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Simple Summary: The activation of wild-type p53 protein in human lymphoma is a promising therapeutic strategy. A precise interruption of a protein–protein interaction, such as the p53-MDM2 interaction by nutlin-3a may lead lymphoma cells to apoptotic death through the stabilization and activation of p53. Despite this well-characterized scientific fact, there are lymphoma types that prevent or delay apoptosis by still unknown or purely understood mechanisms. Even lymphoma types that were initially affected by this interruption, finally managed to avoid death. An in vitro integrative comparative multi-omics analysis of different lymphoma types, before and after, wild-type p53 activation is a powerful way to shed light on such molecular mechanisms. Our findings provide deep holistic qualitative and quantitative molecular screening that highlights the putative role of specific proteins and pathways in lymphoma pathogenesis and shed light on the global effect of nutlin-3a. This information is the basis for a targeted and detailed study on specific proteins and pathways involved in lymphoma progression and resistance. In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis. [ABSTRACT FROM AUTHOR]

The MDM2–p53 protein–protein interaction is a promising model for researchers to design, study, and discover new anticancer drugs. The design of therapeutically active compounds that can maintain or restore the binding of MDM2 to p53 has been found to limit the oncogenic activities of both. This led to the current development of a group of xanthone-core and cis-imidazoline analogs compounds, among which γ-Mangostin (GM), α-Mangostin (AM), and Nutlin exhibited their MDM2–p53 interaction inhibitory effects. Therefore, in this study, we seek to determine the mechanisms by which these compounds elicit MDM2–p53 interaction targeting. Unique to the binding of GM, AM, and Nutlin, from our findings, they share the same three active site residues Val76, Tyr50, and Gly41, which represent the top active side residues that contribute to high electrostatic energy. Consequently, the free binding energy contributed enormously to the binding of these compounds, which culminated in the high binding affinities of GM, AM, and Nutlin with high values. Furthermore, GM, AM, and Nutlin commonly interrupted the stable and compact conformation of MDM2 coupled with its active site, where Cα deviations were relatively high. We believe that our findings would assist in the design of more potent active anticancer drugs. [ABSTRACT FROM AUTHOR]

Summary: Aim The oral MDM2 antagonist idasanutlin inhibits the p53-MDM2 interaction, enabling p53 activation, tumor growth inhibition, and increased survival in xenograft models. Methods We conducted a Phase I study of idasanutlin (microprecipitate bulk powder formulation) to determine the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, food effect, and clinical activity in patients with advanced malignancies. Schedules investigated were once weekly for 3 weeks (QW × 3), once daily for 3 days (QD × 3), or QD × 5 every 28 days. We also analyzed p53 activation and the anti-proliferative effects of idasanutlin. Results The dose-escalation phase included 85 patients (QW × 3, n = 36; QD × 3, n = 15; QD × 5, n = 34). Daily MTD was 3200 mg (QW × 3), 1000 mg (QD × 3), and 500 mg (QD × 5). Most common adverse events were diarrhea, nausea/vomiting, decreased appetite, and thrombocytopenia. Dose-limiting toxicities were nausea/vomiting and myelosuppression; myelosuppression was more frequent with QD dosing and associated with pharmacokinetic exposure. Idasanutlin exposure was approximately dose proportional at low doses, but less than dose proportional at > 600 mg. Although inter-patient variability in exposure was high with all regimens, cumulative idasanutlin exposure over the whole 28-day cycle was greatest with a QD × 5 regimen. No major food effect on pharmacokinetic exposure occurred. MIC-1 levels were higher with QD dosing, increasing in an exposure-dependent manner. Best response was stable disease in 30.6% of patients, prolonged (> 600 days) in 2 patients with sarcoma. Conclusions Idasanutlin demonstrated dose- and schedule-dependent p53 activation with durable disease stabilization in some patients. Based on these findings, the QD × 5 schedule was selected for further development. Trial registration: NCT01462175 (ClinicalTrials.gov), October 31, 2011. [ABSTRACT FROM AUTHOR]

The use of p53-MDM2/X inhibitors is a prospective strategy in anti-cancer therapy for tumors with wild type p53 protein. In our study, new low-molecular-weight inhibitors of the p53-mdm2 interaction have been proposed. The two-step synthesis of the imidazoline core with subsequent modifications for the nitrogen atom was carried out. New molecules caused the accumulation of p53 protein levels more than seven times than in comparison with the control. The accumulation of proapoptotic proteins such as p21 and PUMA has also been investigated, and the mechanism of cell death has been shown.

