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Glioblastoma multiforme (GBM), also known as grade IV astrocytoma, represents the most aggressive primary brain tumor. The complex genetic heterogeneity, the acquired drug resistance, and the presence of the blood-brain barrier (BBB) limit the efficacy of the current therapies, with effectiveness demonstrated only in a small subset of patients. To overcome these issues, here we propose an anticancer approach based on ultrasound-responsive drug-loaded organic piezoelectric nanoparticles. This anticancer nanoplatform consists of nutlin-3a-loaded ApoE-functionalized P(VDF-TrFE) nanoparticles, that can be remotely activated with ultrasound-based mechanical stimulations to induce drug release and to locally deliver anticancer electric cues. The combination of chemotherapy treatment with chronic piezoelectric stimulation resulted in activation of cell apoptosis and anti-proliferation pathways, induction of cell necrosis, inhibition of cancer migration, and reduction of cell invasiveness in drug-resistant GBM cells. Obtained results pave the way for the use of innovative multifunctional nanomaterials in less invasive and more focused anticancer treatments, able to reduce drug resistance in GBM. Statement of significance Piezoelectric hybrid lipid-polymeric nanoparticles, efficiently encapsulating a non-genotoxic drug (nutlin-3a) and functionalized with a peptide (ApoE) that enhances their passage through the BBB, are proposed. Upon ultrasound stimulation, nanovectors resulted able to reduce cell migration, actin polymerization, and invasion ability of glioma cells, while fostering apoptotic and necrotic events. This wireless activation of anticancer action paves the way to a less invasive, more focused and efficient therapeutic strategy.

Nutlin is a family of p53-targeting drugs. It is able to bind to Mdm2, thereby accelerate the accumulation of p53 that is a prominent tumor suppressor. An integrated module of the Nutlin PBK and p53 pathway is composed of positive feedback mediated by Mdm2 mRNA as well as the drug Nutlin and negative feedback mediated by Mdm2 protein. The main research content of our paper is how the time delay of protein synthesis, response time delay of Nutlin drug, the degradation rate of Mdm2, the degradation rate of p53 depended on Mdm2 and the actual dose of Nutlin in the cell affect the oscillatory behavior caused by Hopf bifurcation in the integrated network system of Nutlin PBK and p53 pathways. The stability of the unique positive equilibrium point and the existence of Hopf bifurcation are studied by taking the time delays as the bifurcation parameters and applying bifurcation theory. Based on the normal form theory and central manifold theorem, explicit criteria to determine the Hopf bifurcation direction and stability of the bifurcated periodic solution are established. It is found that the time delays and key parameters in the integrated network system of Nutlin PBK and p53 pathways play an important role in the amplitude and period of p53 oscillation according to the results from the numerical simulation and theoretical calculation. These results may provide us with a better understanding of the biological functions of the p53 pathway and some clues for cancer treatment.

Activation of the P53 pathway through inhibition of MDM2 using nutlins has shown clinical promise in the treatment of solid tumors and hematologic malignancies. There is concern, however, that nutlin therapy might stimulate the emergence or expansion of TP53-mutated subclones. We recently published the results of a phase 1 trial of idasanutlin in patients with polycythemia vera (PV) that revealed tolerability and clinical activity. Here, we present data indicating that idasanutlin therapy is associated with expansion of TP53 mutant subclones. End-of-study sequencing of patients found that 5 patients in this trial harbored 12 TP53 mutations; however, only 1 patient had been previously identified as having a TP53 mutation at baseline. To identify the origin of these mutations, further analysis of raw sequencing data of baseline samples was performed and revealed that a subset of these mutations was present at baseline and expanded during treatment with idasanutlin. Follow-up samples were obtained from 4 of 5 patients in this cohort, and we observed that after cessation of idasanutlin, the variant allele frequency (VAF) of 8 of 9 TP53 mutations decreased. Furthermore, disease progression to myelofibrosis or myeloproliferative neoplasm blast phase was not observed in any of these patients after 19- to 32-month observation. These data suggest that idasanutlin treatment may promote transient TP53 mutant clonal expansion. A larger study geared toward high-resolution detection of low VAF mutations is required to explore whether patients acquire de novo TP53 mutations after idasanutlin therapy.

