The MDMX (MDM4) oncoprotein as a therapeutic target and determinant of response to MDM2-p53 binding antagonists in human cancer

Abstract

The tumour suppressor p53 is activated by cellular stress to induce cell cycle arrest and/or apoptosis. Despite being frequently mutated, theTP53 gene is wild-type and functional in approximately 50% of human cancers. Targeting the p53 tumour suppressor pathway by inhibition of its negative factors MDM2 and MDMX is central to many cancer therapies. Small molecule antagonists have been developed to inhibit p53-MDM2 binding to release p53 and reactivate p53 function. However, previous studies have indicated that MDMX amplification or expression may be associated with resistance to MDM2-p53 binding inhibitors. MDMX could also play a significant role in the response to other therapeutic agents that act by a p53- dependent mechanism. The effects of MDM2-p53 binding antagonists (Nutlin-3 and RG7388) and the MDM2/X–p53 binding co-inhibitor (RO5963) were compared in a panel of cell lines of different TP53 and MDMX(MDM4) status. The endpoints tested included expression of p53 and its downstream transcriptional targets, growth inhibition, cell cycle distribution changes and caspase 3/7 apoptotic activity. Moreover, the effect of suppression of MDMX expression by lentiviral shRNA and siRNA systems on the response to MDM2 inhibitors and co-inhibitors was tested in a panel of cell lines. Affymetrix Human Transcriptome Array 2.0 was used to detect differences in the expression of full-length genes and alternatively spliced forms after suppression of MDMX expression in MDM4-amplified MRK-nu-1 cells. The results showed that cells with wild-type p53 respond to both MDM2-p53 and MDM2/X-p53 antagonists by growth inhibition. TP53 mutational status is the main factor governing resistance to MDM2-p53 binding antagonists. In TP53 wild-type cells, MDMX expression is associated with sensitivity to the RO5963 MDM2/X coninhibitor and has only minor impact on resistance to MDM2-p53 binding antagonists. Knockdown of MDMX reduced cell growth by induction of cell cycle arrest in both p53 dependent and independent ways, while the effect of MDMX suppression had a modest effect on the efficacy of MDM2-p53 binding antagonists which was cell line dependent. Reduction of MDMX expression slightly increased TP53-dependent downstream transcriptional activity measured by Affymetrix Human transcriptome ii array 2.0. The gene showing the greatest increase in response to MDMX knockdown was VGLL1, which is an oncogene associated with the Hippo pathway that regulates organ size. Suppression of MDMX may activate VGLL1-TEAD dependent transcriptional activity, thereby regulating cell proliferation via an increase of VGLL1 expression, linking to the Hippo signalling pathway. In summary, TP53 status has a much greater impact on the response to pure MDM2- p53 binding antagonists compared with MDMX expression. In wild-type TP53 cell lines, MDMX amplification and high expression was modestly associated with resistance to MDM2-p53 binding antagonists.