Murine Double Minute 2 Antagonist Research Articles

A structure-based virtual screening identifies a novel MDM2 antagonist in the activation of the p53 signaling and inhibition of tumor growth.

p53, a tumor suppressor protein, has a vital role in the regulation of the cell cycle, apoptosis, and DNA damage repair. The degradation of p53 is predominantly controlled by the murine double minute 2 (MDM2) protein, a ubiquitin E3 ligase. The overexpression or amplification of MDM2 is commonly observed in various human cancers bearing wild-type p53 alleles, leading to the rapid degradation of the p53 protein and the attenuation of p53 tumor suppression functions. Thus, a major effort in p53-based cancer therapy has been to research MDM2 antagonists that specifically stabilize and activate p53, leading to the suppression of tumor growth. However, despite numerous efforts to develop MDM2 antagonists, to date they have failed to reach clinical use, largely because of the cytotoxicity associated with these small molecules. This study used our newly designed structure-based virtual screening approach on a commercial compound library to identify a novel compound, CGMA-Q18, which directly binds to MDM2, leading to the activation of p53, the induction of apoptosis, and cell cycle arrest in cancer cells. Notably, CGMA-Q18 significantly inhibited tumor xenograft growth in nude mice without observable toxicity. These findings highlight our useful virtual screening protocol and CGMA-Q18 as a putative MDM2 antagonist.

Abstract CT066: Phase 1 study of MDM2 antagonist ASTX295 in patients with solid tumors with wild-type TP53

Abstract Background: ASTX295 is a novel small molecule antagonist of human Murine Double Minute 2 (MDM2), which tightly regulates the level and activity of the p53 tumor suppressor. To minimize potential thrombocytopenia previously seen with antagonism of this target, ASTX295 was designed to have a short half-life (t1/2) with decreased bone marrow (BM) exposure. ASTX295 has proapoptotic and tumor growth inhibitory activity in preclinical cancer (CA) models. We report here the safety and preliminary efficacy of the phase 1 and dose expansion portions of Study ASTX295-01. Methods: ASTX295-01 is a first-in-human, open-label, multicenter, phase 1/2 study to assess safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity of ASTX295 (NCT03975387). Eligible patients (advanced solid tumors with Wild-Type [WT] TP53, ECOG PS 0-2, adequate organ and bone marrow function, etc.) had oral administration of ASTX295 starting at a daily dose of 15 mg. Dose escalation was by standard 3 + 3 with cohorts testing multiple regimens. Safety was assessed by CTCAE and disease was assessed by RECIST 1.1. Safety and efficacy and dosing recommendations were overseen by the Data Safety Review Committee. Results:83 subjects were enrolled in over 14 cohorts, evaluating daily (QD) and intermittent dosing regimens and the effect of food. The mean age of subjects on study was 60.1 year, 56.9 were male, with 2.94 (0-15) prior chemotherapy regimens. For PK exposure, the total weekly AUC for 400 mg QD and 660 twice a week (BIW) are similar. The median tmax is 3 hours and mean t1/2 is 4-6 hours. PD demonstrates p53 pathway modulation. Nausea, vomiting, diarrhea, and fatigue were dose limiting. RP2D regimens were 660 mg BIW and 400 mg QD and these regimens were expanded with a total of 25 additional subjects. The most common treatment related AEs for both regimens were nausea, diarrhea, and vomiting at 76.2%,76.2%, and 33.3% respectively, for daily and 71.4%, 47.6%, and 33.3%, for intermittent. Grade 3 GI AEs were reported in 14.3% of subjects in the daily cohort vs 9.5% in the intermittent; none had Grade ≥ 4. One subject had grade 2 thrombocytopenia. Based on decreased incidence and duration of GI AEs, 660 mg BIW was selected as the RP2D.Multiple objective responses were seen, including 1 subject with non-small cell lung CA and 3 subjects with liposarcoma (LPS), a tumor with frequent MDM2 amplification. For LPS the ORR is 7.9% (de-differentiated 7.7%, well differentiated 8.0%), with a PFS of 7.95 and 9.66 months respectively, along with a 16-week disease control rate of 69.2% and 72.0%. Five subjects with GBM were enrolled, 4 with MDM2 amplification, 3 of whom showed evidence of tumor regression, with one subject remaining on study for > 34 cycles. Conclusion:ASTX295 was well tolerated at doses producing p53 pathway modulation with manageable toxicities, notably avoiding significant thrombocytopenia. Preliminary single agent efficacy was observed in heavily pretreated subjects across multiple solid tumor types with WT TP53. Citation Format: Ecaterina E. Dumbrava, Fabio M. Iwamoto, Mark Agulnik, Mohammed Milhem, Anthony W. Tolcher, Rashmi Chugh, Michael J. Demeure, Alain Mita, Kurt Demel, Mark Diamond, Beloo Mirakhur, Laksmi Wilson, Aram Oganesian, Danna Chan, Harold N. Keer, Jason Taylor, Martin J. Sims, Andrea Biondo, Simone Jueliger, Alexander Spira. Phase 1 study of MDM2 antagonist ASTX295 in patients with solid tumors with wild-type TP53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT066.

