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In recent years, the strategy of inhibiting the interactions of p53 with murine double minute 2 (MDM2) and murine double minute X (MDMX) has been proved to be a promising approach for tumor therapy. However, the poor proteolytical stability and low intracellular delivery efficiency of peptide inhibitors limit their clinical application. Here, we designed and synthesized the bicyclic stapled peptides based on p53 by combining all-hydrocarbon stapling and lactam stapling strategies. We demonstrated that bicyclic stapled peptide p53-16 significantly improved α-helicity and proteolytic stability. Especially, p53-16 showed nanomolar binding affinity for MDM2 and MDMX. In addition, p53-16 could penetrate the cell membrane, and selectively inhibited the activity of tumor cells via activating p53 pathway in vitro. Our data suggest that p53-16 is a potential dual inhibitor of MDM2 and MDMX interactions. The bicyclic stapling strategy is a promising drug design strategy for protein–protein interactions inhibitors.

Introduction: E3 (ubiquitin) ligases play a major role in the ubiquitin-proteasome system (UPS), responsible for the ubiquitination and degradation of various proteins. The UPS has many roles, including regulation of the cell cycle. To mediate these functions, there are many different types of E3s, each with different substrates. A major E3 system involved in oncogenesis is the Mdm2/MdmX system, which acts as a heterodimer to degrade the tumour suppressor, p53, responsible for inducing cell cycle arrest and/or apoptosis in cancer cells, as needed. Upon overexpression/hyperactivation through mutation, the Mdm2/MdmX system can promote carcinogenesis through increasing degradation of p53, preventing necessary cell cycle arrest/apoptosis in cancer cells. Methods: A literature review was conducted to synthesize and analyze research on Mdm2/MdmX E3 overexpression/hyperactivation, and the treatment options available for cancers in which overexpression/hyperactivation plays a role. Results: There are many types of mutations that may be present in cancer cells, however mutations leading to the inactivation of p53 are some of the most common. Inactivation of p53 can be achieved by direct gene mutation, or overexpression/hyperactivation of Mdm2/Mdmx. Current drugs target the expression of MdmX/Mdm2 or their binding interactions with p53. Inhibition of these interactions triggers apoptosis in cancer cells due to increased p53 activity. Therapies that have been developed to target the Mdm2/MdmX system include small molecule inhibitors such as Nutlins and MI compounds, as well as peptide drugs. Discussion: Although direct mutations of p53 are commonly found in cancer, mutated p53 is not a viable drug target, so instead many treatment options specifically target a dysregulated Mdm2/MdmX system. Future studies should investigate novel drug targets, minimization of side effects, and treatment in the presence of mutations to other DNA repair systems. Conclusion: This literature review aids in establishing an interdisciplinary perspective on the types of oncogenic mutations in the Mdm2/MdmX pathway, combining biochemical and mechanistic research with clinical applications and pharmacology, as well as identifying future drug targets involved in this system.

Most well studied as proteins that restrain the p53 tumor suppressor protein, MDM2 and MDMX have rich lives outside of their relationship to p53. There is much to learn about how these two proteins are regulated and how they can function in cells that lack p53. Regulation of MDM2 and MDMX, which takes place at the level of transcription, post-transcription, and protein modification, can be very intricate and is context-dependent. Equally complex are the myriad roles that these two proteins play in cells that lack wild-type p53; while many of these independent outcomes are consistent with oncogenic transformation, in some settings their functions could also be tumor suppressive. Since numerous small molecules that affect MDM2 and MDMX have been developed for therapeutic outcomes, most if not all designed to prevent their restraint of p53, it will be essential to understand how these diverse molecules might affect the p53-independent activities of MDM2 and MDMX.

Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.

We hypothesized that murine double minute X (MDMX), a negative p53-regulator, may be involved in dysfunctional p53-signaling in anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive and ALK-negative, characterized frequently by non-mutated

Nanomedicines generally suffer from poor accumulation in tumor cells, low anti-tumor efficacy, and drug resistance. In order to address these problems, we introduced a novel nanomedicine based on dual anti-cancer drugs, which showed good cell nuclear accumulation properties. The novel nanomedicine consisted of three components: (1) dual anti-cancer drugs, 10-hydroxycamptothecin (HCPT) and chlorambucil (CRB), whose targets are located in the cell nucleus, (2) a nuclear localizing dodecapeptide, PMI peptide (TSFAEYWNLLSP), which could activate p53 by binding with MDM2 and MDMX located in the cell nucleus, and (3) an efficient self-assembling tripeptide FFY. Our nanomedicine exhibited enhanced cellular uptake and nuclear accumulation properties, thus achieving an excellent anti-cancer capacity both in vitro and in vivo. Our study will provide an inspiration for the development of novel multifunctional nanomaterials for cancer diagnosis and therapy.

As alternatives to small-molecular proteolysis-targeting chimeras (PROTAC), peptide-based molecular glues (MG) are a broad range of dual-functional ligands that simultaneously bind with targetable proteins and E3 ligases by mimicking proteinprotein interaction (PPI) partners. Methods: Herein, we design a peptide-derived MG to target a tumor-driving protein, MDMX, for degradation, and nanoengineered it into a supramolecular gold(I)-thiol-peptide complex (Nano-MP) to implement the proteolysis recalcitrance, cellular internalization, and glutathione-triggered release. To optimize the tumor targeting, a pH-responsive macromolecule termed polyacryl sulfydryl imidazole (PSI) was synthesized to coat Nano-MP. Results: As expected, Nano-MP@PSI induced the MDMX degradation by ubiquitination and subsequently restored the anti-cancer function of p53 and p73. Nano-MP@PSI revealed potent anti-cancer activities in an orthotopic xenograft mouse model of retinoblastoma by intraocular injection and a patient-derived xenograft model of malignant pancreatic cancer by systemic injection, while maintaining a favorable safety profile and showing a highly favorable clearable profile of excretion from the living body. Conclusion: Collectively, this work not only provided a clinically viable paradigm for the treatment of a wide variety of tumors by multiple administration types, but, more importantly, it bridged the chasm between peptides and PROTACs, and likely reinvigorated the development of peptide-derived proteolysis-targeting chimeras for a great variety of diseases.

