Topoisomerase IIb binding delineates localized mutational processes and driver mutations in cancer genomes.

Type-II topoisomerases resolve topological stress in DNA through double-strand breaks. While topoisomerases are chemotherapy targets linked to therapy-related genotoxicity, TOP2B is uniquely positioned to influence mutagenesis through its activity in non-dividing cells and sensitivity to topoisomerase poisons. To investigate this, we generated DNA-binding maps of TOP2B, CTCF, and RAD21 in human cancer samples and analyzed these for driver mutations and mutational processes across 6500 whole cancer genomes. TOP2B-CTCF-RAD21 and TOP2B-RAD21 sites are enriched in somatic mutations and structural variants, particularly at sites with evolutionary conservation, high transcription and long-range chromatin interactions. TOP2B binds driver genes such as TP53, MYC, FOXA1, and VHL, and many frequently mutated non-coding regions. We show that one non-coding TOP2B-bound element at the non-coding RNA gene RMRP drives tumor initiation and growth in vivo. Our study highlights TOP2B as a safeguard of genome integrity and a marker of mutational processes and hotspots in cancer, underscoring implications for cancer genomics research.