Targeting MDM2 in Soft-Tissue Sarcomas (and Other Solid Tumors): The Revival?

Summary:Murine double minute 2 (MDM2) inhibitors represent an opportunity to target p53 in solid tumors with wild-type TP53 and particularly in some sarcomas characterized by a consistent amplification of the MDM2 gene. In this issue of Cancer Discovery, LoRusso and colleagues and Koyama and colleagues report the results of two early-phase studies investigating two different TP53–MDM2 antagonists in patients with soft-tissue sarcomas and other solid tumors.See related article by LoRusso et al., p. 1802 (5) .See related article by Koyama et al., p. 1814 (8) .

232 (PB112) - Low SKP2 expression is predictive of sensitivity to an MDM2 antagonist in p53 wild-type AML

Background: Non-mutational aberrations of p53 function are a common event in AML. Using fragment-based drug discovery we have identified a Murine double minute 2 (MDM2) antagonist that potently activates p53 pathway. Here, we present the discovery of a novel patient selection strategy for MDM2 antagonist-induced apoptosis in wild-type p53 AML. Material and Methods: Cell panel screen and bioinformatics analyses of p53 wild-type AML cell lines that undergo apoptosis following Compound 1 (MDM2 antagonist) treatment versus those that do not were employed to identify novel patient selection strategy. Correlation between low levels of SKP2 (S-Phase Kinase Associated Protein 2) and induction of apoptosis following Compound 1 treatment was tested in vitro using Western Blot and flow cytometry analysis. Genetic manipulation of SKP2 levels using knockdown and over-expression approaches in wild-type p53 AML cells were used to validate the role of SKP2 expression in in vitro and in vivo experiments. Finally, bone marrow-derived primary AML blasts were tested to confirm the novel patient selection strategy. Results: Cell panel screen of p53 wild-type cell lines showed a wide range of sensitivity to Compound 1 suggesting that additional biomarkers are required to ensure patient stratification. Further bioinformatics analysis of apoptotic and non-apoptotic AML cancer cell lines following treatment with Compound 1 demonstrated differential gene expression levels and identified several potential predictive biomarkers. Western blot and flow cytometry analyses demonstrated that basal SKP2 expression level is lower in apoptotic AML cell lines than in non-apoptotic cells. Over-expression of SKP2 levels in MV4-11 cells reduced MDM2 antagonist-dependent cell death in previously apoptotic cell line. Similarly, SKP2 knockdown in OCI-AML3 cells increased MDM2 antagonist-induced cytotoxicity. Studies performed in mouse systemic AML model in vivo also demonstrated that high levels of SKP2 reduced p53 response to Compound 1. Finally, the correlation of SKP2 expression and Compound 1 sensitivity was confirmed in primary AML blasts isolated from patients. Conclusions: This work demonstrates in vitro and in vivo activity of MDM2 antagonist in AML models with low basal SKP2 levels. These pre-clinical data validate low levels of SKP2 as a novel patient selection strategy in AML and support the clinical investigation of MDM2 antagonist as therapeutic strategy for the treatment of SKP2low p53wt AML. No conflict of interest.

Considering the Role of Murine Double Minute 2 in the Cardiovascular System?

The E3 ubiquitin ligase Murine double minute 2 (MDM2) is the main negative regulator of the tumor protein p53 (TP53). Extensive studies over more than two decades have confirmed MDM2 oncogenic role through mechanisms both TP53-dependent and TP53-independent oncogenic function. These studies have contributed to designate MDM2 as a therapeutic target of choice for cancer treatment and the number of patents for MDM2 antagonists has increased immensely over the last years. However, the question of the physiological functions of MDM2 has not been fully resolved yet, particularly when expressed and regulated physiologically in healthy tissue. Cardiovascular complications are almost an inescapable side-effect of anti-cancer therapies. While several MDM2 antagonists are entering phase I, II and even III of clinical trials, this review proposes to bring awareness on the physiological role of MDM2 in the cardiovascular system.