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

Objective: To identify the p53 binding pocket as well as active residues in mdm2 protein, and search for similar (or) better compounds to inhibit mdm2-p53 interactions in comparison to FDA approved drug (Nutlin) by ligand and structure based virtual screening methods, docking, and molecular dynamic simulation studies. Methods: A structure and ligand based virtual screening for mdm2 protein; targeting the key residues involved in their active binding of p53 peptide was conducted after obtaining structurally suitable compounds similar to Nutlin from ZINC database. These compounds are virtually screened onto the mdm2 protein targeting its active binding site where p53 binds. The best compound with highest binding affinity was taken up for further analysis using molecular dynamic simulations for further validating the docking studies and to reveal interactions during the conformational changes Results: We discovered several compounds that are potentially able to block the interaction between active residues of mdm2 and p53 complex, suggesting their capability to act as anti-cancer agents. As proven by our structure based virtual screening studies coupled with semi-flexible and flexible docking studies, compound ZINC59256947 was found to be the strongest inhibitors for mdm2 protein amongst all of those isolates from ZINC database. Conclusion: Our results suggest that, a simple, selective and reliable inhibition assay can be performed to search for novel inhibitors of p53-mdm2 interaction. Therefore this study provides a rationalization to the ability of a ZINC59256947, an isolate from ZINC database with strong binding affinity towards mdm2 protein, for future implications as anti-cancer agent.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway (PPP) produces NADPH that can protect cells from potentially lethal reactive oxygen species (ROS). We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a. [ABSTRACT FROM AUTHOR]

The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs. [ABSTRACT FROM AUTHOR]

As an anticancer therapeutic, Interferon-alpha (IFNα) is used to treat a number of malignancies. However, the application of IFNα is restricted mostly due to its high toxicity. Therefore, novel combination therapeutic regimens are required to decrease the toxicity of IFNα and enhance its efficacy. Here we show that the treatment of p53-deficient human non-small lung carcinoma H1299 cells with IFNα in combination with an inhibitor of MDM2, Nutlin-3a, synergistically affects the proliferation of cancer cells. Importantly, Nutlin-3a was able to reduce the effective dose of IFNα about 3.4 times. Strikingly, this phenomenon is p53-independent, because H1299 cells lack p53, but is highly dependent on MDM2 because its ablation makes tumor cells completely insensitive to IFNα alone or in combination with Nutlin-3a. On the contrary, overexpression of MDM2 makes H1299 cells more susceptible to both IFNα and IFNα/Nutlin-3a treatments. Mechanistically, treatment with combination of IFNα and Nutlin-3a attenuates cyclin D1/CDK4 on the protein level and hence blocks cell cycle progression. This mechanism may be responsible, at least in part, for the anti-proliferative effects on H1299 cells observed. Our data suggest that the expression of MDM2 confers sensitivity of cancer cells to IFNα/Nutlin-3a treatment. Moreover, our data also confirm positive effect of Nutlin even on p53-deficient neoplasms. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

The human Mediator complex regulates RNA polymerase II transcription genome-wide. A general factor that regulates Mediator function is the four-subunit kinase module, which contains either cyclin-dependent kinase 8 (CDK8) or CDK19. Whereas CDK8 is linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. We discovered that osteosarcoma cells (SJSA) are naturally depleted of CDK8 protein. Whereas stable CDK19 knockdown was tolerated in SJSA cells, proliferation was reduced. Notably, proliferation defects were rescued upon the reexpression of wild-type or kinase-dead CDK19. Comparative RNA sequencing analyses showed reduced expression of mitotic genes and activation of genes associated with cholesterol metabolism and the p53 pathway in CDK19 knockdown cells. SJSA cells treated with 5-fluorouracil, which induces metabolic and genotoxic stress and activates p53, further implicated CDK19 in p53 target gene expression. To better probe the p53 response, SJSA cells (shCDK19 versus shCTRL) were treated with the p53 activator nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state after nutlin-3 treatment, and this effect was kinase independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a role for CDK19 in cellular resistance to nutlin-3. [ABSTRACT FROM AUTHOR]

A virtual screening protocol involving docking and molecular dynamics has been tested against the results of fluorescence polarization assays testing the potency of a series of compounds of the nutlin class for inhibition of the interaction between p53 and Mdmx, an interaction identified as a driver of certain cancers. The protocol uses a standard docking method (AutoDock) with a cutoff based on the AutoDock score (ADscore), followed by molecular dynamics simulation with a cutoff based on root-mean-square-deviation (RMSD) from the docked pose. An analysis of the experimental and computational results shows modest performance of ADscore alone, but dramatically improved performance when RMSD is also used. [ABSTRACT FROM AUTHOR]