The aim of this study was to investigate the potential biological activities of nutlin-3 in the regulation of growth and proliferation of non-small cell lung cancer (NSCLC) stem cells (CSCs), which may help in sensitizing to axitinib-induced apoptosis. Nutlin-3 induction of p53 expression was used to test its role in controlling the cell division pattern and apoptosis of NSCLC cells. A549 cells and H460 cells were pretreated with nutlin-3 and then treated with either an Akt1 activator or shRNA-GSK3β, to investigate the potential role of p53 sensitization in the biological effects of axitinib. We also determined the expression levels of GSK3β and p-Akt1 in patients with NSCLC and determined their potential association with survival data using Kaplan‐Meier plots and CBIOTAL. Increased p53 expression stimulated the induction of apoptosis by axitinib and promoted asymmetric cell division (ACD) of NSCLC CSCs. The repression of Akt phosphorylation induced by nutlin-3 promoted the ACD of lung CSCs, decreasing the proportion of the stem cell population. In addition to the induction of apoptosis by axitinib through inhibition of Wnt signaling, nutlin-3 treatment further enhanced axitinib-induced apoptosis by inhibiting Akt1/GSK3β/Wnt signaling. The low expression of GSK3β and increased expression of p-Akt in patients with NSCLC were closely associated with the development of NSCLC. TP53 stimulates the induction of apoptosis in NSCLC by axitinib and the ACD of lung CSCs through its regulatory effects on the p53/Akt/GSK3β pathways.

Cutaneous melanoma, the most aggressive form of skin cancer, is characterized by activating BRAF mutations. Despite the initial success of selective BRAF inhibitors, only few patients exhibited complete responses, whereas many showed disease progression. Melanoma is one of the few types of cancer in which p53 is not frequently mutated, but p53 inactivation can be indirectly achieved by a stable activation of MDM2 induced by a deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) locus, encoding for p16INK4A and p14ARF, two tumor suppressor genes. In this study, we tested the efficacy of the previously synthesized tetra-substituted pyrrole derivatives, 8 g, 8 h and 8i, in melanoma cell lines, and we compared the effects of the most active of these, the 8i compound, with that exerted by Nutlin 3, a well-known inhibitor of p53-MDM2 interaction. The obtained results showed that 8i potentiates the inhibitory effect of Nutlin 3 and the combined use of 8i and Nutlin 3 triggers apoptosis and significantly impairs melanoma viability. Finally, the 8i compound reduces p53-MDM2 interaction and induces p53-HSP90 complex formation, suggesting that the observed raise in p53 transcriptional activity could be mediated by HSP90. Because the main feature of melanoma is the resistance to most chemotherapeutics, our studies suggest that the 8i tetra-substituted pyrrole derivative, restoring p53 functions and its transcriptional activities, may have potential application, at least as adjuvant, in the treatment of human melanoma.

Acute myeloid leukemia (AML) is a clonal heterogenous malignancy of the myeloid cells with a poor prognosis lending itself to novel treatment strategies. TP53 is a critical tumor suppressor and plays an essential role in leukemogenesis. Although TP53 is relatively unusual in de novo AML, inactivation of wild-type p53 (WT-p53) is a common event. Murine double minute 2 (MDM2) is a key negative regulator of p53 and its expression; inhibition of MDM2 is postulated to reactivate WT-p53 and its tumor suppressor functions. Nutlins were the first small molecule inhibitors that bind to MDM2 and target its interaction with p53. RG7388 (idasanutlin), a second-generation nutlin, was developed to improve upon the potency and toxicity profile of earlier nutlins. Preliminary data from early phase trials and ongoing studies suggest clinical response with RG7388 (idasanutlin) both in monotherapy and combination strategies in AML. We herein briefly discuss currently approved therapies in AML and review the clinical data for RG7388 (idasanutlin) and MDM2 inhibition as novel treatment strategies in AML. We further describe efficacy and toxicity profile data from completed and ongoing trials of RG7388 (idasanutlin) and other MDM2-p53 inhibitors in development. Many targeted therapies have been approved recently in AML, with a focus on the older and unfit population for intensive induction therapy and in relapsed/refractory disease. The "nutlins", including RG7388 (idasanutlin), merit continued investigation in such settings.