Expansion of Prior Existing TP53 Mutated Clones in Polycythemia Vera Patients Treated with Idasanutlin

Expansion of Prior Existing TP53 Mutated Clones in Polycythemia Vera Patients Treated with Idasanutlin

MDM2 antagonists as a novel treatment option for acute myeloid leukemia: perspectives on the therapeutic potential of idasanutlin (RG7388).

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.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

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.

Abstract B47: Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells

Abstract Neuroblastoma (NB) arises in the sympathetic nervous system and is the most common extracranial solid tumor of childhood. While treatment outcome for NB has improved over the years, the survival of children with disseminated, high-risk NB tumors is less than 50%. To improve treatment outcome in these patients, inhibition of murine double minute-2 (MDM2) function in the context of front-line therapy used to treat NB (cisplatin and etoposide) is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth, cell migration, and the DNA damage response. Moreover, MDM2 is a downstream target of MYCN which can be commonly amplified in high-risk NB. The objective of the present study was to evaluate the extent to which the MDM2 antagonist nutlin3a alone or in combination with front-line therapies, could augment cell death in NB cells with clinically relevant molecular profiles. Cisplatin and etoposide are currently used in the clinic as front-line therapy, and when administered alone produced dose-related increases in cell death in SK-N-SH (wtp53, MYCN-nonamplified), IMR32 (wtp53, MYCN-amplified), SK-N-FI (mtp53, MYCN-nonamplified) and SK-N-DZ (mtp53, MYCN-amplified) NB cell lines. Nutlin3a also produced dose-related increases in cell death irrespective of p53 status, suggesting that p53-independent mechanisms, may play a role in increasing cell death produced by blockade of MDM2-mediated signaling. However, IC50 values for nutlin3a administered alone in the absence of DNA damage were >10x higher in the mtp53 cell lines compared with the wtp53 cell lines, suggesting that mechanisms other than MDM2-blockade could also be involved in promoting death in mtp53 NB cells. Using isobolographic analysis and broad ranges of compound ratios, we found that nutlin3a and cisplatin were moderately synergistic in wtp53 MYCN-nonamplified SK-N-SH and markedly synergistic in wtp53 MYCN-amplified IMR32 cells. Moreover, nutlin3a and etoposide were also moderately synergistic in these wtp53 cell lines. In mtp53 NB cells, nutlin3a was also synergistic with both cisplatin and etoposide irrespective of MYCN status. Interestingly, cisplatin and etoposide, which are frequently used together clinically, were strictly additive in all four cell lines by isobolographic analysis. Pharmacodynamic analysis was conducted in IMR32 cells (wtp53, MYCN-amplified) since this molecular profile is commonly found in high-risk NB. IMR32 cells were treated with vehicle, nutlin3a, cisplatin, and the combination of cisplatin/nutlin3a, and activation of p53 and the status of signaling pathways that regulate cell growth were assessed by Western analyses. Optimal activation of the p53 pathway was observed by 24-48 hours post-treatment which correlated with a robust downregulation of MYCN and the anti-apoptotic protein survivin. These data suggest that a combination therapy that incorporates efficient modulation of the MDM2 pathway and builds upon front-line therapies enhanced cell death of neuroblastoma cells that are highly resistant to conventional therapies. This combinatorial approach that builds upon front-line therapy is now under evaluation in orthotopic NB xenograft models. Citation Format: Karen E. Pollok, Barbara J. Bailey, Kania Katarzyna, Lawrence M. Gelbert, Harlan E. Shannon. Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B47.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Abstract 3435: MDM2 as a therapeutic target: improving upon front-line chemotherapy for nervous system tumors.

