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

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.

A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo

“…S3, A to D ). Consistent with both our iRFP data and published reports for p53 signalling after TBI 33 – 35 , we observed sustained p21 staining in the spleen, small intestine, and colon of PG13-iRFP mice at 3 days after both 6 and 8 Gy TBI treatments. Notably, tissues were histologically similar and exhibited similar induction of p21 in both PG13-iRFP+ and matched PG13-iRFP reporter-negative mice, suggesting that the presence of the p53 reporter did not alter the tissue response to TBI treatment.…”

“…S2F ). This observation is consistent with the established rapid activation of p53 after irradiation in vivo 6 , 31 – 35 , as well as the persistence of downstream p53 targets in irradiated tissues within the first 72 hours after TBI 33 – 35 . We were not able to resolve significantly increased IRFP signal in PG13-iRFP reporter mice earlier than 6 hours ( Fig.…”

“…For total body irradiation (TBI) experiments, mice were treated as previously described 33 with either 6 or 8 Gy TBI from an Xstrahl RS225 Cabinet X-ray Irradiator (Xstrahl) as indicated, imaged at the time points indicated, and sampled as noted in the manuscript.…”

It’s Getting Complicated—A Fresh Look at p53-MDM2-ARF Triangle in Tumorigenesis and Cancer Therapy

“…Multiple mouse models have also been utilized to suggest delicate distinctions between MDM2's ubiquitin ligase activity and ability to control p53 function. Two separate mutants of Mdm2 (Y487A-Y489A in human; I438K-I440K in human) have been demonstrated to partially restrain p53 response to DNA damage despite losing its ability to promote p53 degradation (Tollini et al, 2014;Humpton et al, 2021). Interestingly, while Mdm2 Y487A causes no developmental defect yet promotes p53-dependent mortality in response to sub-lethal stress in adult mice, Mdm2 I438K leads to embryonic lethality but is tolerated when only switched on in adult mice to allow enhanced p53 response to DNA damage.…”

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function

“…MDM2 inhibits p53’s transcriptional activity by directly binding p53 and promoting p53 ubiquitination and subsequent degradation by the proteasome ( Momand et al, 1992 ; Oliner et al, 1993 ; Haupt et al, 1997 ; Honda et al, 1997 ; Kubbutat et al, 1997 ; Shi & Gu, 2012 ). The importance of MDM2 E3 activity in p53 regulation is underscored by mouse studies, where expression of catalytically inactive Mdm2 mutants results in embryonic lethality that is rescued by Trp53 deletion ( Itahana et al, 2007 ; Humpton et al, 2021 ). The core of MDM2 E3 activity resides in its C-terminal RING domain.…”