We propose a new paradigm in the drug design for the revival of the p53 pathway in cancer cells. It is shown that the current strategy of using small molecule based Mdm2 inhibitors is not enough to adequately revive p53 in cancerous cells, especially when it comes to the extracting pulsating behavior of p53. This fact has come to notice when a novel method for the drug dosage design is introduced using system oriented concepts. As a test case, small molecule drug Mdm2 repressor Nutlin 3a is considered. The proposed method determines the dose of Nutlin to revive p53 pathway functionality. For this purpose, PBK dynamics of Nutlin have also been integrated with p53 pathway model. The p53 pathway is the focus of researchers for the last thirty years for its pivotal role as a frontline cancer suppressant protein due to its effect on cell cycle checkpoints and cell apoptosis in response to a DNA strand break. That is the reason for finding p53 being absent in more than 50% of tumor cancers. Various drugs have been proposed to revive p53 in cancer cells. Small molecule based drugs are at the foremost and are the subject of advanced clinical trials. The dosage design of these drugs is an important issue. We use control systems concepts to develop the drug dosage so that the cancer cells can be treated in appropriate time. We investigate by using a computational model how p53 protein responds to drug Nutlin 3a, an agent that interferes with the MDM2-mediated p53 regulation. The proposed integrated model describes in some detail the regulation network of p53 including the negative feedback loop mediated by MDM2 and the positive feedback loop mediated by Mdm2 mRNA as well as the reversible represses of MDM2 caused by Nutlin. The reported PBK dynamics of Nutlin 3a are also incorporated to see the full effect. It has been reported that p53 response to stresses in two ways. Either it has a sustained (constant) p53 response, or there are oscillations in p53 concentration. The claimed dosage strategy achieves the p53 response in the first case. However, for the induction of oscillations, it is shown through bifurcation analysis that to achieve oscillating behavior of p53 inhibition of Mdm2 is not enough, rather antirepression of the p53-Mdm2 complex is also needed which leads to the need of a new drug design paradigm. [ABSTRACT FROM AUTHOR]

Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53. [Display omitted] • A CRISPR screen identifies FAM193A as required for the response to MDM2 inhibition • FAM193A is necessary for p53 stabilization and downstream transcriptional responses • FAM193A interacts with the RING domain of MDM4 and destabilizes MDM4 • FAM193A expression is associated with better prognosis in multiple cancer types Szwarc et al. identify FAM193A as a positive regulator of the tumor suppressor p53. Using genetic screening, FAM193A is found to be required for the cellular response to targeted p53 activation. FAM193A interacts with the p53 repressors MDM2 and MDM4, destabilizes MDM4, and enhances p53 activity as a transcription factor. [ABSTRACT FROM AUTHOR]

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis -stilbene diamines: A platform for the preparation of single-enantiomer cis -imidazolines for protein–protein inhibition. J. Org. Chem. 79, 6913–6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity ( K d value of 150 nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly ( K d values from 600 nM to 12 μM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21 Cip1 expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure–activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation. [ABSTRACT FROM AUTHOR]

MDM2 and MDMX are the primary negative regulators of p53, which under normal conditions maintain low intracellular levels of p53 by targeting it to the proteasome for rapid degradation and inhibiting its transcriptional activity. Both MDM2 and MDMX function as powerful oncogenes and are commonly over-expressed in some cancers, including sarcoma (~20%) and breast cancer (~15%). In contrast to tumors that are p53 mutant, whereby the current therapeutic strategy restores the normal active conformation of p53, MDM2 and MDMX represent logical therapeutic targets in cancer for increasing wild-type (WT) p53 expression and activities. Recent preclinical studies suggest that there may also be situations that MDM2/X inhibitors could be used in p53 mutant tumors. Since the discovery of nutlin-3a, the first in a class of small molecule MDM2 inhibitors that binds to the hydrophobic cleft in the N-terminus of MDM2, preventing its association with p53, there is now an extensive list of related compounds. In addition, a new class of stapled peptides that can target both MDM2 and MDMX have also been developed. Importantly, preclinical modeling, which has demonstrated effective in vitro and in vivo killing of WT p53 cancer cells, has now been translated into early clinical trials allowing better assessment of their biological effects and toxicities in patients. In this overview, we will review the current MDM2- and MDMX-targeted therapies in development, focusing particularly on compounds that have entered into early phase clinical trials. We will highlight the challenges pertaining to predictive biomarkers for and toxicities associated with these compounds, as well as identify potential combinatorial strategies to enhance its anti-cancer efficacy. [ABSTRACT FROM AUTHOR]

