Genome Instability induced in cancer cells carrying mutations in Type II topoisomerases

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DNA topoisomerase II (Top2) alters DNA topology by making a double-strand break in DNA and passing an intact duplex through the break. The transient double strand break is generated through a protein/DNA covalent intermediate termed the cleavage complex. Mammalian cells contain two Top2 isoforms termed Top2α and Top2β. The two enzymes have distinct biological functions with Top2α playing a key role in chromosome segregation and Top2β having unique roles in transcription. While the DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks, a variety of mechanisms can interfere with the cleavage/religation equilibrium of the enzyme. For example, anti-cancer drugs like etoposide generate elevated levels of cleavage complexes leading to cytotoxicity and genome instability. Drugs targeting Top2 also cause secondary malignancies that arise from enzyme induced DNA damage. Since Top2 can cause genome instability, a hallmark of cancer, it is plausible that Top2 failure could be a driver of genome instability leading to cancer. We identified unique mutants in yeast Top2 and the two human isoforms that generated high levels of DNA damage in the absence of inhibitors of the enzyme. These mutant enzymes have a unique characteristic: while they can be expressed in yeast cells proficient for repairing DNA damage, their expression in DNA repair deficient cells, such as rad52- cells leads to cell killing. We examined whether one of these mutants, (top2-F1025Y,R1128G) induced mutations in yeast and found that expression of this allele led to a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Recently, it has been shown that human cancer cells that express Top2α-K743N are associated with a mutational signature closely related to that induced by yeast top2-F1025Y,R1128G suggesting that Top2α- K743N is a mutator that gives rise to genome instability. In this application, we will study the biochemical characteristics of Top2α-K743N. In addition, a small number of recurrent mutations have been found in cancer cells in Top2α or Top2β. Notably, Top2β-R656H has been recovered from more than 16 tumors of diverse origin. We will apply the tools we developed for studying top2-F1025Y,R1128G to the recurrent mutations found in both Top2α and Top2β. We will use expression of the mutant enzymes in rad52- cells and induction of etoposide hypersensitivity as a preliminary screen to determine whether the mutant proteins induce cytotoxic DNA damage. Putative DNA damaging Top2 proteins will be will be purified and characterized for the ability to generate DNA damage in vitro. Finally, we will begin to generate mammalian cell lines that express the mutant alleles by either CRISPR/Cas9 editing or ectopic expression to characterize how the mutant enzymes can lead to genome instability. Our results will establish whether Top2 alterations found in tumors can be oncogenic.