Chromothripsis and ecDNA initiated by N4BP2 nuclease fragmenting cytoplasm exposed chromosomes

Genome instability, including chromothripsis, is a hallmark of cancer. Cancer cells frequently contain micronuclei - small, nucleus-like structures formed by chromosome missegregation - that are susceptible to rupture, exposing chromatin to cytoplasmic nucleases. Through an unbiased imaging-based siRNA screen targeting all 204 known and putative human nucleases, we identify a previously uncharacterized cytoplasmic endonuclease, NEDD4-binding protein 2, N4BP2, that enters ruptured micronuclei and initiates DNA damage, leading to chromosome fragmentation. N4BP2 promoted formation of extrachromosomal DNA in drug-induced gene amplification. N4BP2 promoted DNA fragmentation, tumorigenesis, and tumor cell proliferation in an induced model of human high-grade human glioma. Analysis of over 10,000 human cancer genomes revealed elevated N4BP2 expression to be predictive of chromothripsis and copy number amplifications, including ecDNA. The dataset here are DLD-1 cell genome sequences used in this study.