Discovery of MDM2 and MDM2-p53 interaction inhibitors changed the direction of anticancer research as it is involved in about 50% of cancer cases globally. Not only the inhibition of MDM2 but also its interaction with p53 proved to be an effective strategy in anticancer drug design and development. Various molecules of natural as well as synthetic origin have been reported to possess excellent MDM2 inhibitory potential. The present review discusses the pathophysiology of the MDM2-p53 interaction loop and MDM2/MDM2-p53 interaction inhibitors from literature covering recent patents. Focus has also been put on characteristic features of the active site of the target and its desired interactions with the currently FDA-approved inhibitor. The designing approach of previously reported MDM2/MDM2-p53 interaction inhibitors, their SAR studies, in silico studies, and the biological efficacy of various inhibitors from natural as well as synthetic origins are also elaborated. An attempt is made to cover recently patented MDM2/MDM2- p53 interaction inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We present the mechanism of interaction of Wnt network module, which is responsible for periodic somitogenesis, with p 53 regulatory network, which is one of the main regulators of various cellular functions, and switching of various oscillating states by investigating p 53–Wnt model. The variation in Nutlin concentration in p 53 regulating network drives the Wnt network module to different states, stabilized, damped and sustain oscillation states, and even to cycle arrest. Similarly, the change in Axin 2 concentration in Wnt could able to modulate the p 53 dynamics at these states. We then solve the set of coupled ordinary differential equations of the model using quasi steady state approximation. We, further, demonstrate the change of p 53 and GSK 3 interaction rate, due to hypothetical catalytic reaction or external stimuli, can able to regulate the dynamics of the two network modules, and even can control their dynamics to protect the system from cycle arrest (apoptosis). [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. [ABSTRACT FROM AUTHOR]

TP53 (p53) is the most frequently mutated gene in cancer, being altered in approximately 50% of human malignancies. In most, if not all, cancers lacking mutation, wild-type (WT) p53 is inactivated by interaction with cellular (MDM2/MDM4) or viral proteins, leading to its degradation. Because of its near universal alteration in cancer, p53 is an attractive target for the development of new targeted therapies for this disease. However, until recently, p53 was widely regarded as “undruggable”. This situation has now changed, as several compounds have become available that can restore wild-type properties to mutant p53 (e.g., PRIMA-1 and PRIMA-1MET). Other compounds are available that prevent the binding of MDM2/MDM4 to WT p53, thereby blocking its degradation (e.g., nutlins). Anti-mutant p53 compounds are potentially most useful in cancers with a high prevalence of p53 mutations. These include difficult-to-treat tumors such as high grade serous ovarian cancer, triple-negative breast cancer and squamous lung cancer. MDM2/4 antagonists, on the other hand, are likely to be efficacious in malignancies in which MDM2 or MDM4 is overexpressed such as sarcomas, neuroblastomas and specific childhood leukemias. Presently, early clinical trials are ongoing evaluating the anti-mutant p53 agent, PRIMA-1MET, and specific MDM2–p53 nutlin antagonists. [ABSTRACT FROM AUTHOR]

Abstract: A peculiar feature of several types of childhood cancer is that loss-of-function mutations of the TP53 (p53) tumor suppressor gene are uncommon, in contrast to many adult tumors. As p53 needs to be inactivated in order for tumor cells to survive and thrive, pediatric tumors typically make use of other mechanisms to keep p53 in check. One of the critical negative regulators of p53 is the MDM2 oncoprotein. Many anticancer drug development efforts in the past decade have therefore been devoted to the discovery and optimization of small molecules that selectively disrupt the interaction between MDM2 and p53, which could provide, in principle, a potent means to restore p53 function in tumor cells with wild-type p53. The nutlins are the class of selective inhibitors of the p53–MDM2 interaction that are currently most advanced in their clinical development. We review here the preclinical data that support the potential therapeutic use of nutlin drugs in the treatment of various pediatric tumors, including neuroblastoma, retinoblastoma, osteosarcoma, Ewing’s sarcoma, rhabdomyosarcoma, medulloblastoma, and childhood acute lymphoblastic leukemia. [Copyright &y& Elsevier]

Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7- tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53+/+ and p53-/- cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53+/+ cells but not in p53-/- cells. Among up-regulated genes in HCT p53+/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53+/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach. [ABSTRACT FROM AUTHOR]

p53 is a powerful tumor suppressor and is an attractive cancer therapeutic target. A breakthrough in cancer research came from the discovery of the drugs which are capable of reactivating p53 function. Most anti-cancer agents, from traditional chemo- and radiation therapies to more recently developed non-peptide small molecules exert their effects by enhancing the anti-proliferative activities of p53. Small molecules such as nutlin, RITA, and PRIMA-1 that can activate p53 have shown their anti-tumor effects in different types of hematological malignancies. Importantly, nutlin and PRIMA-1 have successfully reached the stage of phase I/II clinical trials in at least one type of hematological cancer. Thus, the pharmacological activation of p53 by these small molecules has a major clinical impact on prognostic use and targeted drug design. In the current review, we present the recent achievements in p53 research using small molecules in hematological malignancies. Anticancer activity of different classes of compounds targeting the p53 signaling pathway and their mechanism of action are discussed. In addition, we discuss how p53 tumor suppressor protein holds promise as a drug target for recent and future novel therapies in these diseases. [ABSTRACT FROM AUTHOR]

