Shabro, A. | bioRxiv Science

bioRxiv2026

A multi-species toolkit of TOP2 hypercleavage mutants for studying topoisomerase II-mediated DNA damage

DNA topoisomerase II (TOP2) generates transient DNA double-strand breaks that are trapped as TOP2-DNA covalent complexes (TOP2cc) by antibiotic and chemotherapy drugs. Here, we characterize tools for study of cellular responses to TOP2cc, exploiting a Saccharomyces cerevisiae TOP2 mutant (TOP2-F1025Y,R1128G) that generates spontaneous and inhibitor-induced covalent complexes at elevated frequencies. This Top2-hc (for "hypercleavage") mutant protein inhibits yeast cell growth when expressed alone or with endogenous Top2, and growth defects are exacerbated in DNA-repair-deficient genetic backgrounds and/or in the presence of low doses of the Top2 poison mAMSA. We generated analogous mutations in human and mouse TOP2A and TOP2B that gave increased TOP2cc, hypersensitization to topoisomerase poisons, increased DNA damage, and decreased cell survival in cultured cells. We further established knock-in mouse models with inducible, tissue-specific expression of each TOP2-hc isoform, demonstrating overt organismal toxicity and cellular markers of DNA damage responses. To illustrate the potential of these genetic tools, we carried out proof-of-principle screens in yeast and cultured human cells for sensitivity to TOP2-hc. The yeast screen revealed strong requirements for homologous recombination, moderate roles for sister chromatid cohesion and kinetochore function, and dependencies on vesicle and vacuolar functions. The pilot shRNA screen in human cells revealed shared requirements for resistance to expression of either TOP2A-hc or TOP2B-hc as well as examples of isoform specificity. These findings establish hypercleavage mutant proteins as effective tools for studying topoisomerase isoform-specific DNA damage and offer a foundation for exploring TOP2cc toxicity and tolerance in vivo. Significance statementDNA topoisomerase II enzymes untangle chromosomes by cutting DNA, but incomplete resealing creates toxic damage that is the basis of antibacterial and chemotherapy drugs. Here we describe toolkits in yeast, mammalian cells, and mice that take advantage of mutant topoisomerase II enzymes that trap on DNA without drugs, creating powerful genetic systems to better study how cells deal with this type of DNA damage. We provide benchmarking data to validate these tools and to illustrate how they can be used for screens in cultured cells or tissue-specific experiments in vivo. These toolkits overcome longstanding technical barriers and enable new ways to study topoisomerase II-mediated DNA damage.

genetics↗

bioRxiv2023

Anaplastic Thyroid Cancer Cells Upregulate Mitochondrial One-Carbon Metabolism To Meet Purine Demand, Eliciting A Critical Targetable Vulnerability

Anaplastic thyroid cancer (ATC) is one of the most aggressive and lethal tumor types, characterized by loss of differentiation, epithelial-to-mesenchymal transition, extremely high proliferation rate, and generalized resistance to therapy. To identify novel relevant, targetable molecular alterations, we analyzed gene expression profiles from a genetically engineered ATC mouse model and from human patient datasets, and found consistent upregulation of genes encoding enzymes involved in the one-carbon metabolic pathway, which uses serine and folates to generate both nucleotides and glycine. Genetic and pharmacological inhibition of SHMT2, a key enzyme of the mitochondrial arm of the one-carbon pathway, rendered ATC cells glycine auxotroph and led to significant inhibition of cell proliferation and colony forming ability, which was primarily caused by depletion of the purine pool. Notably, these growth-suppressive effects were significantly amplified when cells were grown in the presence of physiological types and levels of folates. Genetic depletion of SHMT2 dramatically impaired tumor growth in vivo, both in xenograft models and in an immunocompetent allograft model of ATC. Together, these data establish the upregulation of the one-carbon metabolic pathway as a novel and targetable vulnerability of ATC cells, which can be exploited for therapeutic purposes.

cancer biology↗