nutlin-3a and Disease-Models--Animal

Fragile X syndrome (FXS), the most prevalent inherited intellectual disability and one of the most common monogenic forms of autism, is caused by a loss of fragile X messenger ribonucleoprotein 1 (FMR1). We have previously shown that FMR1 represses the levels and activities of ubiquitin ligase MDM2 in young adult FMR1-deficient mice, and treatment by a MDM2 inhibitor Nutlin-3 rescues both hippocampal neurogenic and cognitive deficits in FMR1-deficient mice when analyzed shortly after the administration. However, it is unknown whether Nutlin-3 treatment can have long-lasting therapeutic effects.. We treated 2-month-old young adult FMR1-deficient mice with Nutlin-3 for 10 days and then assessed the persistent effect of Nutlin-3 on both cognitive functions and adult neurogenesis when mice were 6-month-old mature adults. To investigate the mechanisms underlying the persistent effects of Nutlin-3, we analyzed the proliferation and differentiation of neural stem/progenitor cells isolated from these mice and assessed the transcriptome of the hippocampal tissues of treated mice.. We found that transient treatment with Nutlin-3 of 2-month-old young adult FMR1-deficient mice prevents the emergence of neurogenic and cognitive deficits in mature adult FXS mice at 6 months of age. We further found that the long-lasting restoration of neurogenesis and cognitive function might not be mediated by changing intrinsic properties of adult neural stem cells. Transcriptomic analysis of the hippocampal tissue demonstrated that transient Nultin-3 treatment leads to significant expression changes in genes related to the extracellular matrix, secreted factors, and cell membrane proteins in the FMR1-deficient hippocampus.. Our data indicates that transient Nutlin-3 treatment in young adults leads to long-lasting neurogenic and behavioral changes likely through modulating adult neurogenic niche that impact adult neural stem cells. Our results demonstrate that cognitive impairments in FXS may be prevented by an early intervention through Nutlin-3 treatment.

Topics: Animals; Cognition; Cognitive Dysfunction; Crisis Intervention; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Imidazoles; Mice; Mice, Knockout; Neurogenesis; Piperazines

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Topics: Animals; Apoptosis; Cell Differentiation; Cell Separation; Coronary Stenosis; Coronary Vessels; Disease Models, Animal; DNA Damage; Genes, p53; Graft Survival; Imidazoles; Induced Pluripotent Stem Cells; Ligation; Mice; Mice, SCID; Myocytes, Cardiac; Piperazines

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Humans; Imidazoles; Indoles; Mice; Mice, Transgenic; Mutation; N-Myc Proto-Oncogene Protein; Neuroblastoma; para-Aminobenzoates; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Radiation, Ionizing; Signal Transduction; Spiro Compounds; Tumor Suppressor Protein p53

Adrenocortical carcinoma (ACC) is a rare malignancy, and CTNNB1 is frequently mutated in ACC. Our study aims to screen for effective agents with antineoplastic activity against ACC with CTNNB1 mutation. In-silico screening of the Genomics of Drug Sensitivity in Cancer (GDSC) database was conducted. Drug sensitivity in cells with CTNNB1 mutation was analyzed and further in vitro and in vivo studies were performed using the compound. Only one compound, Nutlin-3a, an MDM2 inhibitor, was significantly sensitive in 18 cancer cells with CTNNB1 mutation. Further analysis of the 18 cells revealed no significant efficacy between cells with both CTNNB1 and TP53 mutations indicating concomitant TP53 mutation did not impact on drug efficacy. We verified that Nutlin-3a inhibited cellular proliferation in ACC cell line NCI-H295R which harbored CTNNB1 mutation but not in SW13 cells which did not. Nutlin-3a induced cell apoptosis and G1 cell-cycle arrest in NCI-H295R cells. Nutlin-3a also decreased cellular migration and inhibited epithelial-to-mesenchymal transition (EMT) process in terms of EMT index. Nutlin-3a resulted in decreased β-catenin level independent of p53 level in NCI-H295R but not SW13 cells. We also evaluated the effect of Nutlin-3a on hormonal secretion of NCI-H295R cells and found it resulted in decreased levels of cortisol, androgen, and progesterone. Nutlin-3a treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo. Our study has revealed that Nutlin-3a potently inhibits ACC with CTNNB1 mutation. How p53/MDM2 axis coordinates with Wnt/beta-Catenin signaling in ACC warrants further study.

