Extrachromosomal DNA Associates with Nuclear Condensates and Reorganizes Chromatin Structures to Enhance Oncogenic Transcription [ChIA-Drop]
Extrachromosomal, circular DNA (ecDNA) is a prevalent and significant oncogenic alteration in cancer genomes. Its frequency is associated with aggressive tumor behavior and poor patient outcome. To understand how ecDNA promotes oncogenesis, we map ecDNA-associated chromatin complexes at single-molecule resolution in three well-characterized ecDNA+ cancer models that harbor different oncogenes. Using a suite of 3D genome mapping and imaging analyses, we determined ecDNA nuclear organization and uncovered that ecDNA hubs are part of MED1-mediated condensates that can be rapidly disrupted by agents perturbing liquid-liquid phase separation (LLPS). The functional relevance of ecDNA condensates was demonstrated by inhibition of LLPS which led to hub dispersal, the reduction of MED1 co-activator binding, inhibition of oncogenic transcription and selectively increase cell death of ecDNA-containing cells across a broad range of cancer models. Robust epigenetically silencing of the regulatory modules within ecDNAs effectively disrupts ecDNA's mode of actions and demonstrates the causality between ecDNA enhancer function, chromosomal contacts, transcription and cellular proliferation, which directly corroborate ecDNA's trans-activator activity. Our findings reveal that ecDNA drives tumor promotion activity through nuclear condensate and suggests that exploiting these nuclear structures may serve as a universal strategy against malignant tumors. Our work elucidates the structures, functions and regulation of ecDNA which is expected to transform the paradigm for studying cancer genomes and expand the current views of how oncogenes are regulated, from which new clinical strategies can be realized. Overall design: Perform chromatin interaction assays including WGS, ChIA-Drop, ChIA-PET, ChIATAC, ChIP-seq, CUT&Tag, and RNA-seq to define genome-wide ecDNA-mediated chromatin interactomes and characterize their functional impact on transcription programs following 1,6-HD treatment and CRISPR-based epigenomic perturbations in cancers.