The C terminus of p53 binds the N-terminal domain of MDM2

Cell Health and Cytoskeleton, 2010

The tumor suppressor p53 functions as an important defense against the development of cancer, and is negatively regulated by interaction with the oncogene and E3 ligase MDM2. In a tightly controlled system of feedback, MDM2 is, in turn, inhibited by the tumor suppressor p14 ARF. The inhibition of MDM2-p53 interaction is an appealing therapeutic strategy for the treatment of cancer, and significant advances have been made in the development of small-molecule inhibitors which block this interaction and reactivate wild-type p53. However, the p53 gene is frequently mutated or deleted in cancer, or the wild-type p53 function inhibited by high levels of MDM2. Neuroblastoma (NB) is one such cancer and has presented a major therapeutic challenge in pediatric oncology. Although most NB tumors have wild-type p53, the p14 ARF /MDM2/p53 pathway is often altered, leading to resistance to many mainstay chemotherapeutics and a high incidence of relapse. In preclinical studies, the MDM2/p53 interaction inhibitor nutlin-3a has shown effectiveness in the treatment of chemoresistant NB with wild-type, mutant or null-p53 status, indicating that nutlin-3a has potential for the treatment of a broad range of chemoresistant and relapse tumors.

Journal of Molecular Biology, 2002

We have investigated the kinetic and thermodynamic basis of the p53 -MDM2 interaction using a set of peptides based on residues 15 -29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580 nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53 -MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19 -26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.

Proceedings of the National Academy of Sciences, 2006

The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.

Oncotarget, 2016

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

Cell Death and Disease, 2015

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

BMC Cancer, 2011

Background The mammalian DNA-damage response (DDR) has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs), mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+) with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37). Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

Annual Review of Pharmacology and Toxicology, 2009

Tumor suppressor p53 is an attractive cancer therapeutic target because it can be functionally activated to eradicate tumors. Direct gene alterations in p53 or interaction between p53 and MDM2 proteins are two alternative mechanisms for the inactivation of p53 function. Designing small molecules to block the MDM2-p53 interaction and reactivate the p53 function is a promising therapeutic strategy for the treatment of cancers retaining wild-type p53. This review will highlight recent advances in the design and development of small-molecule inhibitors of the MDM2-p53 interaction as new cancer therapies. A number of these small-molecule inhibitors, such as analogs of MI-219 and Nutlin-3, have progressed to advanced preclinical development or early phase cinical trials.

Genes and Development, 2018

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.

Proceedings of the National Academy of Sciences, 2000

p300 acetylates and activates the tumor suppressor p53 after DNA damage. Here, we show that MDM2, a negative-feedback regulator of p53, inhibited p300-mediated p53 acetylation by complexing with these two proteins. First, we purified a p300 -MDM2-p53 protein complex from HeLa nuclear extracts, which was inactive in p53 acetylation, but active in histone acetylation. Also, wild-type, but not N-terminally deleted, MDM2 inhibited p53 acetylation by p300 in vitro and in vivo. This inhibition was specific for p53, because MDM2 did not affect acetylation of histones or the C terminus of p73 by p300. Consequently, wild-type, but not the mutant, MDM2 repressed the p300-stimulated sequence-specific DNA-binding and transcriptional activities of p53. These results demonstrate that an additional mechanism of p53 inactivation by MDM2 is to inhibit p53 acetylation by p300.

2023

Supplementary Figure S1. Analysis of p53 ubiquitination and binding A, p53-/-Mdm2-/-Mdm4-/mouse embryonic fibroblasts (TKO-MEFs) were transiently transfected with indicated plasmids and after 24hrs treated with 10µM of the proteasome inhibitor MG132 for 7hrs. Cell lysates were analysed by WB for the indicated proteins. The mutant MDM2C464G, null for ubiquitin ligase activity, was used as negative control of ubiquitinating activity. B, C, TKO-MEFs were transiently transfected with p53 and indicated MDM2 mutants (B) or MDM4 mutants (C). After 16 hours, 200 µg of cell lysate were immunoprecipitated with mAb Anti-p53 (Ab1) and coimmuonocomplexes analysed by WB. WCE is 1/5 of immunorecipitated lysate. D, analysis of p53 ubiquitination in HCT116 untreated (NT) or treated with 0,1% DMSO, 10µM Pep3 or Pep3M for 24 hours. 30µM MG132 was used for 8hrs. E, analysis of in vitro p53 ubiquitination in the presence of GST-MDM2 and of the indicated peptides (20µM). In vitro translated p53-flag was reacted with 100 ng human recombinant GST-MDM2 in the presence of 200nM UBE1, 1µM UbcH5c, 500µM Ubiquitin, 1X Ubiquitin conjugation reaction buffer, 5mM ATP for 30' at 37°. Each peptide prereacted with MDM2 for 15'.