Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells.

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species ( EGFR, CDK4, PDGFRA ). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR -containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.