Background/aim Despite advances in treatment modalities, the visual prognosis of retinoblastoma still remains unsatisfactory, underscoring the need to develop novel therapeutic approaches. Materials and methods The effect on the growth of six human retinoblastoma cell lines and a normal human fibroblast cell line of CEP1347, a small-molecule kinase inhibitor originally developed for the treatment of Parkinson's disease and therefore with a known safety profile in humans, was examined. The role of the P53 pathway in CEP1347-induced growth inhibition was also investigated. Results CEP1347 selectively inhibited the growth of retinoblastoma cell lines expressing murine double minute 4 (MDM4), a P53 inhibitor. Furthermore, CEP1347 reduced the expression of MDM4 and activated the P53 pathway in MDM4-expressing retinoblastoma cells, which was required for the inhibition of their growth by CEP1347. Conclusion We propose CEP1347 as a promising candidate for the treatment of retinoblastomas, where functional inactivation of P53 as a result of MDM4 activation is reportedly common.

Many high-affinity peptide antagonists of MDM2 and MDMX have been reported as activators of the tumor suppressor protein p53 with therapeutic potential. Unfortunately, peptide activators of p53 generally suffer poor proteolytic stability and low membrane permeability, posing a major pharmacological challenge to anticancer peptide drug development. We previously obtained several potent dodecameric peptide antagonists of MDM2 and MDMX termed PMIs, one of which, TSFAEYWALLSP, bound to MDM2 and MDMX at respective affinities of 0.49 and 2.4 nM. Here we report the development of gold nanoparticles (Np) as a membrane-traversing delivery vehicle to carry PMI for anticancer therapy. Np-PMI was substantially more active in vitro than Nutlin-3 in killing tumor cells bearing wild-type p53, and effectively inhibited tumor growth in metastasis in a mouse homograft mode of melanoma and a patient-derived xenograft model of colon cancer with a favorable safety profile. This clinically viable drug delivery strategy can be applied not only to peptide activators of p53 for cancer therapy, but also to peptide therapeutics in general aimed at targeting intracellular protein-protein interactions for disease intervention.

Via regulation of cellular stress responses, p53 contributes to the maintenance of homeostasis. Contrary to its well-established pro-death function, p53 is also implicated in promoting cell survival by mediating the adaptive stress response. Emerging data reveal that the adaptive stress response is coupled with p53 decline that is a prerequisite for the induction of pro-survival pathways augmenting cell fitness. However, if the adaptive stress responses persist or become chronic, the sustained p53 downregulation would result in a permanent loss of p53 function and p53-dependent homeostasis. The available information suggests a model in which cells respond to different levels of stress by governing the activity and abundance of p53 that, in turn, determines the cell fate dependent on not only the intensity but also the duration of stress.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2–MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2–MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2–MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Chemotherapy resistance represents a major obstacle for the treatment of patients with breast cancer (BC) and greatly restricts the therapeutic effect of the first-line chemotherapeutic agent doxorubicin (DOX). The present study aimed to investigate the feasibility of the recombinant dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor in reversing the DOX resistance of BC. Both DOX-resistant human breast carcinoma cell lines exhibited a multidrug resistance (MDR) phenotype. The ability of the dual-target MDM2/MDMX inhibitor in reversing doxorubicin resistance was subsequently verified, (9.15 and 13.92 - fold reversal indexes) respectively. We observed that the MDM2/MDMX inhibitor in combination with DOX could suppress proliferation, promote cell cycle arrest and induce apoptosis. In addition, it was capable of reducing rhodamine123 efflux in DOX-resistance BC cell lines and further played a key role in BC nude mice model. The groups that were treated with the combination of the drugs had decreased P-glycoprotein/multidrug resistance-associated protein/cdc 2/Bcl-2 expression and increased CyclinB1/Bax expression. These effects were caused due to activation of the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)/TAK1/p38 mitogen-activated protein kinase (MAPK) signaling pathway, as shown by small interfering RNA (siRNA) silencing and immumohistochemical staining of BC tissue sections. Furthermore, high MDM2/MDMX expression was positively associated with weak TAB1 expression in BC patients. Therefore, the recombinant dual-target MDM2/MDMX inhibitor could reverse doxorubicin resistance via the activation of the TAB1/TAK1/p38 MAPK pathway in wild-type p53 multidrug-resistant BC.

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2–Murine Double Minute X (MDM2–MDMX) heterodimer. The role of MDM2–MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

Inhibition of the MDM2/X-p53 interaction is recognized as a potential anti-cancer strategy, including the treatment of glioblastoma (GB). In response to cellular stressors, such as DNA damage, the tumor suppression protein p53 is activated and responds by mediating cellular damage through DNA repair, cell cycle arrest and apoptosis. Hence, p53 activation plays a central role in cell survival and the effectiveness of cancer therapies. Alterations and reduced activity of p53 occur in 25-30% of primary GB tumors, but this number increases drastically to 60-70% in secondary GB. As a result, reactivating p53 is suggested as a treatment strategy, either by using targeted molecules to convert the mutant p53 back to its wild type form or by using MDM2 and MDMX (also known as MDM4) inhibitors. MDM2 down regulates p53 activityviaubiquitin-dependent degradation and is amplified or overexpressed in 14% of GB cases. Thus, suppression of MDM2 offers an opportunity for urgently needed new therapeutic interventions for GB. Numerous small molecule MDM2 inhibitors are currently undergoing clinical evaluation, either as monotherapy or in combination with chemotherapy and/or other targeted agents. In addition, considering the major role of both p53 and MDM2 in the downstream signaling response to radiation-induced DNA damage, the combination of MDM2 inhibitors with radiation may offer a valuable therapeutic radiosensitizing approach for GB therapy. This review covers the role of MDM2/X in cancer and more specifically in GB, followed by the rationale for the potential radiosensitizing effect of MDM2 inhibition. Finally, the current status of MDM2/X inhibition and p53 activation for the treatment of GB is given.

