EcDNA hubs drive cooperative intermolecular oncogene expression

Abstract

Extrachromosomal DNAs (ecDNAs) are prevalent in human cancers and mediate high oncogene expression through elevated copy number and altered gene regulation1. Gene expression typically involves distal enhancer DNA elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs, comprised of ~10-100 ecDNAs clustered in the nucleus of interphase cells, drive intermolecular enhancer input for amplified oncogene expression. Single-molecule sequencing, single-cell multiome, and 3D enhancer connectome reveal subspecies of MYC-PVT1 ecDNAs lacking enhancers that access intermolecular and ectopic enhancer-promoter interactions in ecDNA hubs. ecDNA hubs persist without transcription and are tethered by BET protein BRD4. BET inhibitor JQ1 disperses ecDNA hubs, preferentially inhibits ecDNA oncogene transcription, and kills ecDNA+ cancer cells. Two amplified oncogenes MYC and FGFR2 intermix in ecDNA hubs, engage in intermolecular enhancer-promoter interactions, and transcription is uniformly sensitive to JQ1. Thus, ecDNA hubs are nuclear bodies of many ecDNAs tethered by proteins and platforms for cooperative transcription, leveraging the power of oncogene diversification and combinatorial DNA interactions. We suggest ecDNA hubs, rather than individual ecDNAs, as units of oncogene function, cooperative evolution, and new targets for cancer therapy.

Extrachromosomal circular DNA in cancer: history, current knowledge, and methods

“…Analysis of the 3D topology of ecDNA also showed novel contacts between oncogenes and regulatory elements compared with genes localized on chromosomes [14,15]. ecDNA-bound promoters and enhancers can also make contact with chromosomal DNA and other ecDNA in trans to stimulate transcription [77,99]. ecDNA can reinsert in chromosomes, creating HSRs and preserving the amplification of the oncogene in a more stable form [8,16,34,100,101].…”

“…There is significant correlation between gene copy number and oncogene transcription from both ecDNA and chromosomal DNA [16,17,76]. However, oncogenes on ecDNA show even higher expression than can be explained by copy number amplification [17,77]. This copy number-independent transcription increase can be partially explained by enhanced chromatin accessibility on ecDNA compared to linear amplicons [17,76].…”

“…OPEN ACCESS techniques such as FISH and electron microscopy used to confirm their extrachromosomal status and circular structure [8,19,76]. Further features of ecDNA have been studied by overlapping the genomic coordinates of circular amplicons with whole-genome assays of chromatin accessibility, methylation and 3D contacts and comparing to the features of linear amplicons [14,15,76,77].…”

Principles of ecDNA random inheritance drive rapid genome change and therapy resistance in human cancers

“…Emerging data demonstrate that the altered topology of ecDNA drives enhanced chromatin accessibility and rewires gene regulation to drive oncogenic transcription 4 . Further, the unique higher-level organization of ecDNA particles into hubs 26 further contributes to ecDNA-mediated pathogenesis. The findings presented here reveal that ecDNA uniquely shapes each of the foundational principles of Darwinian evolution – random inheritance by descent, enhanced variation through random segregation, and selection, thereby accelerating tumour cell evolution to maximize adaptation.…”

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

“…As validation, we reconstructed the 1.258 Mb circular ecDNA circle encoding EGFR in GBM39 cells using this workflow, yielding a structure fully consistent with previous reports using WGS and optical mapping 7,8 (Extended Data Figure 3). To further demonstrate the utility of this tool, we applied this pipeline to a stomach cancer cell line, SNU16, which contains multiple ecDNA species with MYC, FGFR2 and additional sequences connected by complex structural rearrangements (Extended Data Figure 4a) 21 . CRISPR-CATCH using guides targeting the MYC or FGFR2 amplicon resulted in multiple visible bands in PFGE (Figure 4b), revealing extensive molecular heterogeneity of ecDNAs.…”