BackgroundThe topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α.Methodology/Principal FindingsWe show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.Conclusions/SignificanceThe discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.
Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of 1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ϳ2-and 4-fold-lower levels of 1 fiber than did T3wt virions. Importantly, using RNA interference to reduce 1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of 1 per virion. As 1 levels were reduced, virion infectivity increased by 2-to 5-fold per cellbound particle, demonstrating a causal relationship between virion 1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins 3 and 1C at similar rates. However, having started with fewer 1 molecules, a complete loss of 1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid 3 and 1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and 1 levels. IMPORTANCEWild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein 1 became more infectious toward transformed cells. Reduced 1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of 1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer 1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy. M ammalian orthoreovirus (reovirus) is a nonenveloped, icosahedral virus in the Reoviridae family (1). Reovirus is nonpathogenic in humans and has long served as a safe model system for understanding icosahedral virus structure and replication. Although reovirus encodes...
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