Inactivation of the tumor suppressor p53 has been generally accepted as a hallmark of tumor. MDM2 and MDMX, the two closely related proteins are considered to be critical for negatively regulating p53 activity through inhibitory binding to and post-translational modification of the p53 protein. We have demonstrated that MDMX facilitates MDM2-mediated p53 ubiquitination and degradation via recruitment of the ubiquitin-conjugating enzyme UbcH5c to the MDM2-MDMX heterooligomers. Here, we discuss our new findings from genetically engineered mouse models and a potential therapeutic strategy.

Somatic missense mutations of the CSNK1A1 gene encoding casein kinase 1 alpha (CK1α) occur in a subset of myelodysplastic syndrome (MDS) with del(5q) karyotype. The chromosomal deletion causes CSNK1A1 haplo-insufficiency. CK1α mutations have also been observed in a variety of solid and hematopoietic tumors at low frequency. The functional consequence of CK1α mutation remains unknown. Here we show that tumor-associated CK1α mutations exclusively localize to the substrate-binding cleft. Functional analysis of recurrent mutants E98K and D140A revealed enhanced binding to the p53 inhibitor MDMX, increased ability to stimulate MDMX-p53 binding, and increased suppression of p21 expression. Furthermore, E98K and D140A mutants have reduced ability to promote phosphorylation of β-catenin, resulting in enhanced Wnt signaling. The results suggest that the CK1α mutations observed in tumors cause gain-of-function in cooperating with MDMX and inhibiting p53, and partial loss-of-function in suppressing Wnt signaling. These functional changes may promote expansion of abnormal myeloid progenitors in del(5q) MDS, and in rare cases drive the progression of other tumors.

MDMX (MDM4) is emerging as an important breast cancer (BC) biomarker, and oncoprotein, that can be targeted in combination with its well-known family member MDM2. While MDM2 has previously been implicated in driving BC metastasis, information about the role of MDMX in driving circulating tumor cells (CTCs) and BC metastasis is lacking. BCs often have alterations of MDM2, MDMX, and mutant p53 (mtp53). Therefore, the role of MDM2 and MDMX in the context of mtp53 in BCs requires further clarification. Our group has recently reported that triple negative breast cancer (TNBC) metastasis is dependent on both MDM2 and MDMX, and depleting MDM2 results in increased MDMX, but depleting MDMX does not cause an increase in MDM2. In the context of human TNBC expressing mtp53 in an orthotopic mouse model the down-regulation of MDMX virtually cleared CTCs from the blood. Contemplations, using the available literature, suggest that disrupting the stability and/or function of MDMX protein (and its downstream targets), in the context of mtp53 expressing BCs, might be beneficial for patient survival. It remains to be determined if blocking mtp53-MDMX pathways can inhibit early stage TNBC and eliminate CTCs that have the potential to form metastatic lesions.

The p53 gene, a well-known tumor suppressor gene, plays a crucial role in cell cycle regulation, DNA repair, cell differentiation, and apoptosis. MDM2 exerts p53-dependent activity mainly by binding to p53 protein to form MDM2-p53 negative feedback loop. In addition, MDM2 is involved in a number of pathways that regulate cell proliferation and apoptosis, playing a p53-independent role. The p53 binding domain of MDMX bind to p53 transcriptional activation domain, inhibiting the transcriptional activity of p53 on its downstream genes, but does not mediate the degradation of p53. The anti-tumor effect is exerted by inhibiting the interaction between the MDM2/MDMX protein and the p53 protein by a small-molecule or by restoring the activity of the p53 protein. This review describes in the structural features, biological functions and mechanisms of p53-MDM2/MDMX, and summarizes small-molecule targeting p53-MDM2/MDMX.

p53 is the major tumor suppressor and the most frequently inactivated gene in cancer. p53 could be disabled either by mutations or by upstream negative regulators, including, but not limited to MDM2 and MDMX. p53 activity is required for the prevention as well as for the eradication of cancers. Restoration of p53 activity in mouse models leads to the suppression of established tumors of different origin. These findings provide a strong support to the anti-cancer strategy aimed for p53 reactivation. In this review, we summarize recent progress in the development of small molecules, which restore the tumor suppressor function of wild-type p53 and discuss their clinical advance. We discuss different aspects of p53-mediated response, which contribute to suppression of tumors, including non-canonical p53 activities, such as regulation of immune response. While targeting p53 inhibitors is a very promising approach, there are certain limitations and concerns that the intensive research and clinical evaluation of compounds will hopefully help to overcome.

Nanostructures formed with bioactive peptides offer an exciting prospect in clinical oncology as a novel class of therapeutic agents for human cancers. Despite their therapeutic potential, however, peptide-based nanomedicines are often inefficacious in vivo due to low cargo-loading efficiency, poor tumor cell-targeting specificity and limited drug accumulation in tumor tissues. Here, we describe the design, via assembly of a p53-activating peptide termed PMI, functionalized PEG and fluorescent lanthanide oxyfluoride nanocrystals, of a novel nanotheranostic shaped in flexible rods. This lanthanide-peptide nanorod or LProd of bionic nature exhibited significantly enhanced tumor-targeting and -imaging properties compared to its spherical counterpart. Importantly, LProd potently inhibited tumor growth in a mouse model of human colon cancer through activating tumor suppressor protein p53 via MDM2/MDMX antagonism, while maintaining a highly favorable biosafety profile. Our data demonstrate that LProd as a multifunctional theranostic platform is ideally suited for tumor-specific peptide drug delivery with real-time disease tracking, thereby broadly impacting clinical development of antitumor peptides.