Abstract There is a significant unmet medical need to develop more effective treatments for cancers of the central and peripheral nervous systems. Glioblastoma multiforme (GBM) is the most common form of brain cancer, and successful long-term treatment has not been realized. Neuroblastomas (NB) arise in the sympathetic nervous system and are the most common extracranial solid tumor of childhood. Survival of children with disseminated, high-risk NB tumors is less than 50%, even with intensive multimodal treatment. To improve treatment outcome, inhibition of murine double minute 2 (MDM2) function in the context of front-line therapy is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth and migration. MDM2 is a negative regulator of p53 and p73 and, under low stress conditions, sequesters them in the cytoplasm. However, inhibition of MDM2 interactions with p53 and p73 by the MDM2 antagonist nutlin3a can lead to activation of p53/p73-mediated cell-death in tumor cells. The objective of the present study was to evaluate the extent to which blockade of MDM2-p53/p73 interactions by nutlin3a, alone or in combination with front-line therapies, could augment cell death in GBM and NB cells with a variety of molecular profiles. Using the median effect model, we found that temozolomide (TMZ) and nutlin3a were synergistic in increasing cell death in wtp53 U87 and U373 but were predominantly additive in mtp53 U118 cells. Primary patient GBM cells were typically resistant to TMZ relative to established cell lines. However, resistance to TMZ was decreased in the presence of nutlin3a. Further, TMZ and nutlin3a were synergistic in primary GBM10 and GBM43 but not in recurrent primary IU-GBM16, IU-GBM23, IU-GBM27, and IU-GBM32 cells. Moreover, in NB cells with wtp53, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-SH and additive in MYCN amplified IMR5. In mtp53 NB cells, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-FI as well as in MYCN amplified SK-N-DZ. Nutlin3a alone produced dose-related increases in cell death of all GBM and NB cell lines irrespective of p53 status, suggesting that p53-independent mechanisms play a role in inhibition of cell growth produced by blockade of MDM2-mediated signaling. Consistent with this hypothesis, nutlin3a increased p73 protein levels in mtp53 GBM43 cells that were exposed to TMZ. Inhibition of MDM2 function also inhibited repair of TMZ-mediated DNA damage in GBM. In comet assays, nutlin3a+TMZ decreased DNA repair relative to TMZ alone in wtp53 U87 and GBM10 and in mtp53 GBM43 cells. The present results demonstrate that MDM2 is a valid therapeutic target in GBM and NB cells with varying genetic profiles. In conclusion, inhibition of MDM2 signaling by nutlin3a is generally synergistic to additive in combination with front-line chemotherapeutics used to treat cancers of the central and peripheral nervous systems. Citation Format: Harlan E. Shannon, Haiyan Wang, Shanbao Cai, Wei Michael Liu, Lawrence M. Gelbert, Aaron A. Cohen-Gadol, Jann N. Sarkaria, Lindsey D. Mayo, Karen E. Pollok. MDM2 as a therapeutic target: improving upon front-line chemotherapy for nervous system tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3435. doi:10.1158/1538-7445.AM2013-3435

Correlation of TP53 and MDM2 genotypes with response to therapy in sarcoma

Relatively few sarcomas harbor TP53 (tumor protein p53) mutations, but in many cases, amplification of MDM2 (murine double minute 2) effectively inactivate p53. The p53 pathway activity can also be affected by normal genetic variation. The mutation status of TP53 and expression of MDM2, TP53, and their genetic variants SNP309 and R72P (Arg72Pro) were investigated in 125 sarcoma patient samples and 18 sarcoma cell lines. Association of the different genotypes and gene aberrations with chemotherapy response and survival, as well as response to MDM2 antagonists in vitro was evaluated. Twenty-two percent of the tumors had mutant TP53 and 20% MDM2 gene amplification. Patients with wild-type TP53 (TP53(Wt) ) tumors had improved survival (P < .001) and TP53(Wt) was an independent prognostic factor (hazard ratio = 0.41; 95% confidence interval = 0.23-0.74; P = .03). Interestingly, there was a trend toward longer time to progression after chemotherapy for tumors with the apoptosis-prone p53 variant R72 (P = .07), which was strongest with doxorubicin/ifosfamide-based regimens (P = .01). Liposarcomas had low R72 frequency (33% versus 56%), but increased levels of MDM2 and MDM4 (51% and 11%, P < .001). MDM2 overexpression on a TP53(Wt) background predicted better response to MDM2 antagonist Nutlin-3a, irrespective of R72P or SNP309 status. Improved survival after chemotherapy was found in patients with TP53(Wt) tumors harboring the R72 variant. MDM2 overexpression in TP53(Wt) tumors predicted good response to MDM2 antagonists, irrespective of R72P or SNP309 status. Thus, detailed TP53 and MDM2 genotype analyses prior to systemic therapy are recommended.