Abstract: Hypoxia is part of the tumor microenvironment favoring cancer resistance to chemotherapy mediated by mutations in the tumor suppressor p53 gene (TP53), or by conformational wt TP53 dysfunction. Since it is important to suppress tumor adaptation to hypoxia, irrespective of p53 status, we compared the efficacy of nutlin-3 which prevents MDM2-wt p53 interactions and PRIMA-1 which promotes mutant p53 reactivation and induction of massive apoptosis, under normoxia and hypoxia, against (a) SKBR3 breast carcinoma harboring a mutant p53R175H and over-expressing erbB2; and (b) genetically matched breast cancer ERα positive MCF-7 cells harboring either wt p53 or mutant p53 R175H. Under normoxia, PRIMA-1 was active against breast cancer cells harboring mutant p53. However, hypoxia further increased the susceptibility of mutant p53 breast cancer SKBR3 cells to lower PRIMA-1 levels, possibly through oxidative stress since this was counteracted by N-acetylcysteine. When using MCF-7 cells over-expressing mutant p53, PRIMA-1 synergized with exogenous peroxidase to increase apoptosis concomitantly with induction of PUMA and Mn-SOD, under normoxia. Wt p53 MCF-7 cells responded to hypoxia by increasing superoxide dismutase and their reactivity with the PAb240 antibody, known to recognize conformationally-inactive p53. This correlated with sensitization of wt p53 MCF-7 cells to PRIMA-1 but not to nutlin-3. PRIMA-1 toxicity against normoxic wt p53 MCF-7 cells was also decreased by Mn-SOD over-expression or when added with the glutathione antagonist, buthionine sulfoximine. This report shows for the first time that hypoxia increases PRIMA-1 toxicity in human breast cancer cells, partly by modulating p53 conformation and by inducing superoxide turnover. Our results suggest that PRIMA-1 may help to prevent hypoxia-mediated tumor chemoresistance. [Copyright &y& Elsevier]

More than two-thirds of breast cancers express the estrogen receptor (ER) and depend on estrogen for growth and survival. Therapies targeting ER function, including aromatase inhibitors that block the production of estrogens and ER antagonists that alter ER transcriptional activity, play a central role in the treatment of ER+ breast cancers of all stages. In contrast to ER- breast cancers, which frequently harbor mutations in the p53 tumor suppressor, ER+ breast cancers are predominantly wild type for p53. Despite harboring wild-type p53, ER+ breast cancer cells are resistant to chemotherapy-induced apoptosis in the presence of estrogen. Using genome-wide approaches, we have addressed the mechanism by which ER antagonizes the proapoptotic function of p53. Interestingly, both ER agonists such as estradiol and the selective ER modulator (SERM) tamoxifen promote p53 antagonism. In contrast, the full ER antagonist fulvestrant blocks the ability of ER to inhibit p53-mediated cell death. This inhibition works through a mechanism involving the modulation of a subset of p53 and ER target genes that can predict the relapse-free survival of patients with ER+ breast cancer. These findings suggest an improved strategy for the treatment of ER+ breast cancer using antagonists that completely block ER action together with drugs that activate p53-mediated cell death. [ABSTRACT FROM AUTHOR]

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2 - an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction. [ABSTRACT FROM AUTHOR]

Abstract: p53 is a powerful anti-tumoral molecule frequently inactivated by mutations or deletions in cancer. However, half of all human tumors expresses wild-type p53, and its activation, by antagonizing its negative regulator Mdm2, might offer a new strategy for therapeutic protocol. In this work, we present a molecular dynamics study on Mdm2 structure bound to two different known inhibitors with the aim to investigate the structural transitions between apo-Mdm2 and Mdm2-inhibitor complexes. We tried to gain information about conformational changes binding a benzodiazepine derivative inhibitor with respect the known nutlin and the apo form. The conformational changes alter the size of the cleft and were mainly in the linker regions, suggesting that the overall dynamic nature of Mdm2 is related to dynamic movements in these regions. [Copyright &y& Elsevier]