The mouse double minute 4 (MDM4) is emerging from the shadow of its more famous relative MDM2 and is starting to steal the limelight, largely due to its therapeutic possibilities. MDM4 is a vital regulator of the tumor suppressor p53. It restricts p53 transcriptional activity and also, at least in development, facilitates MDM2’s E3 ligase activity toward p53. These functions of MDM4 are critical for normal cell function and a proper response to stress. Their importance for proper cell maintenance and proliferation identifies them as a risk for deregulation associated with the uncontrolled growth of cancer. MDM4 tails are vital for its function, where its N-terminus transactivation domain engages p53 and its C-terminus RING domain binds to MDM2. In this review, we highlight recently identified cellular functions of MDM4 and survey emerging therapies directed to correcting its dysregulation in disease.

Triple-negative breast cancer (TNBC) has a poor prognosis mainly due to insensitivity or resistance to standard anthracycline- and taxane-based chemotherapy, urgently calling for new adjuvants to reverse drug resistance. Dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor has been proved to play a critical part against cancer, particularly focusing on the tremendous potential to enhance the efficacy of doxorubicin (DOX), however little was reported in TNBC. In the present study, we investigated the synergistic antitumor effect of the MDM2/MDMX inhibitor with DOX using three TNBC cell lines, two in situ transplantation tumor models and 214 clinical samples. We observed that the MDM2/MDMX inhibitor combined with DOX could not only inhibit cell vitality and migration and invasion abilities, but also highly inhibit tumor growth in TNBC nude mice. Besides, co-treatment of MDM2/MDMX inhibitor and DOX suppressed epithelial to mesenchymal transition (EMT) through increasing the TAK1-binding protein 1 (TAB1), transforming growth factor β-activated kinase 1 (TAK1) and p38 mitogen-activated protein kinase (MAPK) expression. Small interfering RNA-mediated TAB1 knockdown induced the EMT, desensitized cells to DOX and enhanced the migration and invasion abilities. High MDM2/MDMX expression was positively associated with weak TAB1 expression in 214 TNBC tumor tissues confirmed by immumohistochemical staining and MDM2/MDMX/TAB1 expression was significantly related to TNBC patient survival. These findings indicate that dual-target MDM2/MDMX inhibitor could increase the sensitization of doxorubicin and inhibit migration and invasion abilities in TNBC cells through p38 MAPK pathway activation caused EMT suppression and hence could be useful in TNBC treatments in future.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/β-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of β-Catenin in a p53-independent manner. Wnt/β-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/β-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/β-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Ueda et al. report that overexpression of MDMX in pre-leukaemic stem cells provides these cells with a competitive advantage and promotes the progression of various pre-leukaemic conditions to acute myeloid leukaemia in several mouse models.

MDM2 is an oncogenic E3 ligase found to be overexpressed in a number of human cancers, leading to poor prognosis. MDM2 overexpression inhibits the function of the tumour suppressor p53, which plays a critical role in safeguarding the integrity of the genome. MDM2 ubiquitinates p53 and tags it for proteasomal degradation. MDM2 also exhibits p53-independent oncogenic activities through targeting other tumour suppressor proteins, such as Foxo3a and Rb. Thus, aberrant regulation of Mdm2 is a key factor in promotion of tumor formation and progression and represents an important cancer therapeutic target. Here, we describe an integrated structure- based approach to develop potential lead compounds for inhibition of MDM2 oncogenic activity. We established a structure-based virtual screening strategy and used the crystal structure of the MDM2:MDMX RING domain heterodimer to predict the potential “druggable” pocket on Mdm2. An in silico screening of a small molecule compound library was employed to identify the candidate compounds that could interact with the MDM2:MDMX heterodimer RING domain. Additionally, using biochemical and cellular assays, the candidate compounds were examined for their ability to inhibit MDM2 E3 ligase activity, to induce apoptosis and to inhibit cell proliferation in cancer cell lines. This study reveals that inhibition of the MDM2:MDMX RING heterodimer could be a plausible approach for the development of MDM2 inhibitors as potential anti-cancer therapeutic agents.

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

p53 and p73 are critical tumor suppressors often inactivated in human cancers through various mechanisms. Owing to high structural homology, the proteins have many common functions and recognize the same set of genes involved in apoptosis and cell cycle regulation. p53 is known as the ‘guardian of the genome’ and together with p73 form a barrier against cancer development and progression. The TP53 is mutated in more than 50% of all human cancers and the germline mutations in TP53 predispose to the early onset of multiple tumors in Li-Fraumeni Syndrome (LFS), the inherited cancer predisposition. In cancers where TP53 gene is intact, p53 is degraded. Despite the ongoing efforts, the treatment of cancers remains challenging. This is due to late diagnoses, the toxicity of current standard of care and marginal benefit of newly approved therapies. Presently, the endeavours focus on reactivating p53 exclusively, neglecting the potential of the restoration of p73 protein for cancer eradication. Taken that several small molecules reactivating p53 failed in clinical trials, there is a need to develop new treatments targeting p53 proteins in cancer. This review outlines the most advanced strategies to reactivate p53 and p73 and describes drug repurposing approaches for the efficient reinstatement of the p53 proteins for cancer therapy.

