The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the thirteen different cancer types evaluated, 6–90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

The Mdm2 oncogene is a negative regulator of the p53 tumor suppressor and recently identified inhibitor of DNA break repair. Nutlin-3 is a small molecule inhibitor of Mdm2/p53 interaction that can induce apoptosis in cancer cells through activation of p53. While this is promising therapy for those cancers with wild-type p53, half of all human cancers have inactivated p53. Here, we reveal a previously unappreciated effect of Nutlin is inhibition of DNA break repair, stemming from its ability to increase Mdm2 protein levels. The Nutlin-induced increase in Mdm2 inhibited DNA double-strand break (DSB) repair and prolonged DNA damage response signaling independent of p53. Mechanistically, this effect of Nutlin required Mdm2 and acted through Nbs1 of the Mre11/Rad50/Nbs1 DNA repair complex. In ovarian cancer cells where >90% have inactivated p53, Nutlin combined with the genotoxic agents, cisplatin or etoposide, had a cooperative lethal effect resulting in increased DNA damage and apoptosis. Therefore, these data demonstrate an unexpected consequence of pharmacologically increasing Mdm2 levels that when utilized in combination with genotoxic agents induces synthetic lethality in ovarian cancer cells, and likely other malignant cell types, that have inactivated p53. Implications Data reveal a therapeutically beneficial effect of pharmacologically increasing Mdm2 levels combined with chemotherapeutic agents for malignancies that have lost functional p53.

<p>Supplemental Figures S1-5. Figure S1. Nutlin inhibits γH2AX foci formation and prolongs resolution. Figure S2. Inhibition of pS/TQ foci formation and resolution by Nutlin. Figure S3. Nutlin inhibits γH2AX foci formation and resolution in ovarian cancer cells. Figure S4. Nutlin cooperates with genotoxic agents in p53-inactivated ovarian cancer cells. Figure S5. Nutlin and DNA damaging drugs combine to kill ovarian cancer cells that harbor mutant p53.</p>

<p>Supplemental Figures S1-5. Figure S1. Nutlin inhibits γH2AX foci formation and prolongs resolution. Figure S2. Inhibition of pS/TQ foci formation and resolution by Nutlin. Figure S3. Nutlin inhibits γH2AX foci formation and resolution in ovarian cancer cells. Figure S4. Nutlin cooperates with genotoxic agents in p53-inactivated ovarian cancer cells. Figure S5. Nutlin and DNA damaging drugs combine to kill ovarian cancer cells that harbor mutant p53.</p>