Mitotic transcription ensures ecDNA inheritance through chromosomal tethering

SUMMARY Extrachromosomal DNA (ecDNA) are circular DNA bodies that play critical roles in tumor progression and treatment resistance by amplifying oncogenes across a wide range of cancer types. ecDNA lack centromeres and are thus not constrained by typical Mendelian segregation, enabling their unequal accumulation within daughter cells and associated increases in copy number. Despite intrinsic links to their oncogenic potential, the fidelity and mechanisms of ecDNA inheritance are poorly understood. Here, we show that ecDNA are protected against cytosolic mis-segregation through mitotic clustering and by tethering to the telomeric and subtelomeric regions of mitotic chromosomes. ecDNA-chromosome tethering depends on BRD4 transcriptional co-activation and mitotic transcription of the long non-coding RNA PVT1 , which is co-amplified with MYC in colorectal and prostate cancer cell lines. Disruption of ecDNA-chromosome tethering through BRD4 inhibition, PVT1 depletion, or inhibiting mitotic transcription results in cytosolic mis-segregation, ecDNA reintegration, and the formation of homogeneously staining regions (HSRs). We propose that nuclear inheritance of ecDNA is facilitated by an RNA-mediated physical tether that links ecDNA to mitotic chromosomes and thus protects against cytosolic mis-segregation and chromosomal integration.