Ionizing Radiation Shapes Genome Evolution Through Nuclear Abnormalities That Trigger Delayed Proliferative Death

Ionizing radiation (IR) is widely used in cancer therapy, yet the mechanisms by which IR-induced DNA damage translates into delayed proliferative death and secondary malignancy risk remain incompletely defined. Radiation generates double-strand breaks (DSBs) that are frequently misrepaired, giving rise to micronuclei and chromosome bridges--abnormal nuclear structures known to drive chromosomal instability. Here, a modified live-cell imaging and single-cell whole-genome sequencing (WGS) approach was used to directly connect early cytological responses to genome-wide outcomes in cells exposed to graded IR doses. Tracking asynchronously cycling cells revealed that micronuclei, chromosome bridges, and nuclear fragmentation form at high frequency during the first mitosis after irradiation, in a strongly cell-cycle-dependent manner: micronuclei were most frequent after S-phase irradiation, whereas chromosome bridges predominated when damage occurred in G1 or S phase. Lineage tracing combined with clonogenic assays showed that formation of abnormal nuclear structures in the first post-irradiation division is a major determinant of long-term proliferative capacity, with centromere-containing (centric) fragments and bridges exerting a stronger anti-proliferative effect than acentric micronuclei. Single-cell WGS of daughter and granddaughter cells demonstrated that these structures generate reciprocal copy-number changes, clustered rearrangements, and chromothripsis-like events that are largely incompatible with sustained proliferation. In contrast, bulk WGS of surviving single-cell-derived clones revealed only modest increases in single-nucleotide variants and indels but an enrichment of nonhomologous end-joining-associated deletion signatures and selected structural variants, including amplification of a pre-existing 12p gain. These findings identify abnormal nuclear structures as key intermediates linking IR-induced DNA lesions to mitotic catastrophe and provide insight into selective tolerance of specific rearrangements.