Abstract: Three series of novel imidazoline derivatives were designed, synthesized, and evaluated for their p53–MDM2 binding inhibitory activities, and anti-proliferation activities against PC3, A549, KB, and HCT116 cancer cell lines. Five of the tested compounds showed enhanced p53–MDM2 binding inhibitory potency and anti-proliferation activities in comparison with that of Nutlin-1. Flow cytometric analysis indicated that compound 7c, one of the most potent p53–MDM2 binding inhibitors with a K i value of 0.6μM, showed its ability to arrest cell cycle progression. [Copyright &y& Elsevier]

Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti-VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuroblastoma and other p53 wild-type tumors. We orthotopically implanted SH-SY5Y neuroblastoma cells into nude mice ( n = 40) and treated as follows: control, bevacizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1α and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, αSMA). Both angiogenesis and metastatic burden were digitally quantified. In vitro, Nutlin-3a suppresses HIF-1α expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control ( P < 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells ( P < 0.0001), perivascular basement membrane ( P < 0.04), and vascular mural cells ( P < 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis ( P < 0.0001 for both) and significant decrease in incidence of metastasis ( P < 0.05) and metastatic burden ( P < 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly targeting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical development of this approach. [ABSTRACT FROM AUTHOR]

p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53-MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-μ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53-MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21). [ABSTRACT FROM AUTHOR]

Chemotherapeutics (e.g., aurora kinase inhibitors) designed to target proliferative cells are often nonspecific for tumor cells as normal cycling cells are also susceptible. Indeed, one of the major dose-limiting toxicities of aurora kinase inhibitors is a dangerous depletion of neutrophils in patients. In this study we proposed a strategy to selectively target p53 mutant cells while sparing normal ones. The strategy is based on the understanding that normal cells have an intact p53 pathway but not tumor cells carrying p53 mutations. Nongenotoxic activation of p53 using nutlin led to a reversible activation of G1 and G2 arrest in normal cells, which prevents them from entering mitosis, thus protecting them from the side effects of aurora kinase inhibition (VX-680), namely endoreduplication and apoptosis. Cells carrying mutant p53 are selectively killed by the nutlin/VX-680 combination, whereas p53 wild-type cells retain their proliferative capacity. The major implications drawn from these results are: (1) reversible nongenotoxic activation of p53 may be used as a strategy for the chemoprotection of normal tissues, and (2) aurora kinase inhibitors may have alleviated side effects when used in combination with nutlin-like inhibitors. We highlight the distinct roles of p53 and p73 in mediating the cellular responses to VX-680 and suggest that dual protection by p53 and p73 are needed to guard against endoreduplication and polyploidy. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor protein is frequently mutated in human tumors. It is thought that the p53 pathway is indirectly impaired in the remaining tumors, for example by overexpression of its important regulators Mdm2 and Mdm4, making them attractive targets for the development of anti-cancer agents. Recent studies have suggested that Mdm4 levels determine the sensitivity of tumor cells for anti-cancer therapy. To investigate this possibility, we studied the drug sensitivity of several breast cancer cell lines containing wild-type p53, but expressing different Mdm4 levels. We show that endogenous Mdm4 levels can affect the sensitivity of breast cancer cells to anti-cancer agents, but in a cell line-dependent manner and depending on an intact apoptotic response. Furthermore, treatment with the non-genotoxic agent Nutlin-3 sensitizes cells for doxorubicin, showing that activation of p53 by targeting its regulators is an efficient strategy to decrease cell viability of breast cancer cells. These results confirm a function of Mdm4 in determining the efficacy of chemotherapeutic agents to induce apoptosis of cancer cells in a p53-dependent manner, although additional undetermined factors also influence the drug response. Targeting Mdm4 to sensitize tumor cells for chemotherapeutic drugs might be a strategy to effectively treat tumors harboring wild-type p53. [ABSTRACT FROM AUTHOR]

Abstract: It has long been known that traditional anticancer radio- and chemotherapies in part work through direct or indirect activation of the p53 tumour suppressor pathway. However, many of these strategies are nonselective and genotoxic. The emerging understanding of the pathways that regulate p53 has led to the notion that it should be possible to activate the p53 pathway in ways that are inherently nongenotoxic. Important targets for pharmacological interference in this respect are MDM2 and MDMX, key negative regulators of p53. Genetic and pharmacologic studies suggest that blocking the physical interaction of these proteins with p53, or inhibiting the catalytic role of MDM2 in tagging p53 for proteasomal degradation, both of which lead to an increase in the transcriptional activity of p53, may indeed be an efficient and safe way to eradicate tumour cells that retain wild-type p53. Here we review the rationale for such strategies, as well as the current state in the discovery and development of drugs that reactivate p53 by inhibiting its inhibitors MDM2 and MDMX. The first compounds that have been shown in model systems to be able selectively to kill cancer cells in this way are now entering clinical trials and the promise of MDM2 inhibitors as a new therapeutic anticancer modality should therefore become clear in the not-too-distant future. [Copyright &y& Elsevier]

