Profiling Inhibitors Of Mdm2: P53 And Mdmx: P53 In Relation To Mdmx Protein Levels

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Pharmacological antagonism of the interaction between MDM2 and/or MDMX with p53 has been pursued as a therapeutic strategy to restore p53 tumour suppressor activity in wild type TP53 tumours with amplification or over-expression of these negative regulators. The majority of small molecule approaches described to date inhibit MDM2 binding to p53, which has E3 ligase activity and through ubiquitination targets p53 for degradation. However, MDMX, although devoid of E3 ligase activity itself, forms heterodimers with MDM2 and augments its activity to induce polyubiquitination of p53. Further complexity in this regulatory network arises from the fact that MDM2 is a target gene of p53 transcription, and that MDM2 can also control the stability of MDMX. Since MDMX is widely reported to confer resistance to established MDM2:p53 antagonists, this study aimed to examine the activity of MDM2:p53 selective inhibitors across a panel of wild-type TP53 cell lines with variable MDM2 and MDMX protein expression, and to contrast this with recently described inhibitors with mixed MDM2:p53 and MDMX:p53 activity. Growth inhibition studies (72h) were performed using XTT assays, but with verification by orthogonal methods (SRB, clonogenic assays). Gene expression was determined at the level of mRNA and protein. The selectivity of all inhibitors was confirmed initially in p53 wild-type and non-functional paired cell lines. As anticipated, MDM2:p53 selective inhibitors (nutlin-3a, MI-773, RG7112) were active in MDM2 amplified cell lines with low MDMX expression (SJSA-1 and T778; GI50 range 0.2 to 1 uM). However, a similar level of activity was also evident in cell lines with low/medium MDM2 in the presence of medium/high MDMX expression (Pre B 697, A375, HCT116, MRK-NU1, and NGP cells: GI50 range 0.2 to 2 uM). Appreciable resistance was only evident in Jeg3 and MCF-7 cells (low MDM2 and high MDMX), averaged compound GI50 values ranging from 9 - 10 uM in Jeg3 and 6 - 14 uM in MCF-7. Whilst a concentration dependent degradation of MDMX was observed in some sensitive cell lines with medium/high MDMX expression, in others no change was evident, suggesting that there are multiple determinants of cellular sensitivity to such inhibitors. Importantly, the mixed MDM2:p53 and MDMX:p53 antagonist RO-5963, displayed a different spectrum of activity, with least growth inhibition evident in SJSA-1 and T778 MDM2 amplified cell lines, and activity being retained in MDMX amplified Jeg3 cells. These studies support the concept that targeting MDMX may provide a different spectrum of antitumour activity to selective MDM2:p53 inhibition. However, MDMX protein levels per se do not constitute a universal resistance factor to selective MDM2:p53 inhibitors and, beyond MDM2 amplification, the identification of additional markers of sensitivity is warranted for this class of agent. Citation Format: Sue Tudhope, Yan Zhao, Anita Wittner, Elaine Wilmore, Annalisa Bertoli, Santosh Adhikari, Suzannah J. Harnor, Fabio Del Bello, Alessandro Piergentili, John Lunec, Ian R. Hardcastle, Roger J. Griffin, Bernard R. Golding, Celine Cano, David R. Newell, Stephen R. Wedge. Profiling inhibitors of MDM2:p53 and MDMX:p53 in relation to MDMX protein levels. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2014-5451