Abstract Introduction: Extrachromosomal DNA (ecDNA) contributes to oncogene amplification in human cancers, yet the mechanisms underlying the formation and function of human-viral hybrid ecDNA (hybrid ecDNA), circular elements harboring both HPV and human sequences, remain unclear. We recently identified hybrid ecDNA in human papillomavirus-associated oropharyngeal cancer (HPVOPC), a rapidly increasing malignancy. Here, we show that de novo enhancer formation at viral-human junctions activates viral oncogenes and promotes hybrid ecDNA self-amplification, revealing a tractable therapeutic vulnerability. Methods: Hybrid ecDNA was identified in HPVOPC cell lines and patient-derived xenografts (PDX) using whole-genome sequencing and AmpliconArchitect. Validation was performed by multicolor FISH. Chromatin accessibility and enhancer activity were profiled by ATAC-seq and H3K27ac ChIP-seq. Hi-C sequencing was used to assess 3D chromatin interactions and potential contacts among distinct ecDNA species. Functional significance was evaluated by CRISPR interference (CRISPRi) targeting ecDNA-derived enhancers. Therapeutic relevance was assessed by BET inhibition in vitro and in vivo. Results: Hybrid ecDNA was detected in both cell lines and PDX tumors and validated by overlapping human and HPV FISH signals. Epigenomic profiling revealed de novo active enhancers flanking HPV sequences exclusively in hybrid ecDNA(+) tumors. Hi-C demonstrated enhancer-promoter loops linking host enhancers to HPV oncogenes. CRISPRi targeting these enhancers significantly inhibited proliferation in hybrid ecDNA(+) models only (P = 0.006). BET inhibition selectively suppressed hybrid ecDNA(+) tumor growth in vivo (P = 2×10-5). Importantly, Hi-C contacts also indicated enriched interaction patterns among distinct ecDNA species, suggesting potential extrachromosomal cooperation that enhances viral oncogene expression. Conclusions: Hybrid ecDNA is a functionally critical structure in HPVOPC by generating de novo enhancers and reorganizing chromatin interactions to activate viral oncogenes, creating a therapeutic vulnerability. Contact enrichment among distinct ecDNA species further suggests participation in a broader extrachromosomal regulatory network in HPV-driven malignancies. Citation Format: Takuya Nakagawa, Jens Luebeck, Kaiyuan Zhu, Sasik Roman, Brin Rosenthal, Kathleen Fisch, Toyoyuki Hanazawa, Atsushi Kaneda, Paul S. Mischel, Vineet Bafna, Joseph A. Califano. De novo enhancer formation at human-viral junctions drives hybrid extrachromosomal DNA amplification in HPV-associated 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 1925.
Nakagawa et al. (Fri,) studied this question.