunosuppressive secretomes including MIF and MDK to impair CD8⁺T cell cytotoxicity. In vivo, ENY2 knockdown attenuated 5-Aza-dC-accelerated tumor growth and restored CD8⁺T cell infiltration. Our findings establish ENY2 as an important epigenetic-immune bridge and a therapeutic vulnerability in BC.