ecDNA hubs drive cooperative intermolecular oncogene expression [ChIP]

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high oncogene expression through gene amplification and altered gene regulation. Gene induction typically involves cis regulatory elements that contact and activate genes on the same chromosome. Here we show that ecDNA hubs, clusters of ~10-100 ecDNAs within the nucleus, enable intermolecular enhancer-gene interactions to promote oncogene overexpression in trans. ecDNAs encoding multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumors. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the BET protein BRD4 in a MYC-amplified colorectal cancer cell line. BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-based oncogene transcription. A BRD4-bound promoter in PVT1 is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent MYC expression. PVT1 promoter on a heterologous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic CRISPRi silencing of ecDNA enhancers reveal intermolecular enhancer-gene activation among multiple oncogene loci amplified on distinct ecDNAs. Together, these results demonstrate that ecDNA hubs are protein-tethered clusters of ecDNAs which enable intermolecular transcriptional regulation. ecDNA hubs may act as units of oncogene function, cooperative evolution, and potential targets for cancer therapy. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for BRD4 and H3K27ac in COLO320-DM, COLO320-HSR and SNU16 cell lines, with or without JQ1 treatment.