Topics: Adrenocortical Carcinoma; Animals; Antineoplastic Agents; beta Catenin; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression; Hormones; Humans; Imidazoles; Male; Mice; Mutation; Piperazines; Xenograft Model Antitumor Assays

The great majority of sporadic vestibular schwannomas (VSs) are due to the mutations of the NF2 gene encoding merlin. Sporadic VSs exhibit variable growth patterns and only a small fraction of the tumours are fast-growing; however, the underlying mechanisms remain undefined.. DNA sequencing and dosage analysis were used to identify the NF2 mutation status in sporadic schwannomas. The expression and sub-cellular localization of merlin and p53-MDM2 were assessed by immunoblotting, qRT-PCR and immunofluorescence. In vitro and in vivo studies were performed to reveal the effects of Nutlin-3 (a MDM2 inhibitor) and/or MG-132(a proteasome inhibitor) on schwannomas. The proliferation of schwannoma cells was assessed by CCK-8 assay, EdU staining and Flow cytometry analysis.. Double genetic hits of NF2 tended to occur in fast-growing tumours, characterized by the absence of merlin. The deregulation of p53-MDM2 was demonstrated to mediate merlin-deficient tumour growth, characterized by a nuclear accumulation of stabilized MDM2, contributing to a nuclear export of p53 for degradation. Nutlin-3 blocked the proliferation of schwannoma cells via a cooperative recovery of merlin and p53, accompanied by the shuttling of both proteins from the cytoplasm to the nucleus. We further demonstrated a difference in the sensitivity to Nutlin-3 between schwannoma cells with and without merlin expression. Nutlin-3 combined with MG-132 narrowed this between-group difference and triggered stronger inhibitory effects on the growth of schwannomas through coordinated reactivation of p53.. These findings present treatment strategies directed on the pathogenesis of sporadic schwannomas. FUND: National Natural Science Foundation of China.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, Neurofibromatosis 2; High-Throughput Nucleotide Sequencing; Humans; Imidazoles; Mice; Mutation; Neurilemmoma; Piperazines; Proteasome Endopeptidase Complex; Protein Binding; Protein Stability; Proteolysis; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genotype; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Imidazoles; Lung Neoplasms; Mice; Molecular Targeted Therapy; Mutation; Piperazines; Simvastatin; Tumor Suppressor Protein p53

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Combined Modality Therapy; Disease Models, Animal; Glioblastoma; Humans; Imidazoles; Piperazines; Proto-Oncogene Proteins c-mdm2; Temozolomide; Xenograft Model Antitumor Assays

Inflammation has been implicated in myocardial infarction (MI). MDM2 associates with nuclear factor-κB (NF-κB)-mediated inflammation. However, the role of MDM2 in MI remains unclear. This study aimed to evaluate the impacts of MDM2 inhibition on cardiac dysfunction and fibrosis after experimental MI and the underlying mechanisms. Three-month-old male C57BL/6 mice were subjected to left anterior descending (LAD) coronary artery ligation for induction of myocardial infarction (MI). Immediately after MI induction, mice were treated with Nutlin-3a (100 mg/kg) or vehicle twice daily for 4 weeks. Survival, heart function and fibrosis were assessed. Signaling molecules were detected by Western blotting. Mouse myofibroblasts under oxygen and glucose deprivation were used for in vitro experiments. MDM2 protein expression was significantly elevated in the mouse heart after MI. Compared with vehicle-treated animals, Nutlin-3a treatment reduced the mouse mortality. Nutlin-3a treatment improved heart function and decreased the infarct scar and fibrosis compared with vehicle. Furthermore, MDM2 inhibition restored IκB and inhibited NF-κB activation, leading to suppressed production of proinflammatory cytokines in the heart after MI. The consistent results were obtained in vitro. MDM2 inhibition reduced cardiac dysfunction and fibrosis after MI. These effects of MDM2 inhibition is mediated through modulating NF-κB activation, resulting in inhibition of inflammatory response.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Fibrosis; Heart Diseases; I-kappa B Proteins; Imidazoles; Inflammation; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myofibroblasts; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Survival Rate

