A mitotic restriction point for extrachromosomal DNA hubs in cancer cells

Abstract

Extrachromosomal DNA (ecDNA) is widespread in human cancers and promotes tumour evolution, intratumour heterogeneity and poor clinical outcomes. ecDNA molecules tend to cluster into micron-scale nuclear hubs that enhance oncogene transcription. However, how these higher-order assemblies form and persist remains largely unknown. Here we discover mitosis as the critical window for ecDNA hub organisation across cell generations. We develop an optical screen that identifies the mitotic-entry regulator WEE1 as an important regulator of hub integrity and oncogene expression. Analysis of WEE1 perturbation reveals that CDK1- and CDK2-regulated programmes cooperate to carry unresolved damage into mitosis, promoting reorganisation of ecDNA tethered to mitotic chromosomes. Long-term live-cell imaging shows that, in unperturbed cells, ecDNA hub architecture is remodelled primarily during mitosis and subsequently maintained throughout interphase. This coupling between mitotic and interphase organisation is conserved across diverse cancer types and ecDNA-associated oncogenes. Together, these findings define a mitotic restriction point that governs ecDNA hub organisation, oncogene expression and ecDNA inheritance through successive cell generations, revealing a potential window for therapeutic intervention in ecDNA-driven cancers.