Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice.

Timothy J Humpton,Douglas Strathdee,Karen H Vousden,David Stevenson,Koji Nomura,Andreas K Hock,Karen Blyth,Julia Weber,Helge M Magnussen,Colin Nixon,Sandeep Dhayade,Danny T Huang

doi:10.1101/gad.341875.120

Timothy J Humpton, Douglas Strathdee + Show 10 more

Open Access

https://doi.org/10.1101/gad.341875.120

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Abstract

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.

Highlights

The p53 transcription factor is a critical regulator of the cellular response to an extensive range of genotoxic, metabolic, and environmental stressors (Levine 2020)
To examine the importance of complete loss of MDM2 E3 activity, we previously demonstrated that single point mutations in the MDM2 RING finger domain at amino acid I440 or R479 prevented the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2 E3 activity without otherwise disrupting the structure of the MDM2 RING finger domain (Nomura et al 2017)
To understand the in vivo consequences of loss of E3 activity in MDM2 in mice, we first generated mutations in mouse Mdm2 at residues analogous to human MDM2 C464, I440, and R479 (C462A, I438K, I438E, and R477P), which were previously shown to abrogate E3 function. Transfection of these mutants with p53 into mouse embryo fibroblasts (MEFs) confirmed a similar defect in the ability to degrade p53 compared with wild-type MDM2 (Supplemental Fig. S1A,B)

Summary

Introduction

The p53 transcription factor is a critical regulator of the cellular response to an extensive range of genotoxic, metabolic, and environmental stressors (Levine 2020). Two related RING finger E3-ligase proteins, MDM2 and MDMX (MDM4 in mice), act as the main negative regulators of p53 stability (Karni-Schmidt et al 2016; Moyer et al 2017). Uncovering mechanisms that would lead to the reactivation of the latent p53 in these tumors is an area of therapeutic interest and has resulted in the development of many compounds that block the MDM2-p53 interaction (Cheok and Lane 2017) These compounds have been of limited efficacy in clinical trials due to dose-limiting on-target toxicities arising from p53-mediated neutropenia and thrombocytopenia (Andreeff et al 2016; Kastenhuber and Lowe 2017)

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