PO.MCB06.01 · 分子与细胞生物学
人-病毒连接处的从头增强子形成驱动HPV相关癌症中的杂合染色体外DNA扩增
De novo enhancer formation at human-viral junctions drives hybrid extrachromosomal DNA amplification in HPV-associated cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：染色体外DNA（ecDNA）促进人类癌症中的癌基因扩增，然而人-病毒杂合ecDNA（杂合ecDNA，即同时携带HPV和人类序列的环状元件）形成和功能的潜在机制仍不清楚。我们最近在HPV相关口咽癌（HPVOPC，一种快速增加的恶性肿瘤）中鉴定出杂合ecDNA。在此，我们表明病毒-人连接处的从头增强子形成激活病毒癌基因并促进杂合ecDNA自我扩增，揭示了一个可干预的治疗弱点。
方法：使用全基因组测序和AmpliconArchitect在HPVOPC细胞系和患者来源的异种移植物（PDX）中鉴定杂合ecDNA。通过多色FISH进行验证。通过ATAC-seq和H3K27ac ChIP-seq分析染色质可及性和增强子活性。使用Hi-C测序评估三维染色质相互作用以及不同ecDNA物种之间的潜在接触。通过靶向ecDNA来源增强子的CRISPR干扰（CRISPRi）评估功能意义。通过体外和体内的BET抑制评估治疗相关性。
结果：在细胞系和PDX肿瘤中均检测到杂合ecDNA，并通过重叠的人类和HPV FISH信号得到验证。表观基因组分析揭示，从头激活的增强子仅在杂合ecDNA(+)肿瘤中侧翼于HPV序列。Hi-C显示了将宿主增强子与HPV癌基因相连的增强子-启动子环。靶向这些增强子的CRISPRi仅在杂合ecDNA(+)模型中显著抑制增殖（P = 0.006）。BET抑制在体内选择性地抑制杂合ecDNA(+)肿瘤生长（P = 2×10⁻⁵）。重要的是，Hi-C接触还表明不同ecDNA物种之间存在富集的相互作用模式，提示可能存在增强病毒癌基因表达的染色体外协作。
结论：杂合ecDNA在HPVOPC中是一种功能上关键的结构，它通过产生从头增强子和重组染色质相互作用来激活病毒癌基因，从而形成治疗弱点。不同ecDNA物种之间的接触富集进一步提示其参与了HPV驱动恶性肿瘤中更广泛的染色体外调控网络。
查看英文原文 English 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.
利益披露 Disclosure
T. Nakagawa, None..
J. Luebeck, None..
K. Zhu, None..
S. Roman, None..
B. Rosenthal, None..
K. Fisch, None..
T. Hanazawa, None..
A. Kaneda, None.
P. S. Mischel,
Boundless Bio Inc. Employment.
V. Bafna, None..
J. A. Califano, None.