Ferroptosis is a novel form of iron-dependent cell death characterized by lipid peroxidation. While the importance and disease relevance of ferroptosis are gaining recognition, much remains unknown about its interaction with other biological processes and pathways. Recently, several studies have identified intricate and complicated interplay between ferroptosis, ionizing radiation (IR), ATM (ataxia–telangiectasia mutated)/ATR (ATM and Rad3-related), and tumor suppressor p53, which signifies the participation of the DNA damage response (DDR) in iron-related cell death. DDR is an evolutionarily conserved response triggered by various DNA insults to attenuate proliferation, enable DNA repairs, and dispose of cells with damaged DNA to maintain genome integrity. Deficiency in proper DDR in many genetic disorders or tumors also highlights the importance of this pathway. In this review, we will focus on the biological crosstalk between DDR and ferroptosis, which is mediated mostly via noncanonical mechanisms. For clinical applications, we also discuss the potential of combining ionizing radiation and ferroptosis-inducers for synergistic effects. At last, various ATM/ATR inhibitors under clinical development may protect ferroptosis and treat many ferroptosis-related diseases to prevent cell death, delay disease progression, and improve clinical outcomes.

Casein kinase 1 alpha (CK1α) is a serine/threonine kinase with numerous functions, including regulating the Wnt/β‐catenin and p53 pathways. CK1α has a well‐established role in inhibiting the p53 tumor suppressor by binding to MDMX and stimulating MDMX‐p53 interaction. MDMX purified from cells contains near‐stoichiometric amounts of CK1α, suggesting that MDMX may in turn regulate CK1α function. We present evidence that MDMX is a potent competitive inhibitor of CK1α kinase activity (K (i) = 8 nM). Depletion of MDMX increases CK1α activity and β‐catenin S45 phosphorylation, whereas ectopic MDMX expression inhibits CK1α activity and β‐catenin phosphorylation. The MDMX acidic domain and zinc finger are necessary and sufficient for binding and inhibition of CK1α. P53 binding to MDMX disrupts an intramolecular auto‐regulatory interaction and enhances its ability to inhibit CK1α. P53‐null mice expressing the MDMX(W) (200S/W201G) mutant, defective in CK1α binding, exhibit reduced Wnt/β‐catenin target gene expression and delayed tumor development. Therefore, MDMX is a physiological inhibitor of CK1α and has a role in modulating cellular response to Wnt signaling. The MDMX‐CK1α interaction may account for certain p53‐independent functions of MDMX.

The Mdm4 (alias MdmX) oncoprotein, like its paralogue and interaction partner Mdm2, antagonizes the tumor suppressor p53. p53-independent roles of the Mdm-proteins are emerging, and we have reported the ability of Mdm2 to modify chromatin and to support DNA replication by suppressing the formation of R-loops (DNA/RNA-hybrids). We show here that the depletion of Mdm4 in p53-deficient cells compromises DNA replication fork progression as well. Among various deletion mutants, only full-length Mdm4 was able to support DNA replication fork progression. Co-depletion of Mdm4 and Mdm2 further impaired DNA replication, and the overexpression of each partially compensated for the other’s loss. Despite impairing replication, Mdm4 depletion only marginally hindered cell proliferation, likely due to compensation through increased firing of replication origins. However, depleting Mdm4 sensitized p53−/− cells to the nucleoside analogue gemcitabine, raising the future perspective of using Mdm4 inhibitors as chemosensitizers. Mechanistically, Mdm4 interacts with members of the Polycomb Repressor Complexes and supports the ubiquitination of H2A, thereby preventing the accumulation of DNA/RNA-hybrids. Thus, in analogy to previously reported activities of Mdm2, Mdm4 enables unperturbed DNA replication through the avoidance of R-loops.

The oncogene MDMX, also known as MDM4 is a critical negative regulator of the tumor suppressor p53 and has been implicated in the initiation and progression of human cancers. Increasing evidence indicates that MDMX is often amplified and highly expressed in human cancers, promotes cancer cell growth, and inhibits apoptosis by dampening p53-mediated transcription of its target genes. Inhibiting MDMX-p53 interaction has been found to be effective for restoring the tumor suppressor activity of p53. Therefore, MDMX is becoming one of the most promising molecular targets for developing anticancer therapeutics. In the present review, we mainly focus on the current MDMX-targeting strategies and known MDMX inhibitors, as well as their mechanisms of action and in vitro and in vivo anticancer activities. We also propose other potential targeting strategies for developing more specific and effective MDMX inhibitors for cancer therapy.

The p53 tumor suppressor protein and its major negative regulators MDM2 and MDMX oncoproteins form the MDM2/MDMX-p53 circuitry, which plays critical roles in regulating cancer cell growth, proliferation, cell cycle progression, apoptosis, senescence, angiogenesis, and immune response. Recent studies have shown that the stabilities of p53, MDM2, and MDMX are tightly controlled by the ubiquitin-proteasome system. Ubiquitin specific protease 7 (USP7), one of the most studied deubiquitinating enzymes plays a crucial role in protecting MDM2 and MDMX from ubiquitination-mediated proteasomal degradation. USP7 is overexpressed in human cancers and contributes to cancer initiation and progression. USP7 inhibition promotes the degradation of MDM2 and MDMX, activates the p53 signaling, and causes cell cycle arrest and apoptosis, making USP7 a potential target for cancer therapy. Several small-molecule inhibitors of USP7 have been developed and shown promising efficacy in preclinical settings. In the present review, we focus on recent advances in the understanding of the USP7-MDM2/MDMX-p53 network in human cancers as well as the discovery and development of USP7 inhibitors for cancer therapy.