Nutlin3a is a small-molecule antagonist of MDM2 that promotes non-genotoxic activation of p53 through p53 protein stabilization and transactivation of p53 target genes. Nutlin3a is the forerunner of a class of cancer therapeutics that have reached clinical trials. Using transgenic and gene-targeted mouse models lacking the critical p53 target genes, p21, Puma, and Noxa, we found that only loss of PUMA conferred profound protection against Nutlin3a-induced killing in both non-transformed lymphoid cells and Eμ-Myc lymphomas in vitro and in vivo. CRISPR/Cas9-mediated targeting of the PUMA gene rendered human hematopoietic cancer cell lines markedly resistant to Nutlin3a-induced cell death. These results demonstrate that PUMA-mediated apoptosis, but not p21-mediated cell-cycle arrest or senescence, is a critical determinant of the therapeutic response to non-genotoxic p53 activation by Nutlin3a. Importantly, in human cancer, PUMA expression may predict patient responses to treatment with MDM2 antagonists.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; CRISPR-Cas Systems; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Rapidly progressive glomerulonephritis is characterized by glomerular necroinflammation and crescent formation. Its treatment includes unspecific and toxic agents; therefore, the identification of novel therapeutic targets is required. The E3-ubiquitin ligase murine double minute (MDM)-2 is a nonredundant element of NF-κB signaling and the negative regulator of tumor suppressor gene TP53-mediated cell cycle arrest and cell death. We hypothesized that the MDM2 would drive crescentic glomerulonephritis by NF-κB-dependent glomerular inflammation and by p53-dependent parietal epithelial cell hyperproliferation. Indeed, the pre-emptive MDM2 blockade by nutlin-3a ameliorated all aspects of crescentic glomerulonephritis. MDM2 inhibition had identical protective effects in Trp53-deficient mice, with the exception of crescent formation, which was not influenced by nutlin-3a treatment. In vitro experiments confirmed the contribution of MDM2 for induction of NF-κB-dependent cytokines in murine glomerular endothelial cells and for p53-dependent parietal epithelial cell proliferation. To evaluate MDM2 blockade as a potential therapeutic intervention in rapidly progressive glomerulonephritis, we treated mice with established glomerulonephritis with nutlin-3a. Delayed onset of nutlin-3a treatment was equally protective as the pre-emptive treatment in abrogating crescentic glomerulonephritis. Together, the pathogenic effects of MDM2 are twofold, that is, p53-independent NF-κB activation increasing intraglomerular inflammation and p53-dependent parietal epithelial cell hyperplasia and crescent formation. We therefore propose MDM2 blockade as a potential novel therapeutic strategy in rapidly progressive glomerulonephritis.

Topics: Animals; Blotting, Western; Disease Models, Animal; Glomerulonephritis; Imidazoles; Immunohistochemistry; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Piperazines; Proto-Oncogene Proteins c-mdm2; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumor Suppressor Protein p53

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein (FMRP). However, the mechanism remains unclear, and effective treatment is lacking. We show that loss of FMRP leads to activation of adult mouse neural stem cells (NSCs) and a subsequent reduction in the production of neurons. We identified the ubiquitin ligase mouse double minute 2 homolog (MDM2) as a target of FMRP. FMRP regulates Mdm2 mRNA stability, and loss of FMRP resulted in elevated MDM2 mRNA and protein. Further, we found that increased MDM2 expression led to reduced P53 expression in adult mouse NSCs, leading to alterations in NSC proliferation and differentiation. Treatment with Nutlin-3, a small molecule undergoing clinical trials for treating cancer, specifically inhibited the interaction of MDM2 with P53, and rescued neurogenic and cognitive deficits in FMRP-deficient mice. Our data reveal a potential regulatory role for FMRP in the balance between adult NSC activation and quiescence, and identify a potential new treatment for fragile X syndrome.

Topics: Adult Stem Cells; Animals; Cognition Disorders; Disease Models, Animal; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; HEK293 Cells; Humans; Imidazoles; Male; Mice; Mice, Knockout; Neural Stem Cells; Piperazines; Proto-Oncogene Proteins c-mdm2

