EZH2-Associated Hypermethylated Gene Signature Predicts Immunotherapy Response and Implicates DUSP5 in Tumor-Immune Regulation in Triple-Negative Breast Cancer

Background/Objectives: Triple-negative breast cancer (TNBC) is a candidate for immune checkpoint blockade; however, current biomarkers remain insufficient to predict therapeutic response or capture tumor-intrinsic mechanisms. Enhancer of Zeste Homolog 2 (EZH2)-mediated epigenetic repression has been implicated in immune evasion, yet the contribution of EZH2-repressed genes to anti-tumor immunity and clinical outcomes in TNBC remains unclear. We aimed to identify EZH2-associated epigenetically repressed genes in TNBC and evaluate their relevance as tumor-intrinsic regulators and potential predictors of immunotherapy outcome. Methods: We performed integrative in silico analyses of The Cancer Genome Atlas (TCGA) breast cancer cohorts to identify EZH2-associated hypermethylated genes in TNBC. A composite 30-gene signature (30GS) was defined based on transcriptional repression and promoter hypermethylation. Associations with clinical outcomes, tumor- and immune-related programs, and therapeutic response were evaluated, with validation in the I-SPY2 cohort and an independent TNBC patient cohort. Results: The 30GS was significantly reduced in TNBC and basal-like tumors and associated with improved clinical outcomes and enrichment of tumor- and immune-related signatures. In the I-SPY2 cohort, the 30GS predicted pathological complete response in patients receiving chemo-immunotherapy (AUC = 0.7377, p = 0.0007). Gene-level analysis identified Dual Specificity Phosphatase 5 (DUSP5) as the gene most consistently associated with immune-related parameters. In an independent TNBC cohort, DUSP5-high tumors demonstrated transcriptional programs enriched for inflammatory, immune-related, and signaling pathways within the NanoString Breast Cancer 360 panel. Conclusions: This study defines an EZH2-associated epigenetic program linked to tumor-intrinsic immune programs in TNBC and identifies DUSP5 as a candidate gene associated with immune-related transcriptional states.