Loss of retinoblastoma (RB) function in the cone cells during retina development is necessary but not sufficient for retinoblastoma development. It has been reported that in the absence of RB activity, a retinoma is generated, and the onset of retina cancer occurs until the p53 pathway is altered. Unlike other types of cancer, in retinoblastoma the p53 tumour suppressor is mostly wild type, although its two primary regulators, MDMX and MDM2, are commonly dysregulated. A mutated RB form is inherited in around 35% of the cases, but normally two, somatic mutations are needed to alter the RB function. Here we investigated the mRNA levels of RB, p53, MDMX and MDM2 in peripheral blood samples of retinoblastoma patients to monitor the pathway status of p53 in somatic cells. We sought to investigate the involvement of these genes in the development of retina cancer, with the aim of identifying biomarkers for early diagnosis of this disease.

MDM2 regulates p53 degradation by functioning as an E3 ubiquitin ligase. The role of MDMX, an MDM2 homolog that lacks E3 ligase activity, in the regulation of p53 degradation remains incompletely understood and sometime controversial. This confusion is due at least in part to studies of p53 degradation mainly carried out in in vitro settings, as elimination of either MDM2 or MDMX from mice results in p53-dependent embryonic lethality, thus obfuscating in vivo studies of the individual roles of MDM2 and MDMX in p53 degradation. To overcome this problem, we generated mice expressing an inducible p53 allele under various MDM2 and MDMX deletion and mutation statuses and studied in vivo p53 degradation. Degradation of p53 in vivo was largely prevented in mice and mouse embryonic fibroblast retaining MDM2 but lacking MDMX. Although MDM2 and MDMX interacted with p53 in the absence of each other, they bound p53 more efficiently as a heterodimer. MDMX, but not MDM2, interacted with ubiquitin-conjugating enzyme UbcH5c, an interaction that was essential for MDMX to enable MDM2 E3 ligase activity for p53 degradation. Grafting the C-terminal residues of MDMX to the C-terminus of MDM2 allowed MDM2 to interact with UbcH5c and enhanced MDM2-mediated p53 degradation in the absence of MDMX. Together, these data indicate that MDMX plays an essential role for p53 degradation in vivo by recruiting UbcH5c to facilitate MDM2 E3 ligase function. Significance: This study provides the first in vivo evidence of MDMX facilitating MDM2-mediated p53 degradation, clarifying its role in the regulation of this critical tumor suppressor.

A role of tumor-suppressive activity of p53 in the tumor microenvironment (TME) has been implicated but remains fairly understudied. To address this knowledge gap, we leveraged our MdmxS314A mice as recipients to investigate how implanted tumor cells incapacitate host p53 creating a conducive TME for tumor progression. We found that tumor cell-associated stress induced p53 downregulation in peritumor cells via an MDMX-Ser314 phosphorylation-dependent manner. As a result, an immunosuppressive TME was developed, as reflected by diminished immune cell infiltration into tumors and compromised macrophage M1 polarization. Remarkably, ablation of MDMX-Ser314 phosphorylation attenuated p53 decline in peritumor cells, which was associated with mitigation of immunosuppression and significant tumor growth delay. Our data collectively uncover a novel role of p53 in regulating the tumor immune microenvironment, suggesting that p53 restoration in the TME can be exploited as a potential strategy of anticancer therapy.

Background: Cancer is characterized by overexpression of p53 associated proteins, which down-regulate P53 signaling pathway. In cancer therapy, p53 activity can be restored by inhibiting the interaction of MDMX (2N0W) and MDM2 (4JGR) proteins with P53 protein. Objective: In the current, study in silico approaches were adapted to use a natural product as a source of cancer therapy. Methods: In the current study in silico approaches were adapted to use a natural product as a source of cancer therapy. For in silico studies, Chemdraw and Molecular Operating Environment were used for structure drawing and molecular docking, respectively. Flavonoids isolated from D. carota were docked with cancerous proteins. Result: Based on the docking score analysis, we found that compound 7 was the potent inhibitor of both cancerous proteins and can be used as a potent molecule for inhibition of 2N0W and 4JGR interaction with p53. Conclusion: Thus the compound 7 can be used for the revival of p53 signaling pathway function however, intensive in vitro and in vivo experiments are required to prove the in silico analysis.

There is interest in peptide drug design, especially for targeting intracellular protein&ndash, protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled &alpha, helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design.

The tumor-suppressive transcription factor p53 is a master regulator of stress responses. In non-stressed conditions, p53 is maintained at low levels by the ubiquitin ligase Mdm2 and its binding partner Mdmx. Mdmx depletion leads to a biphasic p53 response, with an initial post-mitotic pulse followed by oscillations. The mechanism underlying this dynamical behavior is unknown. Two different roles for Mdmx have been proposed: enhancing p53 ubiquitination by Mdm2 and inhibiting p53 activity. Here, we developed a mathematical model of the p53/Mdm2/Mdmx network to investigate which Mdmx functions quantitatively affect specific features of p53 dynamics under various conditions. We found that enhancement of Mdm2 activity was the most critical role of Mdmx under unstressed conditions. The model also accurately predicted p53 dynamics in Mdmx-depleted cells following DNA damage. This work outlines a strategy for rapidly testing possible network interactions to reveal those most impactful in regulating the dynamics of key proteins.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models., T- and NK-cell lymphomas (TCL) are a group of lymphoid malignancies characterized by poor prognosis, but the absence of appropriate pre-clinical models has hampered the development of effective therapies. Here the authors establish several pre-clinical models and identify vulnerabilities that could be further exploited to treat patients afflicted by these diseases.