p53 is expressed frequently, but is rarely mutated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) tumours. Nutlin-3a is a recently developed small molecule that targets Mdm2, a critical negative regulator of p53, and disrupts the p53-Mdm2 interaction resulting in p53 stabilization and activation. We show that nutlin-3a activates p53 in ALK+ ALCL cells carrying a wild type (wt) or mutated but partially functional p53 gene resulting in p53-dependent cell-cycle arrest and apoptosis. Cell-cycle arrest was associated with upregulation of the cyclin-dependent kinase inhibitor p21. Nutlin-3a-induced apoptotic cell death was accompanied by Bax and Puma upregulation, downregulation of Bcl-xl, survivin, and caspase-3 cleavage, and this was reduced when p53-dependent transactivation activity was inhibited by pifithrin-alpha, or when pifithrin-mu was used to inhibit direct p53 targeting of mitochondria. Nutlin-3a sensitized the activation of the extrinsic apoptotic pathway in wt-p53 ALK+ ALCL cells, in part, through upregulation of DR-5 and downregulation of c-Flip(S/L), and was synergistic with TRAIL in cell death induction. In addition, nutlin-3a treatment enhanced doxorubicin cytotoxicity against ALK+ ALCL cells harbouring mt p53, and this was associated with p73 upregulation. These data suggest that disruption of the p53-mdm2 interaction by nutlin-3a offers a novel therapeutic approach for ALK+ ALCL patients. [ABSTRACT FROM AUTHOR]

It has been reported that the induction of cellular senescence through p53 activation is an effective strategy in tumor regression. Unfortunately, however, tumors including adult T-cell leukemia/lymphoma (ATL) have disadvantages such as p53 mutations and a lack of p16(INK4a) and/or p14(ARF). In this study we characterized Nutlin-3a-induced cell death in 16 leukemia/lymphoma cell lines. Eight cell lines, including six ATL-related cell lines, had wild-type p53 and Nutlin-3a-activated p53, and the cell lines underwent apoptosis or cell-cycle arrest, whereas eight cell lines with mutated p53 were resistant. Interestingly, senescence-associated-beta-galactosidase (SA-beta-gal) staining revealed that only ATL-related cell lines with wild-type p53 showed cellular senescence, although they lack both p16(INK4a) and p14(ARF). These results indicate that cellular senescence is an important event in p53-dependent cell death in ATL cells and is inducible without p16(INK4a) and p14(ARF). Furthermore, knockdown of Tp53-induced glycolysis and apoptosis regulator (TIGAR), a novel target gene of p53, by small interfering RNA(siRNA) indicated its important role in the induction of cellular senescence. As many patients with ATL carry wild-type p53, our study suggests that p53 activation by Nutlin-3a is a promising strategy in ATL. We also found synergism with a combination of Nutlin-3a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), suggesting the application of Nutlin-3a-based therapy to be broader than expected. [ABSTRACT FROM AUTHOR]

Mantle cell lymphoma (MCL) is a clinically aggressive B-cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) and overexpression of cyclin D1. A high proportion of MCL tumors harbor wild-type (wt) and potentially functional p53 gene. We show here that stabilization and activation of wt-p53 using a recently developed potent MDM2 inhibitor, nutlin 3A, results in significant p53-dependent G1-S cell cycle arrest and apoptosis in MCL cells through regulation of p53 target genes. As mTOR signaling is activated in MCL and may control cyclin D1 levels, we show that p53 activation may downregulate the AKT/mTOR pathway through a mechanism involving AMP kinase (AMPK). Despite the non-genotoxic mode of nutlin 3A treatment, we show evidence that stabilization of p53 is associated with its phosphorylation at serine 15 residue and activation of AMPK. Stimulation of AMPK kinase activity using AICAR inhibits phosphorylation of critical downstream effectors of mTOR signaling, such as 4E-BP1 and rpS6. Pharmacologic inhibition of AMPK using compound C in nutlin-3A-treated MCL cells harboring wt-p53 did not affect the level of (ser15)p-p53, suggesting that the (ser15)p-p53 --> AMPK is the direction involved in the p53/AMPK/mTOR cross talk. These data establish a p53 --> AMPK --> mTOR mechanism in MCL and uncover a novel biologic effect of potent MDM2 inhibitors in preclinical models of MCL. [ABSTRACT FROM AUTHOR]

