Abstract CN08-02: Repair of topoisomerase II mediated DNA damage: A potential mechanism for targeting specific Top2 isoforms
Authors: Nitiss John L. , Mishina Margarita , Berk Jeffrey 
Published: May 2019

Abstract
DNA topoisomerases are ubiquitous enzymes that change a property of DNA structure that is termed DNA topology (1). During the processes of DNA replication, transcription, nucleosomes assembly, and chromosome condensation, the double stranded structure of DNA imposes restraints that can lead to overwinding or underwinding of DNA. DNA topoisomerases overcome these constraints by making transient breaks in DNA using a unique mechanism that entails the creation of an enzyme:DNA covalent intermediate. Drugs targeting these enzymes exploit this enzymatic mechanism and increase the level of enzyme covalently bound to DNA. The generation of elevated levels of enzyme:DNA covalent complexes is the key event in cell killing by drugs targeting DNA topoisomerases. Drugs that lead to elevated cleavage are termed topoisomerase poisons (2,3). Protein linked DNA strand breaks are rapidly induced by topoisomerase poisons, and enzyme mediated DNA damage effectively blocks transcription and replication.
Mammalian cells encode two type II topoisomerases that function in distinct biological processes. Top2α is the main isoform involved in DNA replication and chromosome separation, and is required for the viability of all dividing cells (4). While mouse knockouts of Top2β are inviable due to neuromuscular defects (5), viable Top2β- cells can be recovered, and show no effects on replication or cell viability. The two isoforms show similar biochemical properties, and both isozymes are inhibited by most Top2 poisons. Since Top2α is expressed in dividing cells, it has been broadly assumed that the most important target for anti-cancer drugs is Top2α, and the relevance of Top2β to anti-cancer drug action remains an open question (6). However, recent results suggest that Top2β may be a contributor to some of the deleterious effects of drugs that target Top2 enzymes. Treatment regimens that include Top2 poisons, especially etoposide and teniposide, have been shown to cause translocations that can lead to secondary acute myeloid leukemia and other malignancies (7). A novel insight into secondary malignancies induced by Top2 targeting drugs has come from studies using a transgenic mouse model that carried skin specific knockout of Top2β. While etoposide applied to skin can generate melanomas, in skin lacking Top2β, the frequency of etoposide induced tumors was substantially reduced (8). An intriguing hypothesis is that targeting Top2β in non-dividing cells may lead to non-lethal DNA damage that results in oncogenic translocations.
Top2β may also play an important role in anthracycline-induced cardiomyopathy. Anthracyclines affect cells by a variety of mechanisms including acting as Top2 poisons, but also by the generation of free radicals. Bisdioxopiperazines are effective cardioprotectants and the major mechanism had been proposed to be due to chelation of iron that participates in free radical formation. Anthracyclines induce DNA damage signals that are blocked by bisdioxopiperazines. The DNA damage signal induced by anthracyclines is attenuated in MEFs derived from Top2β-null mice compared with MEFs that express Top2β (9). Since bisdioxopiperazines can lead to targeted degradation of Top2β, an important part of the cardioprotective mechanism may be the selective elimination of Top2β, thereby reducing the effects of drug induced DNA damage.
While specific targeting of Top2α may be an effective therapeutic strategy that eliminates some of the toxicity due to targeting both isoforms of Top2, it is less clear how this can be accomplished. While a Top2α specific drug has been recently described (10), the high degree of homology between the two enzymes suggests that it may be difficult to devise a potent and specific inhibitor of Top2α. A second approach may be to enhance specific targeting of Top2α by inhibiting repair of Top2α but not Top2α mediated DNA damage. Is this possible?
Repair of Top2 mediated damage requires recognition of a trapped Top2 complex, removal of the protein that is covalently bound to DNA, and repair of the DNA strand breaks. There are two mechanisms that have been described for initiating repair of Top2 DNA damage. In the first mechanism, covalently bound Top2 is targeted for degradation by the proteasome. Liu and colleagues showed that treatment with Top2 poisons led to a rapid depletion of Top2β (11). Degradation of Top2β could be prevented by inhibitors of transcription, but not by replication inhibitors. Subsequent work has shown that in some cell lines, the α isoform is also degraded in a proteasome dependent fashion. These results suggest that there may be differential recognition of trapped Top2α and Top2β. There is little information concerning effectors of repair after proteolytic degradation of Top2, but if the recognition mechanisms differ, then subsequent repair steps may in part by separate pathways.
An important pathway for removing protein DNA adducts is to excise the “adduct” by nucleolytic digestion of DNA that is covalently bound to the protein. A nucleolytic step is required even if the Top2 complex is partially degraded by proteolysis, since a protease cannot hydrolyze the phosphotyrosyl linkage of the protein bound to DNA. Until recently, there has been little direct information about nucleolytic removal of Top2 covalently bound to DNA. Recent results have implicated CtIP, a nuclease associated with the MRN complex as one protein that may participate in removal of trapped Top2 DNA covalent complexes (6). Other nucleases in yeast may also play important roles in processing Top2 covalent complexes including the structure specific nuclease Slx1/Slx4 and the excision repair nuclease Rad2 (XPG in mammalian cells). Deciphering the pathways involved in repairing Top2α and Top2β complexes will require additional information about genes that function in the repair of Top2 mediated damage, and the development of assays that can assess isoform specific repair pathways.
We have identified a large number of genes in yeast that are required for the repair of Top2 mediated DNA damage. These include genes required for double strand break repair, checkpoint surveillance of DNA damage, and many unanticipated genes. Importantly, several genes encoding nucleases and genes encoding proteins that participate in proteolytic degradation pathways confer high levels of sensitivity to etoposide when mutated. These genes represent a useful starting point for defining pathways that are required for repairing Top2 mediated damage in mammalian cells.
Keeney and colleagues developed a novel assay in yeast to identify potential nucleolytic processing pathways of a Top2 related protein that initiates meiotic recombination (12). We have applied the Keeney assay to identify whether direct nucleolytic processing of Top2α or Top2β occurs in mammalian cells. Our initial results suggest that nucleolytic processing of both Top2 and Top2 occur even in the absence of proteolysis. Interestingly, the products of the processing reactions suggest that there may be important differences in processing of the two isoforms.
DNA topoisomerase II has been a known important target of anti-cancer drugs for more than 25 years. He results described above provide unexpected aspects of reducing side effects of Top2 targeting drugs, and perhaps increasing their clinical efficacy. Substantial efforts will be required both in rational drug discovery, and in the understanding of the cellular responses to Top2 targeting to maximize the clinical usefulness of Top2 targeting agents.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN08-02.