// Ning Kon 1,* , Donglai Wang 1,* , Tongyuan Li 1 , Le Jiang 1 , Li Qiang 2,3,** and Wei Gu 1,3 1 Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA 2 Naomi Berrie Diabetes Center, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York, USA 3 Department of Pathology and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York, USA * These two authors contributed equally to this work ** This author is listed as a co-corresponding author Correspondence to: Wei Gu, email: // Keywords : p53; Mdmx; Mdm4; metabolism; adipose tissues Received : September 26, 2017 Accepted : December 08, 2017 Published : January 02, 2018 Abstract Although cell-cycle arrest, senescence and apoptosis remain as major canonical activities of p53 in tumor suppression, the emerging role of p53 in metabolism has been a topic of great interest. Nevertheless, it is not completely understood how p53-mediated metabolic activities are regulated in vivo and whether this part of the activities has an independent role beyond tumor suppression. Mdmx (also called Mdm4), like Mdm2, acts as a major suppressor of p53 but the embryonic lethality of mdmx-null mice creates difficulties to evaluate its physiological significance in metabolism. Here, we report that the embryonic lethality caused by the deficiency of mdmx , in contrast to the case for mdm2 , is fully rescued in the background of p53 3KR/3KR , an acetylation-defective mutant unable to induce cell-cycle arrest, senescence and apoptosis. p53 3KR/3KR /mdmx -/- mice are healthy but skinny without obvious developmental defects. p53 3KR/3KR /mdmx -/- mice are resistant to fat accumulation in adipose tissues upon high fat diet. Notably, the levels of p53 protein are only slightly increased and can be further induced upon DNA damage in p53 3KR/3KR /mdmx -/- mice, suggesting that Mdmx is only partially required for p53 degradation in vivo . Further analyses indicate that the anti-obesity phenotypes in p53 3KR/3KR /mdmx -/- mice are caused by activation of lipid oxidation and thermogenic programs in adipose tissues. These results demonstrate the specific effects of the p53/Mdmx axis in lipid metabolism and adipose tissue remodeling and reveal a surprising role of Mdmx inhibition in anti-obesity effects beyond, commonly expected, tumor suppression. Thus, our study has significant implications regarding Mdmx inhibitors in the treatment of obesity related diseases.

Background: Myelosuppression is a common sequela of acute radiation injury due to sensitivity of proliferating bone marrow cells to ionizing radiation. ALRN-6924 is a clinical-stage, first-in-class, stabilized cell-permeating alpha-helical peptide drug that disrupts the interaction of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2, to induce transient, dose-dependent cell cycle arrest in p53-wild-type tissues. ALRN-6924 is being evaluated in clinical trials as a selective chemoprotection agent for patients with p53-mutant cancers to protect healthy normal cells from chemotherapy while not protecting p53-mutant cancer cells. We tested whether ALRN-6924 may similarly protect against radiation-induced toxicity in mouse models of acute radiation injury. Materials and methods: Activation of p21 (CDKN1A), a cell cycle regulator under transcriptional control of p53, was measured in formalin-fixed mouse bone marrow by immunohistochemistry analysis (IHC). Proliferation and apoptosis in bone marrow were measured by IHC of Ki67 and cleaved PARP, respectively. Cell cycle arrest was measured in the bone marrow of ALRN-6924-treated C57BL/6 mice by flow cytometry using EdU incorporation. Serum levels of macrophage inhibitory cytokine-1 (MIC-1), a biomarker of p53 activation, were measured by ELISA. As a model of radiation-induced toxicity, C57BL/6 mice (n=7/group) were treated with one or more intravenous 2.4 mg/kg doses of ALRN-6924 at 24, 16, 8, or 1 hour (or combinations thereof) or placebo prior to a 15 Gy shielded-body radiation dose and then monitored for body weight (BW) changes. Results: ALRN-6924 induced cell cycle arrest in mouse bone marrow with a maximum effect at 8 hrs post-dose. Repeated doses of ALRN-6924 every 8 hrs elevated p21 levels in bone marrow that correlated with reduced Ki67 positivity and increased serum MIC-1 levels. Treatment-dependent changes in cPARP expression in bone marrow were evident, but minimal in magnitude. In a nonlethal radiation exposure model, ALRN-6924 yielded significant protection from radiation-induced BW loss in a schedule-dependent manner. Placebo-treated mice showed 10% to 15% BW loss five days after irradiation, while mice receiving one or more ALRN-6924 doses 8 hrs prior to irradiation had an average of 4% BW loss (p=0.008, two-sided t test). Conclusions: ALRN-6924 mitigated toxicity in a mouse model of acute radiation injury. The observed radioprotection effect was correlated with cell cycle arrest in bone marrow after one or more doses of ALRN-6924. These results support further investigation of ALRN-6924 as a radioprotective agent. Disclosures Annis: Aileron Therapeutics, Inc.: Current Employment. Sutton: Aileron Therapeutics, Inc.: Consultancy; Kriya Therapeutics: Consultancy; First Light Pharmaceuticals: Consultancy; Cygnal Therapeutics: Consultancy. Aivado: Aileron Therapeutics, Inc.: Current Employment. Vukovic: Aileron Therapeutics, Inc.: Current Employment.

Castration-resistant prostate cancer (CRPC) frequently develops after initial standard radiation and androgen deprivation therapy, leaving patients with limited further treatment options. Androgen receptor (AR) is a transcription factor that plays a key role in the initiation and progression of prostate cancer. p53, a major tumor suppressor that is rarely mutated in early-stages of prostate cancer, is often deregulated during prostate cancer progression. Here, we report an unusual co-amplification of MDM2 and MDMX, two crucial negative regulators of p53, in CRPC datasets. We demonstrate that combinatorial inhibition of MDM2 and MDMX, with nutlin-3 and NSC207895 respectively, has a profound inhibitory effect on cell proliferation of androgen-responsive, wild-type TP53 gene carrying prostate cancer cells LNCaP and 22Rv1. We further show that the combinatorial inhibition of MDM2 and MDMX not only activates p53, but also decreases cellular levels of AR and represses its function. Additionally, co-expression of MDM2 and MDMX stabilizes AR. Together, our results indicate that combinatorial inhibition of MDM2 and MDMX may offer a novel compelling strategy for prostate cancer therapy.

