Identification and Characterization of the First Small Molecule Inhibitor of MDMX

Abstract

The p53 pathway is disrupted in virtually every human tumor. In ϳ50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. Tumorigenesis is a multistep process that involves dysregulation of several pathways that are crucial for cell growth and survival (1). The p53 pathway regulates cell survival in response to cellular stress (e.g. DNA damage) or oncogenic stress (e.g. Rb pathway dysregulation) (2, 3) and is suppressed in virtually every human cancer by genetic lesions in the p53 gene or other components of the pathway (4). Approximately half of all cancers express wild-type p53, and considerable research over the past decade has focused on inducing p53-mediated cell death in these tumors (4, 5). Most efforts to date have focused on inhibiting MDM2, a negative regulator of p53 (6 -14).Another key regulator of the p53 pathway is a protein related to MDM2 called MDMX (15-17). MDM2 and MDMX share homology in their p53-binding domains, but MDMX is believed to regulate p53 through distinct mechanisms. Specifically, MDM2 primarily regulates p53 stability and subcellular localization, whereas MDMX may directly regulate p53 transcription (17-21). MDMX is genetically amplified in 19% of breast carcinomas, 19% of colon carcinomas, 18% of lung carcinomas, and a smaller percentage of gliomas (17). One of the best characterized tumors with an MDMX amplification is retinoblastoma. Approximately 65% of human retinoblastomas have increased MDMX copy number, which correlates with increased MDMX mRNA and protein (22). Previous studies have demonstrated that the MDMX amplification suppresses p53-mediated cell death in Rb pathway-deficient retinoblasts (22).A general consensus is emerging that to efficiently induce a p53 response in tumor cells that express wild-type p53, it may be necessary to inactivate both MDM2 and MDMX (18,23,24). To date, no screens to identify small molecule inhibitors of MDMX have been reported, and MDM2 inhibitors probably do not bind as efficiently to MDMX because of structural differences in the p53-binding pockets of the two proteins (25-27). Consistent with this theory, nutlin-3a binds MDMX with at least a 40-fold weaker equilibrium binding constant than for MDM2 (22). Therefore, to identify small molecules that bind MDMX and prevent its interaction with p53, we developed biochemical and cell-based assays suitable for high throughput screening (HTS)

Ubiquitylation of p53 by the APC/C inhibitor Trim39

“…Given this, effective elevation of p53 in tumors may well require a mixture of E3 ligase inhibitors tailored to the population of p53-directed ligases present in an individual tumor. Indeed, unique inhibitors that target both MDM2 and MDMX can impede growth and induce apoptosis in cells with high levels of both MDM2 and MDMX (26,31,32).…”

Abrogation of Wip1 expression by RITA-activated p53 potentiates apoptosis induction via activation of ATM and inhibition of HdmX

“…Several small molecules inhibiting Hdm2 have been identified; however, these do not affect HdmX, impeding their efficiency, in particular in tumours with overexpressed HdmX. 16 An inhibitor of the HdmX/p53 interaction, SJ-172550 has been described recently; 19 it does not kill cancer cells on its own, but has an additive effect when combined with Hdm2 inhibitors. However, simultaneous targeting of Hdm2 and HdmX by a single molecule has not yet been reported.…”

“…Recently, the chemical inhibitor of HdmX/p53 interaction, SJ-172550 has been found, which displays additive effects in cancer cells when combined with an Hdm2 inhibitor. 19 Wild-type p53-induced phosphatase 1 (Wip1), encoded by the PPM1D gene, is a serine/threonine phosphatase that exhibits oncogenic features. It is overexpressed in a vast number of tumours 20 and is important for tumour stem cell survival.…”

MDM4 downregulates p53 transcriptional activity and response to stress during differentiation

“…Although they are homologs, it has been shown that Mdm2 and Mdm4 have distinct roles in the regulation of p53, 13 and we show that ES cells are another example where they have non-overlapping functions. Importantly small molecule inhibitors have been developed that can selectively block the p53 Mdm2 interaction and the p53-Mdm4 interaction 28,29 and it is important to understand the differential regulation of p53 by Mdm2 and Mdm4 to optimize their potential clinical use. We also demonstrate that our findings in ES cells are valid in the context of a whole organism.…”