Enlargement of kidney tubules is a common feature of multiple cystic kidney diseases in humans and mice. However, while some of these pathologies are characterized by cyst expansion and organ enlargement, in others, progressive interstitial fibrosis and kidney atrophy prevail. The Kif3a knockout mouse is an established non-orthologous mouse model of cystic kidney disease. Conditional inactivation of Kif3a in kidney tubular cells results in loss of primary cilia and rapid cyst growth. Conversely, loss of function of the gene GLIS2/NPHP7 causes progressive kidney atrophy, interstitial inflammatory infiltration, and fibrosis. Kif3a null tubular cells have unrestrained proliferation and reduced stabilization of p53 resulting in a loss of cell cycle arrest in the presence of DNA damage. In contrast, loss of Glis2 is associated with activation of checkpoint kinase 1, stabilization of p53, and induction of cell senescence. Interestingly, the cystic phenotype of Kif3a knockout mice is partially rescued by genetic ablation of Glis2 and pharmacological stabilization of p53. Thus, Kif3a is required for cell cycle regulation and the DNA damage response, whereas cell senescence is significantly enhanced in Glis2 null cells. Hence, cell senescence is a central feature in nephronophthisis type 7 and Kif3a is unexpectedly required for efficient DNA damage response and cell cycle arrest.

Topics: Animals; Cell Cycle Checkpoints; Cellular Senescence; Checkpoint Kinase 1; Cilia; Cysts; Disease Models, Animal; DNA Damage; Epithelial Cells; Fibrosis; Flow Cytometry; Fluorescent Antibody Technique; Humans; Imidazoles; Kidney Diseases, Cystic; Kidney Tubules; Kinesins; Kruppel-Like Transcription Factors; Mice; Mice, Knockout; Nerve Tissue Proteins; Phenotype; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

Topics: Animals; Antimalarials; Cell Line; Disease Models, Animal; Female; Imidazoles; Indoles; Life Cycle Stages; Liver; Malaria; Malaria, Falciparum; Mice; Mice, Transgenic; Parasite Load; Piperazines; Plasmodium; Plasmodium falciparum; Post-Exposure Prophylaxis; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Tumor Suppressor Protein p53

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.

Topics: Animals; Carboplatin; Cell Death; Clinical Trials as Topic; Disease Models, Animal; DNA Damage; DNA-Binding Proteins; Histones; Humans; Imidazoles; Lung Neoplasms; MCF-7 Cells; Mice; Neoplasm Metastasis; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Triple Negative Breast Neoplasms; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Topics: Animals; Disease Models, Animal; Genes, Recessive; Humans; Ichthyosis, Lamellar; Imidazoles; Keratosis; Organ Culture Techniques; Piperazines; Proto-Oncogene Proteins c-mdm2; Rats; Skin

Translocations involving ETS-transcription factors, most commonly leading to the EWSR1-FLI1 fusion protein, are the hallmark of Ewing sarcoma. Despite knowledge of this driving molecular event, an effective therapeutic strategy is lacking. To test potential treatment regimes, we established a novel Ewing sarcoma zebrafish engraftment model allowing time-effective, dynamic quantification of Ewing sarcoma progression and tumour burden in vivo, applicable for screening of single and combined compounds. In Ewing sarcoma the tumour-suppressor gene TP53 is commonly found to be wild-type, thus providing an attractive target for treatment. Here, we study TP53 wild-type (EW7, CADO-ES1 and TC32) and TP53-deleted (SK-N-MC) Ewing sarcoma cell lines to investigate the potentiating effect of p53 reactivation by Nutlin-3 on treatment with YK-4-279 to block transcriptional activity of EWSR1-FLI1 protein. Blocking EWSR1-FLI1 transcriptional activity reduced Ewing sarcoma tumour cell burden irrespective of TP53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to stabilize p53 resulted in an additive inhibition of TP53 wild-type Ewing sarcoma cell burden, whilst not affecting TP53-deleted Ewing sarcoma cells. Improved inhibition of proliferation and migration by combinatorial treatment was confirmed in vivo by zebrafish engraftments. Mechanistically, both compounds together additively induced apoptosis of tumour cells in vivo by engaging distinct pathways. We propose reactivation of the p53 pathway in combination with complementary targeted therapy by EWSR1-FLI1 transcriptional activity disruption as a valuable strategy against p53 wild-type Ewing sarcoma.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cells, Cultured; Disease Models, Animal; Drug Synergism; Heterografts; Humans; Imidazoles; Indoles; Piperazines; RNA-Binding Protein EWS; RNA-Binding Proteins; Sarcoma, Ewing; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer death in the western world, with a 5-year survival rate below 5%. Murine double minute 2 (Mdm2) is an important negative regulator of the tumor suppressor p53. Reactivation of wild-type p53 is a promising treatment strategy, and inhibitors of Mdm2 have already entered clinical trials. To investigate the effects of Mdm2 inhibitors in PDAC, we used a murine cell line platform with a genetically defined status of p53. Here, we describe that Mdm2 inhibitors can act on a subset of murine PDAC cell lines p53 independently. Furthermore, we observed that Mdm2 inhibitors increase the sensitivity of murine PDAC cell lines toward topoisomerase II inhibitors by inducing effector caspase-independent cell death. The combination of Mdm2 inhibitors with topoisomerase II inhibitors acts independent of the survival factor NFκB/RelA. Mechanistically, Mdm2 inhibitors increase topoisomerase II inhibitor-induced DNA double-strand breaks. We show that Mdm2 binds to Nbs1 of the Mre11-Rad50-Nijmegen breakage syndrome (Nbs) 1 DNA repair complex. In addition, we provide evidence that Mdm2 inhibitors delay DNA repair. These findings may help to design novel therapeutic strategies to overcome therapeutic resistance of PDAC.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Deoxycytidine; Disease Models, Animal; DNA Damage; Doxorubicin; Drug Synergism; Etoposide; Gemcitabine; Imidazoles; Immunohistochemistry; Immunoprecipitation; Indoles; Mice; Pancreatic Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrrolidinones; Topoisomerase II Inhibitors