MDM2 antagonist Nutlin-3a protects wild-type p53 cancer cells from paclitaxel

The p53 pathway has an important role in cell cycle arrest and apoptosis. Downregulated levels of p53 have been shown to increase resistance to the cytotoxic effects of chemotherapy or radiotherapy. MDM2 (murine double minute 2) is able to bind p53 and modulate its transcriptional activity and stability. We studied the effect of Nutlin-3a, an MDM2 antagonist, on the response of non-small cell lung cancer cell lines, A549 (p53+/+) and H1299 (p53−/−), to paclitaxel (Taxol). A549 cells treated with Nutlin-3a plus paclitaxel showed a significant increase in MDM2 and wild-type p53 protein, a marked increase in the number of cells in the G0–G1 and G2-M phases, and a significant decrease in the percentage of cells in the S phase. The percentage of apoptotic A549 cells treated with 10 μmol/L Nutlin-3a plus 10 nmol/L paclitaxel was significantly lower than those treated with paclitaxel alone, and was also lower than that observed in H1299 cells. MTT assays demonstrated that Nutlin-3a plus paclitaxel also significantly reduced the sensitivity of A549 cells to paclitaxel compared with that of H1299 cells. In conclusion, Nutlin-3a mediates the cytotoxic effect of paclitaxel depending on p53 status. It may also protect wild-type p53 cells from mitotic chemotherapeutics.

Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3

Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (e.g. ionizing and UV radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic fashion. Nutlin occupies the p53-binding pocket of MDM2, activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directly nor introduces DNA damage, we hypothesized Nutlin would not induce P53 mutations and therefore not select for p53-mutated cells. To test this, populations of SJSA-1 (p53 wild-type) cancer cells were expanded that survived repeated Nutlin exposures, and individual clones were isolated. Group 1 clones were resistant to Nutlin-induced apoptosis, but still underwent growth-arrest. Surprisingly, while some Group 1 clones retained wild-type p53, others acquired a heterozygous p53 mutation. Apoptosis resistance in Group 1 clones was associated with decreased PUMA induction and decreased caspase 3/7 activation. Group 2 clones were resistant to both apoptosis and growth-arrest induced by Nutlin. Group 2 clones had acquired mutations in the p53 DNA-binding domain and expressed only mutant p53s that were induced by Nutlin treatment, but were unable to bind the P21 and PUMA gene promoters, and unable to activate transcription. These results demonstrate that non-genotoxic p53 activation (e.g. by Nutlin treatment) can lead to the acquisition of somatic mutations in p53 and select for p53-mutated cells. These findings have implications for the potential clinical use of Nutlin and other small molecule MDM2 antagonists.

Abstract 177: Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin resistant human testicular carcinoma cells via the Fas/FasL pathway

Abstract A major role for p53 in the response and execution of apoptosis after chemotherapy has been described for many cancers. Although testicular cancer (TC) often express high levels of p53 and almost no TP53 mutations are found, resistance to chemotherapy is still observed in a subgroup of TC patients. Therefore, the involvement of wild-type p53 in cisplatin sensitivity and resistance of human TC is not clear. In the present study, we demonstrate that in resistant human TC cells (Tera2-CP, Scha, 2102EP) p53 resides in a complex with murine double minute 2 (MDM2) even at higher cisplatin concentrations compared to cisplatin sensitive human TC cells (Tera2). Hyper-activation of the p53 pathway using the MDM2 antagonist Nutlin-3 in TC cells leads to nuclear localization of p53 and effective apoptosis induction as single agent. Targeting MDM2 with short interfering RNA extremely sensitizes TC cells in a similar way. The observed effects are dependent on the presence of wild-type p53, since mutant p53 expressing TC cells (NCCIT) or wild-type p53 suppressed TC cells are resistant to Nutlin-3. Specifically, we show that the Fas death receptor pathway, i.e. enhanced Fas expression (3-fold) and the induction of Fas/FasL interactions, plays an important role in MDM2 antagonist-induced apoptosis in TC cells as demonstrated by using FasL blocking antibodies and FasL small interfering RNA. Importantly, we have identified a similar mechanism in Hodgkin lymphoma cells (KM-H2 and L540) and acute myeloid leukaemia cells (MOLM-13), suggesting a common profile for Nutlin-3-induced apoptosis, which involves the Fas death receptor pathway. Finally, we combined nutlin-3 with cisplatin, which strongly sensitized TC cells, including cisplatin-resistant TC cells, to apoptosis via Fas pathway activation and reduced cell survival more than 8-fold. These results indicate that targeting the p53/MDM2 axis, in combination with standard treatment, appears to be a powerful strategy to pursue in cisplatin-resistant or -refractory testicular cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2011-177

Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation

Tumour protein p53 plays an important role in the vascular remodelling process as well as in oncogenesis. p53 is negatively regulated by murine double minute 2 (MDM2). A recently developed MDM2 inhibitor, nutlin-3, is a non-genotoxic activator of the p53 pathway. So far, the effect of MDM2 inhibition on vascular remodelling has not been elucidated. We therefore investigated the effect of nutlin-3 on neointima formation. Nutlin-3 up-regulated p53 and its downstream target p21 in vascular smooth muscle cells (VSMCs). DNA synthesis assay and flow cytometric analysis revealed that nutlin-3 inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation by cell cycle arrest. This inhibitory effect was abrogated in p53-siRNA-transfected VSMCs. Furthermore, nutlin-3 inhibited PDGF-stimulated VSMC migration. Treatment with nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury in mice, associated with up-regulation of p53 and p21. BrdU incorporation was decreased at 14 days after injury in nutlin-3-treated mice. TUNEL assay showed that nutlin-3 did not exaggerate apoptosis of the injured vessels. Infiltration of macrophages and T-lymphocytes and mRNA expression of chemokine (C-C motif) ligand-5, interleukin-6, and intercellular adhesion molecule-1 were decreased in the injured vessels of nutlin-3-treated mice. Nutlin-3 suppressed NF-κB activation in VSMCs, but not in p53-siRNA-transfected VSMCs. The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Abstract 4530: The anti-tumor effect of MDM2 antagonist nutlin-3 in gastric cancer cell in vitro and in vivo

Abstract Purpose: The mortality of gastric cancer is still high accounting for an estimated 50,000 deaths in Japan. The chemotherapies based on fluoropyrimidine and platinum complexes are widely used for unresectable advanced gastric cancer, however, development of new strategies including molecular targeting therapies are urgently required to obtain further effectiveness. Recent studies demonstrate that 70% of gastric cancers has wild-type p53 gene and its function thought to be inhibited by over-expression of murine double minute 2 (MDM2), a regulator of p53 turnover by binding p53 and acting as a ubiquitin E3 ligase. Nutlin-3 is a small-molecule MDM2 antagonist that efficiently activates p53 and its tumor suppresser function has been shown in retinoblastoma and hematological malignancy but not in gastrointestinal carcinoma. In this study, we investigated the antitumor effects of nutlin-3 in gastric cancer cells in combination with 5-fluorouracil or cisplatin in vitro and in vivo. Experimental Design: Human gastric cancer cell lines carrying wild-type (4 cell lines) or mutated p53 gene (3 cell lines) were incubated with nutlin-3 alone or combination with 5-fluorouracil or cisplatin in time dependent manner. Cell viability was calculated by WST assay. Western blot analysis for MDM2 and p53 are also performed to investigate protein expression. In vivo study, mice xenografted gastric cancer cells were treated with nutlin-3 alone or combination with 5-fluorouracil intraperitoneally. The tumor size was measured and histochemistry was done after 3 weeks administration. Results: Nutlin-3 showed strong cytotoxicity in gastric cancer cells carrying wild-type p53 gene not mutated p53 gene, regardless of expression level of MDM2 protein. Anti-tumor effect of Nutlin-3 was also observed in xenografted mice model. Histochemical analysis indicated nutlin-3 induced apoptotic cell death in tumor tissue. Furthermore, combined treatment with nutlin-3 and 5-fluorouracil or cisplatin revealed enhanced cytotoxicity in gastric cancer cells with wild-type p53 gene. Conclusions: Our results present new option in exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment for gastric cancer patients with wild-type p53 gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4530.

MDM2 Antagonist Nutlin-3 Displays Antiproliferative and Proapoptotic Activity in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL. MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed. Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 micromol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 micromol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO. These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2

In pediatric acute lymphoblastic leukemia (ALL), overexpression of murine double minute 2 (MDM2) protein by leukemic cells is typically associated with a wild-type (wt)-p53 phenotype and chemoresistance. A recently developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In this study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or -null phenotype. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of proapoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. As p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

MDM2 Antagonist Nutlin-3 Suppresses the Proliferation and Differentiation of Human Pre-Osteoclasts Through a p53-Dependent Pathway

Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.