Abstract

BRCA1 promoter methylation predicts sensitivity to PARP inhibitors in high-grade serous ovarian cancer, yet therapeutic resistance is common and mechanistically unresolved. Using long-read direct DNA sequencing of patient-derived xenografts and cell lines, we resolved BRCA1 methylation at single-molecule resolution with structural and transcriptomic analyses. We revealed two convergent PARP inhibitor and platinum resistance mechanisms, validated in patient tumors. First, focal, allele-specific loss of BRCA1 methylation arose through local cis-acting genomic alterations, instead of global epigenetic reprogramming. Engineered in cis sequence alterations near the methylated BRCA1 promoter were sufficient to induce methylation loss, restore homologous recombination, and confer resistance. Similar associations were observed across the genome, suggesting this mechanism extends beyond BRCA1. Second, BRCA1 expression was restored despite intact promoter methylation via structural variant-mediated promoter bypass or alternative transcription initiation. Together, these findings redefine BRCA1 methylation loss as a locus-restricted process and reveal multiple routes by which tumors escape PARP inhibitor therapy.