Shoshani Lab

Gene amplification in the form of extrachromosomal DNA (ecDNA) is a frequent driver in multiple cancer types. As ecDNA lack centromeres, their mitotic segregation does not follow traditional inheritance principles. However, the mechanisms that govern ecDNA fate following mitosis remain unclear. We found that ecDNA undergo numerical and structural optimization under increased selective pressure, with mitotic chromosomal tethering, or detachment, dictating ecDNA fate. When tethered, ecDNA aggregates promote uneven distribution into the newly formed daughter cells, thereby driving inter-cellular numerical heterogeneity and rapid increase of amplification under selective pressure. Mitotically detached ecDNA frequently encapsulate within micronuclei of variable size and content that appear to be highly fragile. Strikingly, ecDNA enclosed in very small micronuclei, which we term nanonuclei, are being actively degraded through autophagy. Together with ongoing structural rearrangements, nanonuclear ecDNA degradation promotes their structural evolution, which facilitates cancer cell adaptation. Our work highlights ecDNA aggregation, micronucleation, and degradation, as pivotal events in directing cancer genome evolution trajectories.Competing Interest StatementA.G.H. is a founder and stock holder in Econic Biosciences. The other authors declare no competing interests.Israel Science Foundation, 686/22Israel Cancer Research FundMinerva Stiftung, 146552

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes¹³, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakagefusionbridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.