Abstract 2838: Modeling putative oncogenic variants of human topoisomerase II by genome editing of a near haploid cell line

DNA topoisomerases (Topos) carry out changes in DNA structure by introducing transient breaks in DNA. These breaks are needed to change DNA conformation, and the enzyme mechanisms minimize possible genome instability from a failure to resolve the cleaved DNA intermediates. Nonetheless, small molecules can interfere with the Topo reaction, leading to elevated DNA cleavage, genome instability and cell death, and this property is the basis of the action of important anti-cancer drugs. Recent work has led to the identification of mutations in eukaryotic Top2 that also result in high levels of Top2 cleavage; including Lys743Asn in the Top2 alpha isoform which was observed in multiple human cancers. Hyper cleavage Top2 mutants are genome destabilizing in yeast and have been shown to induce 2-5 nucleotide de novo duplications, as well as other genetic changes. To generate mammalian cell lines expressing hyper cleavage Top2 mutants, we used Piggybac mediated scarless editing in the haploid mammalian cell line eHap. We accomplished successful editing of both Top2 alpha and Top2 beta in this cell line. Successful editing was assessed by sequencing Top2 cDNAs as well as sequencing of the edited genomic loci. Two independent successfully edited cell lines resulted in changes of Lys600Thr in Top2 beta and Lys579Thr in Top2 alpha; allowing us to compare the effects of mutations at orthologous positions in the two mammalian Top2 isoforms. Both edited cell lines show strong constitutive induction of DNA damage responses with elevated levels of DNA-PKcs and histone H2AX phosphorylation. Interestingly, elevated levels of ubiquitylated H2AX were also seen, suggesting that H2AX ubiquitylation may be indicative of constitutive topoisomerase induced damage. These cell lines are being used to measure genetic changes induced by topoisomerase induced damage and will be a unique tool to identify pathways that repair Topo-induced DNA damage. These cells will also provide insights into topoisomerase mediated mechanisms of oncogenesis. Citation Format: Karin C. Nitiss, Brigid Conroy, Maureen N. McCoy, John L. Nitiss. Modeling putative oncogenic variants of human topoisomerase II by genome editing of a near haploid cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2838.