The cell of origin of tumors and the factors determining the cell of origin remain unclear. In this study, a mouse model of precursor B acute lymphoblastic leukemia/lymphoma (pre-B ALL/LBL) was established by retroviral transduction of Myc genes (N-Myc or c-Myc) into mouse bone marrow cells. Hematopoietic stem cells (HSCs) exhibited the highest susceptibility to N-Myc-induced pre-B ALL/LBL versus lymphoid progenitors, myeloid progenitors and committed progenitor B cells. N-Myc was able to induce pre-B ALL/LBL directly from progenitor B cells in the absence of Ink4a and Arf. Arf was expressed higher in progenitor B cells than Ink4a. In addition, N-Myc induced pre-B ALL/LBL from Arf(-/-) progenitor B cells suggesting that Arf has a predominant role in determining the cell of origin of pre-B ALL/LBL. Tumor cells derived from Ink4a/Arf(-/-) progenitor B cells exhibited a higher rate of proliferation and were more chemoresistant than those derived from wild-type HSCs. Furthermore, the Mdm2 inhibitor Nutlin-3 restored p53 and induced massive apoptosis in mouse pre-B ALL/LBL cells derived from Ink4a/Arf(-/-) cells and human B-ALL cell lines lacking Ink4a and Arf expression, suggesting that Mdm2 inhibition may be a novel therapeutic approach to the treatment of Ink4a/Arf(-/-) B-ALL/LBL, such as is frequently found in Ph(+) ALL and relapsed ALL. Collectively, these findings indicate that Ink4a and Arf are critical determining factors of the cell of origin and the therapeutic sensitivity of Myc-induced lymphoid tumors.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Bone Marrow Cells; Bone Marrow Transplantation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p16; Cytarabine; Disease Models, Animal; Flow Cytometry; Genes, myc; Hematopoietic Stem Cells; Humans; Imidazoles; Mice; Mice, Knockout; Piperazines; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cells, B-Lymphoid; Tumor Suppressor Protein p14ARF

Murine double minute (MDM)-2, an E3 ubiquitin ligase, promotes cancer cell survival and growth, by degrading the cell cycle regulator p53. Antagonism of MDM2 by the small-molecule cis-imidazoline nutlin analogs is under current study for cancer therapy. To test whether MDM2 also promotes regenerative cell growth, we determined the effects of nutlin-3a on tubule cell healing during postischemic acute kidney injury (AKI). Treatment with nutlin-3a impaired tubular cell regeneration during postischemic AKI in wild-type mice in a p53-dependent manner; however, MDM2 blockade also prevented tubular necrosis by suppressing sterile inflammation during the early postischemic phase. This effect also occurred in p53 knockout mice, indicating a second, proinflammatory, p53-independent role for MDM2 in AKI. In vitro experiments confirmed that MDM2 is required to induce mRNA expression and secretion of NFκB-dependent cytokines upon Toll-like receptor stimulation by enhanced binding of NFκB to cytokine promoter-binding sites. Thus, MDM2 links inflammation and epithelial healing during AKI. These additional biological functions need to be regarded when considering MDM2 inhibition therapy.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Gene Expression Regulation; Imidazoles; Inflammation; Inflammation Mediators; Injections, Intraperitoneal; Kidney Tubules; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Regeneration; Reperfusion Injury; RNA, Messenger; Time Factors; Tumor Suppressor Protein p53; Wound Healing

