The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Abstract

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.

Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation

“…29 However, in agreement with various reports showing that Bmi1 is the rate-limiting factor in the ubiquitination reactions mediated by the Ring proteins, 28,29 we demonstrated that Bmi1 overexpression or knockdown was sufficient to, respectively, induce or inhibit p53 polyubiquitination and regulate p53 stability. In vitro, we showed that Bmi1 strongly increased both Ring1A-and Ring1B-mediated p53 mono-and di-ubiquitination, thereby suggesting that an E4 enzyme is necessary in vivo for the elongation of the ubiquitin chain primed by these two E3 ligase complexes, as previously shown for MDM2-mediated p53 polyubiquitination.…”

“…Bmi1 directly binds p53 and induces p53 polyubiquitination Bmi1 is known to enhance the E3 ubiquitin-ligase activity of Ring1A and Ring1B, and control the proteasomal degradation of a non-histone protein, topoisomerase 2a, 28 indicating that the Bmi1-induced phenotype can be mediated via mechanisms that do not involve transcriptional regulation. Consequently, we examined whether Bmi1 could bind p53 and directly induce its polyubiquitination.…”

Polycomb RING1A/RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S phase progression

“…4A) (Guenatri et al, 2004). Chromatin immunoprecipitation (ChIP) in control cells showed H2AK119Ub enrichment at these domains, but it was wiped out in mutant cells after only 48 h of treatment, in agreement with the highly dynamic turnover of this histone mark (Alchanati et al, 2009). In addition, we also found RING1B occupying major satellites in control cells (Fig.…”

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

“…In this study, we have characterized PRT4165, a small molecule identified in a screen for inhibitors of BMI1/RING1A-mediated polyubiquitylation of topoisomerase II-␣ upon trapping the DSB intermediate with the topoisomerase II inhibitor etoposide (7). We demonstrate that PRT4165 inhibits the two E3 ligase paralogues, RING1A and RNF2, in vitro, and that it can be used to inhibit H2A ubiquitylation in cells.…”

“…This inhibits ubiquitylation at DSBs by sequestering ubiquitin in the cytoplasm in lysine 48-linked polyubiquitin chains (6). Recently, an inhibitor of BMI1/RING1-mediated polyubiquitylation of topoisomerase II was identified (7). BMI1/RING1 are subunits of the polycomb repressive complex 1 (PRC1) (8,9).…”