Chromosomal tethering and mitotic transcription promote ecDNA nuclear inheritance

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 mitotic chromosome ends. High fidelity nuclear segregation of MYC-amplifying ecDNA 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 mitotic clustering through BRD4 inhibition, PVT1 depletion, or inhibiting mitotic transcription results in cytosolic mis-segregation, ecDNA chromosomal reintegration, and the formation of homogeneously staining regions (HSRs). We propose that nuclear inheritance of ecDNA is facilitated by an RNA-based mechanism that clusters ecDNA during mitosis and protects against cytosolic missegregation and chromosomal reintegration. Overall design: HiC-sequencing data of asynchronous or mitotic COLO320-DM, COLO320-HSR, or HCT116 cells.