Small molecule drugs bind to a pocket in disease causing target proteins based on complementarity in shape and physicochemical properties. There is a likelihood that other proteins could have binding sites that are structurally similar to the target protein. Binding to these other proteins could alter their activities leading to off target effects of the drug. One such small molecule drug Nutlin binds the protein MDM2, which is upregulated in several types of cancer and is a negative regulator of the tumor suppressor protein p53. To investigate the off target effects of Nutlin, we present here a shape-based data mining effort. We extracted the binding pocket of Nutlin from the crystal structure of Nutlin bound MDM2. We next mined the protein structural database (PDB) for putative binding pockets in other human protein structures that were similar in shape to the Nutlin pocket in MDM2 using our topology-independent structural superimposition tool CLICK. We detected 49 proteins which have binding pockets that were structurally similar to the Nutlin binding site of MDM2. All of the potential complexes were evaluated using molecular mechanics and AutoDock Vina docking scores. Further, molecular dynamics simulations were carried out on four of the predicted Nutlin-protein complexes. The binding of Nutlin to one of these proteins, gamma glutamyl hydrolase, was also experimentally validated by a thermal shift assay. These findings provide a platform for identifying potential off-target effects of existing/new drugs and also opens the possibilities for repurposing drugs/ligands. Agency for Science, Technology and Research (A*STAR) Economic Development Board (EDB) Accepted version Funding was provided by Biomedical Research Council (A*STAR), Singapore. M.S.M. would also like to acknowledge Wellcome Trust-DBT India alliance for a senior fellowship. N.S. would like to acknowledge a CSIR-SPMF fellowship for funding. M.N.N. would like to acknowledge the Biomedical Research Council (BMRC)-Economic Development Board (EDB) Industry Alignment Fund (IAF311017G and H18/01/ a0/B14), A*STAR, Singapore for funding.

Although the number of proteins effectively targeted for posttranslational degradation by PROTAC has grown steadily, the number of E3 ligases successfully exploited to accomplish this has been limited to the few for which small-molecule ligands have been discovered. Although the E3 ligase MDM2 is bound by the nutlin class of small-molecule ligands, there are few nutlin-based PROTAC. Because a nutlin-based PROTAC should both knockdown its target protein and upregulate the tumor suppressor p53, we examined the ability of such a PROTAC to decrease cancer cell viability. A nutlin-based, BRD4-degrading PROTAC, A1874, was able to degrade its target protein by 98% with nanomolar potency. Given the complementary ability of A1874 to stabilize p53, we discovered that the nutlin-based PROTAC was more effective in inhibiting proliferation of many cancer cell lines with wild-type p53 than was a corresponding VHL-utilizing PROTAC with similar potency and efficacy to degrade BRD4. This is the first report of a PROTAC in which the E3 ligase ligand and targeting warhead combine to exert a synergistic antiproliferative effect. Our study highlights the untapped potential that may be unlocked by expanding the repertoire of E3 ligases that can be recruited by PROTAC. Significance: These findings present the first BRD4-targeting MDM2-based PROTAC that possesses potent, distinct, and synergistic biological activities associated with both ends of this heterobifunctional molecule.

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.

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 x 3), once daily for 3 days (QD x 3), or QD x 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 x 3, n = 36; QD x 3, n = 15; QD x 5, n = 34). Daily MTD was 3200 mg (QW x 3), 1000 mg (QD x 3), and 500 mg (QD x 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 x 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 x 5 schedule was selected for further development.

Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies worldwide. Although there have been extensive studies on the molecular mechanisms of its carcinogenesis, FDA-approved drugs for HCC are rare. Side effects, development time, and cost of these drugs are the major bottlenecks, which can be partially overcome by drug repositioning. In this study, we developed a computational framework to study the mechanisms of HCC carcinogenesis, in which drug perturbation-induced gene expression signatures were utilized for repositioning of potential drugs. Specifically, we first performed differential expression analysis and coexpression network module analysis on the HCC dataset from The Cancer Genome Atlas database. Differential gene expression analysis identified 1,337 differentially expressed genes between HCC and adjacent normal tissues, which were significantly enriched in functions related to various pathways, including α-adrenergic receptor activity pathway and epinephrine binding pathway. Weighted gene correlation network analysis (WGCNA) suggested that the number of coexpression modules was higher in HCC tissues than in normal tissues. Finally, by correlating differentially expressed genes with drug perturbation-related signatures, we prioritized a few potential drugs, including nutlin and eribulin, for the treatment of hepatocellular carcinoma. The drugs have been reported by a few experimental studies to be effective in killing cancer cells.

Aim: The purpose of this manuscript is to study the potential characteristics of acquired nutlin-3 resistant non-small cell lung cancer cells (NSCLC). Nutlin-3 is an inhibitor of the murine-double minute 2 protein, the main negative regulator of wild type p53, of which several derivatives are currently in clinical development. Methods: A549 NSCLC cells were exposed to increasing concentrations of nutlin-3 for a period of 18 weeks. Monoclonal derivates were cultured, and the most resistance subclone was selected for whole transcriptome analysis. Gene set enrichment analysis was performed on differentially expressed genes between A549 nutlin-3 resistant cancer cells and the parental A549 p53 wild type cancer cells. Relevant findings were validated at the gene, protein and/or functional level. Results: All nutlin-3 resistant subclones acquired mutations in the TP53 gene, resulting in overexpression of the mutant p53 protein. The most resistant subclone was enriched for genes related to epithelial to mesenchymal transition (EMT), resulting in increased migratory and invasive potential. Furthermore, these cells were enriched in genes related to inflammation, tissue remodelling, and angiogenesis. Importantly, expression of several immune checkpoints, including PD-L1 and PD-L2, was significantly upregulated, and cisplatin-induced cell death was reduced. Conclusion: Transcriptome analysis of a highly nutlin-3 resistant A549 subclone shows the relevance of studying (1) resistance to standard of care chemotherapy; (2) secretion of immunomodulating chemo- and cytokines; (3) immune checkpoint expression; and (4) EMT and invasion in nutlin-3 resistant cancer cells in addition to acquired mutations in the TP53 gene.

Background The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. Methods Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. Results CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. Conclusion Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

The anti-inflammatory activities of P53 in the vasculature have been associated with the enhancement of the endothelial barrier function. In the present study we employed human and bovine lung endothelial cells, to investigate whether P53 expression levels affect the redox status of pulmonary cells. Moreover, we tested the possibility that those events affect the endothelial integrity of the lung microvascular monolayers. Our observations suggest that P53 suppression by LPS, Pifithrin, or small interfering RNA increased the expression of the redox marker malondialdehyde. In contrast, P53 induction by Nutlin or the Hsp90 inhibitor AUY922 exerted the opposite effects, namely suppressed that lipid oxidation marker. The direct measurement of the reactive oxygen species by 2,7-Dichlorodihydrofluorescein diacetate; confirmed the anti-oxidant activity of P53 in the vasculature. Furthermore, the increased reactive oxygen species production due to P53 suppression was associated with lung hyper-permeability responses. In conclusion, P53 supports endothelial barrier function, at least in part, via the modulation of the reactive oxygen species.

Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits. In this study, we report a translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to associate with the enhanced translation of mRNAs carrying multiple copies of an identified 3' UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identify PCBP2 and DHX30 as CGPD-motif interactors. We find that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs increases, and the response to Nutlin shifts toward apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells leads to decreased translation of CGPD-motif mRNAs.

Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candidates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10 melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cytoplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle retention in G0/G1 phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126 was classified as UN GHS category 4 (LD50 > 300–2000 mg/kg), suggesting it has low acute systemic toxicity. LQFM126 can be a promising prototype for anticancer therapy.