Loss of Pten in the Kras(G12D);Amhr2-Cre mutant mice leads to the transformation of ovarian surface epithelial (OSE) cells and rapid development of low-grade, invasive serous adenocarcinomas. Tumors occur with 100% penetrance and express elevated levels of wild-type tumor repressor protein 53 (TRP53). To test the functions of TRP53 in the Pten;Kras (Trp53+) mice, we disrupted the Trp53 gene yielding Pten;Kras(Trp53-) mice. By comparing morphology and gene expression profiles in the Trp53+ and Trp53- OSE cells from these mice, we document that wild-type TRP53 acts as a major promoter of OSE cell survival and differentiation: cells lacking Trp53 are transformed yet are less adherent, migratory, and invasive and exhibit a gene expression profile more like normal OSE cells. These results provide a new paradigm: wild-type TRP53 does not preferentially induce apoptotic or senescent related genes in the Pten;Kras(Trp53+) cancer cells but rather increases genes regulating DNA repair, cell cycle progression, and proliferation and decreases putative tumor suppressor genes. However, if TRP53 activity is forced higher by exposure to nutlin-3a (a mouse double minute-2 antagonist), TRP53 suppresses DNA repair genes and induces the expression of genes that control cell cycle arrest and apoptosis. Thus, in the Pten;Kras(Trp53+) mutant mouse OSE cells and likely in human TP53+ low-grade ovarian cancer cells, wild-type TRP53 controls global molecular changes that are dependent on its activation status. These results suggest that activation of TP53 may provide a promising new therapy for managing low-grade ovarian cancer and other cancers in humans in which wild-type TP53 is expressed.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, Knockout; Ovarian Neoplasms; Piperazines; Tumor Suppressor Protein p53

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).

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Disease Models, Animal; Female; Fluorescent Antibody Technique; Humans; Imidazoles; Immunoprecipitation; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, SCID; Mutation; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Translocation, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Retinoblastoma is a rare childhood cancer of the retina that begins in utero and is diagnosed in the first years of life. The goals of retinoblastoma treatment are ocular salvage, vision preservation, and reduction of short- and long-term side effects without risking mortality because of tumor dissemination. To identify better chemotherapeutic combinations for the treatment of retinoblastoma, several groups have developed genetic mouse models and orthotopic xenograft models of human retinoblastoma for preclinical testing. Previous studies have implicated the MDMX protein in the suppression of the p53 pathway in retinoblastoma and shown that the MDM2/MDMX antagonist, Nutlin-3a, can efficiently induce p53-mediated cell death in retinoblastoma cell lines. However, Nutlin-3a cannot be administered systemically to treat retinoblastoma, because it has poor penetration across the blood-ocular barrier. Therefore, we developed an ocular formulation of Nutlin-3a, Nutlin-3a(OC), and tested the pharmacokinetics and efficacy of this new formulation in genetic and human retinoblastoma orthotopic xenograft models of retinoblastoma. Here, we show that Nutlin-3a(OC) specifically and efficiently targets the p53 pathway and that the combination of Nutlin-3a(OC) with systemic topotecan is a significantly better treatment for retinoblastoma than currently used chemotherapy in human orthotopic xenografts. Our studies provide a new standardized approach to evaluate and prioritize novel agents for incorporation into future clinical trials for retinoblastoma.

Topics: Animals; Conjunctiva; Disease Models, Animal; Humans; Imidazoles; Intraocular Pressure; Mice; Mice, Inbred C57BL; Mice, SCID; Piperazines; Proto-Oncogene Proteins c-mdm2; Retinal Neoplasms; Retinoblastoma; Solubility; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’Mdm4+/− mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Clone Cells; Disease Models, Animal; Disease Progression; Humans; Imidazoles; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Nevus; Pigmentation; Piperazines; Proto-Oncogene Proteins; Skin Neoplasms; Staining and Labeling; Survival Analysis; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G(1) in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G(1) accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G(1)/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3' untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease.

Topics: Animals; Antineoplastic Agents; Apoptosis; Base Sequence; Cyclin D1; Disease Models, Animal; DNA Primers; Drug Screening Assays, Antitumor; Genistein; Imidazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Inbred C57BL; MicroRNAs; Piperazines; RNA, Messenger