Abstract Extrachromosomal DNA (ecDNA) is megabase-sized circular DNA amplified exclusively in tumor cells, driving poor outcomes and therapeutic resistance. EcDNA represents a distinct form of genome instability associated with heightened replication stress and DNA damage. However, how ecDNA manages these elevated damage levels remains unclear. Here, we used long-read sequencing and live-cell imaging to investigate DNA damage repair dynamics and genomic patterns in isogenic cell lines harboring either ecDNA or homogeneously staining region (HSR). We found that damaged ecDNAs are preferentially sequestered into micronuclei following induction of double-strand breaks. This process triggers additional DNA damage beyond the initial lesions and impairs homologous recombination (HR) during knock-in by CRISPR-Cas9. Structural variant analyses further revealed that although ecDNA damage initiates HR repair, the process is frequently incomplete or aberrant. Analysis of whole-genome sequencing data from 14,778 tumors across 39 cancer types in the Genomics England cohort confirmed that ecDNA-positive cancers exhibit enriched HR deficiency (HRD) mutational signatures, even after excluding cases with canonical HRD driver mutations. Together, these findings delineate the dynamic and error-prone repair of ecDNA, linking it to HRD-associated genomic alterations. Our study exposes vulnerabilities of ecDNA-positive cancers that may be therapeutically exploited through synthetic lethality related to HRD. Citation Format: Shu Zhang, Yanbo Wang, Thomas B. Watkins, Xiaowei Yan, Aditi Gnanasekar, Jun Tang, Ivy T. Wong, Howard Y. Chang, Paul S. Mischel. Dynamic and defective repair of extrachromosomal DNA drives genome instability in cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4689.
Zhang et al. (Fri,) studied this question.