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a heterobifunctional all-small molecule PROTAC (PROteolysis TArgeting Chimera) capable of inducing proteasomal degradation of the androgen receptor. This cell permeable PROTAC consists of a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand known as nutlin, connected by a PEG-based linker. The SARM–nutlin PROTAC recruits the androgen receptor to MDM2, which functions as an E3 ubiquitin ligase. This leads to the ubiquitination of the androgen receptor, and its subsequent degradation by the proteasome. Upon treatment of HeLa cells with 10μM PROTAC for 7h, we were able to observe a decrease in androgen receptor levels. This degradation is proteasome dependent, as it is mitigated in cells pre-treated with 10μM epoxomicin, a specific proteasome inhibitor. These results have implications for the potential study and treatment of various cancers with increased androgen receptor levels. [Copyright &y& Elsevier]

Since the discovery of p53, a vast wealth of knowledge on its function and regulation has been accumulated. It is known that it is a key tumour suppressor and that its function is lost in many types of cancers, either by mutation or by excessive negative regulation. Recently, several discoveries have re-energised p53 as a therapeutic target as it has been shown that reintroduction of functional p53 into tumours has a therapeutic benefit. These encouraging results clearly justify the search for small molecules that diminish negative regulation of p53 in tumour cells, where p53 is not mutated as well as compounds that reactivate mutant p53. Important findings have been made to deal with both situations. Additionally, some of the small molecules identified may also help reduce the side effects of commonly used cancer therapeutics. These studies are still in their infancy and require further therapeutic validation, but the future appears bright for finally harnessing p53's tumour suppressing ability. [ABSTRACT FROM AUTHOR]

Nutlin-3, a small molecule inhibitor, activates p53 by disrupting p53-HDM2 association. In this study, we found that Nutlin-3 suppressed cell growth and induced apoptosis in the absence of wild-type p53, suggesting a p53-independent mechanism for Nutlin-3-induced cell death. Like p53, its homolog p73 transactivates proapoptotic genes and induces cell death. Since HDM2, a key negative regulator of p53, also binds to and inhibits p73, we asked whether p73 could mediate Nutlin-3-induced apoptosis. We demonstrate that Nutlin-3 inhibits endogenous binding between the proapoptotic p73 isoform TAp73α and HDM2 in p53-null cells. Dissociation of p73 and HDM2 leads to increased p73 transcriptional activity with upregulation of p73 target genes noxa, puma and p21, as well as enhanced apoptosis. p73 knockdown by siRNA results in rescue of Nutlin-3-treated cells, indicating that Nutlin-3-induced apoptosis is, at least in part, p73 dependent. In addition, Nutlin-3 treatment increases TAp73α protein levels with prolongation of p73 half-life. These results provide the first evidence that Nutlin-3 disrupts endogenous p73–HDM2 interaction and enhances the stability and proapoptotic activities of p73 and thus, provides a rationale for the use of Nutlin-3 in the large number of human tumors in which p53 is inactivated.Oncogene (2008) 27, 997–1003; doi:10.1038/sj.onc.1210707; published online 13 August 2007 [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is the most common solid malignancy in childhood and its prognosis is still generally poor. In contrast to many other cancers, mutations of the p53 tumor suppressor are rare. Instead, significant cytosolic sequestration of wtp53 is one of several mechanisms that attenuate p53 function in this cancer. Here, we report that aberrant p53 hyperubiquitylation contributes to p53 cytoplasmic sequestration in NB. NB lines constitutively harbor an elevated portion of wtp53 as stable ubiquitylated species confined to the cytoplasm. p53 hyperubiquitylation is not due to dysregulation by Hdm2 or proteasomal dysfunction. Instead, the defect lies in p53 regulation by HAUSP, a major p53-deubiquitylating enzyme. In contrast to non-NB cancer cells with nuclear p53 and normal ubiquitylation, p53 from NB cells shows impaired HAUSP interaction. Conversely, interference with p53 hyperubiquitylation in NB cells by Nutlin 3a or by a C-terminal p53 peptide (aa 305–393) results in p53 relocalization from the cytoplasm to the nucleus, and in case of Nutlin, in reactivation of p53's transcriptional and apoptotic functions. Moreover, nutlin and camptothecin act synergistically in inducing NB cell apoptosis. Hence, this study strengthens the rationale for targeting p53 deubiquitylation by drugs like Nutlin as a promising new strategy in NB therapy.Cell Death and Differentiation (2007) 14, 1350–1360; doi:10.1038/sj.cdd.4402166; published online 23 March 2007 [ABSTRACT FROM AUTHOR]