Mdm2 and MdmX are two closely related proteins that have been well-characterized as negative regulators of the tumor suppressor p53. Their interplay and especially respective roles in ubiquitination and subsequent degradation of p53 have lacked clarity. Yang and colleagues now demonstrate an obligate role for MdmX in recruitment of the E2 ubiquitin ligase UbcH5c to the Mdm2-MdmX hetero-oligomer. The use of elegant genetically engineered mouse models ensures the biological relevance of their findings that have important implications for targeted therapies involving these key players in the p53 pathway. See related article by Yang et al., Cancer Res 2021;81:898–909.

The two murine double minute (MDM) family members MDM2 and MDMX are at the center of an intense clinical assessment as molecular target for the management of cancer. Indeed, the two proteins act as regulators of P53, a well-known key controller of the cell cycle regulation and cell proliferation that, when altered, plays a direct role on cancer development and progression. Several evidence demonstrated that functional aberrations of P53 in tumors are in most cases the consequence of alterations on the MDM2 and MDMX regulatory proteins, in particular in patients with hematological malignancies where TP53 shows a relatively low frequency of mutation while MDM2 and MDMX are frequently found amplified/overexpressed. The pharmacological targeting of these two P53-regulators in order to restore or increase P53 expression and activity represents therefore a strategy for cancer therapy. From the discovery of the Nutlins in 2004, several compounds have been developed and reported with the ability of targeting the P53-MDM2/X axis by inhibiting MDM2 and/or MDMX. From natural compounds up to small molecules and stapled peptides, these MDM2/X pharmacological inhibitors have been extensively studied, revealing different biological features and different rate of efficacy when tested in in vitro and in vivo experimental tumor models. The data/evidence coming from the preclinical experimentation have allowed the identification of the most promising molecules and the setting of clinical studies for their evaluation as monotherapy or in therapeutic combination with conventional chemotherapy or with innovative therapeutic protocols in different tumor settings. Preliminary results have been recently published reporting data about safety, tolerability, potential side effects, and efficacy of such therapeutic approaches. In this light, the aim of this review is to give an updated overview about the state of the art of the clinical evaluation of MDM2/X inhibitor compounds with a special attention to hematological malignancies and to the potential for the management of pediatric cancers.

Chemoradiotherapy is the standard treatment modality for advanced non-small cell lung cancer (NSCLC). However, drug and radiation resistance remain major factors influencing its clinical outcome. The purpose of this study was to evaluate whether MDMX can affect the chemosensitivity and clinical outcome of NSCLC. Quantitative real-time PCR (qRT-PCR) was performed to assess MDMX mRNA expression levels in 105 primary NSCLC tissues, its corresponding non-cancerous tissues and two NSCLC-derived cell lines (A549 and SK-MES-1). In addition, immunohistochemistry was carried out to detect MDMX protein expression in the primary NSCLC tissues. The MDMX expression levels were correlated with clinicopathological and survival features. The effects of MDMX expression knockdown on NSCLC cell proliferation and chemosensitivity were evaluated using MTT, flow cytometry and soft agar colony assays. We found that the mRNA expression level of MDMX in NSCLC tissues was significantly higher than that in its corresponding non-tumorous tissues. High MDMX expression was found to be related to poor tumor cell differentiation, advanced TNM stages and the occurrence of lymph node metastases. Patients with a high MDMX expression level exhibited a lower overall survival rate than those with a low expression level. Multivariate analysis showed that a high MDMX protein expression level may serve as an independent prognostic factor for NSCLC patients. In addition, we found that MDMX expression knockdown combined with cisplatin treatment in vitro significantly increased apoptosis and decreased soft agar colony formation in NSCLC-derived cells. Our data indicate that MDMX expression may serve as an independent unfavorable prognostic factor for NSCLC patient outcome, which in turn may at least partly be due to the ability of the MDMX protein to regulate the proliferative capacity and chemosensitivity of NSCLC cells.

Mdm2 and MdmX share high structural similarity in their N-terminal domains, yet dual inhibitors are challenging to design due to differences in the conformations of the binding pockets, and notably of the proposed gatekeeper residue, Y100/99. Analysis of crystal structures and molecular dynamics (MD) simulations of complexes of Mdm2 and MdmX resulted in the identification of a water molecule with a long residence time that appears to be modulated by the conformation of Y100/99. These observations lead us to speculate that dual inhibitors either (i) stabilize both Mdm2 and MdmX with Y100/99 in the open conformation typically seen in complexes of Mdm2 with p53, or (ii) the dual inhibitors are agnostic to the conformation of Y100/99. The recently developed potent dual inhibitory stapled peptide Atsp7041 appears to be agnostic to the conformation of the gatekeeper residue. Proteins 2017; 85:1493-1506. © 2017 Wiley Periodicals, Inc.

Mdm2 and Mdm4 are negative regulators of the tumour suppressor p53; hence, this relationship is the focus of many cancer-related studies. A multitude of experiments across various developmental stages have been conducted to explore the tissue-specific roles of these proteins in the mouse. When Mdm2 or Mdm4 are deleted in the germline or specific tissues, they display different phenotypic defects, some of which lead to embryonic lethality. Mdm2 loss is often more deleterious than loss of its homologue Mdm4. All tissues experience activation of p53 target genes upon loss of Mdm2 or Mdm4; however, the degree to which the p53 pathway is perturbed is highly tissue-specific and does not correlate to the severity of the morphological phenotypes. Therefore, a need for further understanding of how these proteins regulate p53 activity is warranted, as therapeutic targeting of the p53 pathway is rapidly evolving and gaining attention in the field of cancer research. In this review, we discuss the tissue-specificity of Mdm proteins in regulating p53 and expose the need for investigation at the cell-specific level.

Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.