Abstract

Simple SummaryExtrachromosomal circular DNA (eccDNA) contributes significantly to cancer progression through oncogene amplification and increased tumor heterogeneity. However, its regulatory interplay with epigenetic modulators such as EZH2 is not well characterized. Here, the effect of Tazemetostat, a specific EZH2 inhibitor, is examined on both the eccDNA repertoire and gene expression profiles in ovarian cancer. By combining Circle-seq with RNA-seq, we demonstrate that EZH2 suppression leads to substantial reprogramming of eccDNA. Integrated multi-omics revealed 67 genes with changes in eccDNA levels that correlated with altered transcription; among these genes, 11 were identified as potential drivers of drug response. Spatial single-cell RNA sequencing further highlighted thatSFRP1is the only tumor suppressor gene uniformly reactivated following Tazemetostat treatment.SFRP1also showed pronounced expression heterogeneity in cancer-associated fibroblasts, suggesting that its function within the tumor niche is context-dependent. Our results provide initial evidence that targeting EZH2 restructures eccDNA architecture and reinstates SFRP1-mediated tumor suppression. These findings define a previously unrecognized epigenetics–eccDNA pathway that promotes malignant plasticity and treatment resistance, providing a new conceptual basis for addressing eccDNA-driven cancers.Extrachromosomal circular DNA (eccDNA) has emerged as a pivotal contributor to cancer progression, facilitating oncogene amplification, dysregulated gene expression, and tumor heterogeneity. Despite its significance in cancer, the interplay between eccDNA and key epigenetic regulators such as EZH2 remains largely unexplored. In this study, we systematically investigate the correlation between Tazemetostat, a highly selective EZH2 inhibitor, and alterations in the eccDNA landscape and transcriptional prog