Abstract 1930: Unveiling ecDNA spatial organization and epigenetic landscapes through long-read multi-omic sequencing and high-content microscopy.

Abstract Oncogene amplification is one of the most common genetic alterations in cancer, playing a central role in tumorigenesis. A major mechanism of oncogene amplification is extrachromosomal DNA (ecDNA), which is present in nearly 20% of cancer patients and found across half of all cancer types. Patients with ecDNA-positive tumors have significantly shorter survival compared to those without ecDNA. Despite its prevalence and strong association with poor treatment outcomes, there is no approved treatment targeting ecDNA-positive cancers. Developing such treatments requires identifying unique and actionable features of ecDNA. Two unique features of ecDNA warrant special attention because they are seemingly paradoxical. On one hand, proliferation of cancer cells depends on high-level oncogene expression from ecDNA. On the other hand, ecDNA molecules preferentially cluster at the nuclear periphery, a region typically associated with transcriptional repression. Why would cancer cells position most of their ecDNA at the nuclear periphery? What molecular mechanisms control this localization, and what are its functional consequences? These are longstanding questions in the field that have remained unanswered largely due to the lack of experimental approaches capable of deciphering the molecular heterogeneity of ecDNA and linking its spatial organization to oncogene expression. To fill the technical gap and answer these fundamental ecDNA biology questions, we developed DINO-seq, a long-read single-molecule sequencing technology that enables simultaneous profiling protein binding, DNA accessibility and CpG methylation on individual ecDNA molecules. Using DINO-seq, we discovered that ecDNA carries a bivalent epigenetic landscape—coexisting active and repressive epigenetic marks—that is distinct from chromosomal DNA and enables ecDNA to remain transcriptionally active even at the nuclear periphery. Combining a CRISPR screen with high-content microscopy, we further identified molecular anchors that tether ecDNA to the nuclear periphery. Functionally, we show that peripheral ecDNA localization alters replication timing, promotes ecDNA hub formation, and enhances RNA transcription and export. Together, these findings reveal that ecDNA possesses unique spatial and epigenetic properties that distinguish it from chromosomal DNA and represent potential vulnerabilities that could be exploited to target ecDNA-positive cancers. Citation Format: Yanbo Wang, Xiaowei Yan, Natasha E. Weiser, Shu Zhang, Ivy Tsz-Lo Wong, Yung-Hsin Huang, Rui Li, Nicolas Altemose, Paul S. Mischel, Howard Y. Chang. Unveiling ecDNA spatial organization and epigenetic landscapes through long-read multi-omic sequencing and high-content microscopy [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 1930.