The Δ40p53 isoform heterotetramerizes with WTp53 to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with knock-in cell lines that generated homogeneous Δ40p53:WTp53 tetramers from the native TP53 locus, and examined with transcriptomics (PRO-seq, RNA-seq), metabolomics, and other methods. Although phenotypically similar to WTp53 under normal conditions, Δ40p53:WTp53 failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of eRNA transcription and subsequent failure to induce mRNA, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil) also showed Δ40p53:WTp53 defects in mRNA induction; however, other stimulus-specific TFs could then drive the response, yielding similar outcomes vs. WTp53. Our results define how Δ40p53 alters WTp53 function and identify an eRNA requirement for mRNA biogenesis. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest human p53 evolved as a tetramer to support eRNA transcription.

International audience; 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.

Targeted radiotherapy of metastatic neuroblastoma using the somatostatin receptor (SSTR)-targeted octreotide analogue DOTATATE radiolabelled with lutetium-177 (177Lu-DOTATATE) is a promising strategy. This study evaluates whether its effectiveness may be enhanced by combination with radiosensitising drugs. The growth rate of multicellular tumour spheroids, derived from the neuroblastoma cell lines SK-N-BE(2c), CHLA-15 and CHLA-20, was evaluated following treatment with 177Lu-DOTATATE, nutlin-3 and topotecan alone or in combination. Immunoblotting, immunostaining and flow cytometric analyses were used to determine activation of p53 signalling and cell death. Exposure to 177Lu-DOTATATE resulted in a significant growth delay in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Nutlin-3 enhanced the spheroid growth delay induced by topotecan in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Importantly, the combination of nutlin-3 with topotecan enhanced the spheroid growth delay induced by X-irradiation or by exposure to 177Lu-DOTATATE. The efficacy of the combination treatments was p53-dependent. These results indicate that targeted radiotherapy of high risk neuroblastoma with 177Lu-DOTATATE may be improved by combination with the radiosensitising drugs nutlin-3 and topotecan.

Purpose Nutlin is a drug that has been reported to activate p53 in various cell lines. We aim to study the effects of Nutlin in pterygium and compare the effects of Nutlin and mitomycin C (MMC) in pterygium cell lines. Methods Pterygium samples (n = 3) were collected during surgical excision. Normal conjunctival tissues (n = 3) were collected from another quadrant of the same eye. Cell lines were established, and cells from passages 2 to 5 were used. Pterygium and conjunctival cells were treated with different doses of Nutlin and MMC. Cell proliferation and cell migration were measured. Results Cell proliferation was reduced by 39-fold after treatment with 50 μM Nutlin. Cell migration was inhibited with increasing dosages of Nutlin (95% and 28% after treating with 2 and 50 μM Nutlin, respectively). Compared with MMC, Nutlin induced more pterygium cell death and less conjunctival cell death at low doses. At 50% lethal dose for pterygium cells, 95% of conjunctival cells survived after Nutlin treatment, whereas only 63% of conjunctival cells survived after MMC treatment. p21 expression was not detectable in MMC-treated pterygium cells but was detectable after Nutlin treatment. Conclusions In our study, MMC induced cell death in pterygium and conjunctival cell lines, whereas Nutlin had a targeted impact on pterygium cells. Our results implied that MMC inhibited both pterygium cell proliferation and migration through an apoptosis-independent pathway.

The authors have proposed a systems theory-based novel drug design approach for the p53 pathway. The pathway is taken as a dynamic system represented by ordinary differential equations-based mathematical model. Using control engineering practices, the system analysis and subsequent controller design is performed for the re-activation of wild-type p53. p53 revival is discussed for both modes of operation, i.e. the sustained and oscillatory. To define the problem in control system paradigm, modification in the existing mathematical model is performed to incorporate the effect of Nutlin. Attractor point analysis is carried out to select the suitable domain of attraction. A two-loop negative feedback control strategy is devised to drag the system trajectories to the attractor point and to regulate cellular concentration of Nutlin, respectively. An integrated framework is constituted to incorporate the pharmacokinetic effects of Nutlin in the cancerous cells. Bifurcation analysis is also performed on the p53 model to see the conditions for p53 oscillation.

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.