Small-molecule MDM2 Inhibitor Research Articles

P53 can be stabilised by a combination of Nutlin-3 and irradiation in human papillomavirus-positive oropharyngeal carcinoma cells, with no apparent effect on cell growth or clonogenic survival

P53 can be stabilised by a combination of Nutlin-3 and irradiation in human papillomavirus-positive oropharyngeal carcinoma cells, with no apparent effect on cell growth or clonogenic survival

DDEL-15MANIPULATION OF THE BLOOD-BRAIN-BARRIER RESTORES THE EFFICACY OF MDM2 INHIBITION IN ORTHOTOPIC GLIOBLASTOMA MODELS

BACKGROUND: Blood-brain barrier (BBB) integrity is heterogeneous in GBM, and controversy surrounds whether associated heterogeneous delivery may limit efficacy for otherwise highly active drugs. Using a MDM2-amplified patient-derived xenograft model, the small-molecule MDM2 inhibitor SAR405838 was tested for both heterotopic and orthotopic efficacy under BBB-intact and disrupted conditions. METHODS AND RESULTS: A panel of 55 patient-derived GBM xenograft lines was screened for MDM2 expression. Four p53 wild-type lines were selected for in vitro testing with SAR405838. Treatment with 100 nM of SAR405838 induced a significantly higher reduction in neurosphere formation for MDM2 over-expressed lines (GBM108, 143) when compared with lines without MDM2 over-expression (GBM10, 102). SAR405838 also profoundly suppressed flank tumor growth in GBM108 but not GBM102 during in vivo testing. GBM108 tumors regressed to undetectable and remained undetectable for over 3 weeks after cessation of therapy. In contrast to the profound efficacy observed in flank models, the identical SAR405838 dosing regimen was ineffective in GBM108 orthotopic tumors. The impact of the BBB was subsequently tested by over-expressing VEGFA in GBM108 via lentiviral transduction. Consistent with a leakier vasculature, orthotopic GBM108-VEGFA exhibited markedly more pronounced accumulation of Texas Red-3 kDa dextran and extent of contrast enhancement on magnetic resonance imaging when compared to GBM108-EV(empty-vector). Using MALDI-MS, SAR405838 delivery into orthotopic GBM108-VEGFA tumors was also found to be more homogeneous and significantly higher than corresponding GBM108-EV controls. Enhanced drug delivery in GBM108-VEGFA models translated into a marked enhancement in orthotopic treatment efficacy in comparison to GBM108-EV with a median survival prolongation of 37 vs 4 days (p = 0.0055.) CONCLUSIONS: SAR405838 provided remarkable efficacy in heterotopic but not orthotopic tumors. BBB disruption by VEGFA expression markedly enhanced drug delivery and treatment efficacy in orthotopic tumors, demonstrating the critical importance of BBB penetration for targeted therapies in gliomas.

SAR405838: A Novel and Potent Inhibitor of the MDM2:p53 Axis for the Treatment of Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is an aggressive malignancy that can recur locally or disseminate even after multidisciplinary care. Genetically amplified and expressed MDM2, often referred to as a "hallmark" of DDLPS, mostly sustains a wild-type p53 genotype, substantiating the MDM2:p53 axis as a potential therapeutic target for DDLPS. Here, we report on the preclinical effects of SAR405838, a novel and highly selective MDM2 small-molecule inhibitor, in both in vitro and in vivo DDLPS models. The therapeutic effectiveness of SAR405838 was compared with the known MDM2 antagonists Nutlin-3a and MI-219. The effects of MDM2 inhibition were assessed in both in vitro and in vivo. In vitro and in vivo microarray analyses were performed to assess differentially expressed genes induced by SAR405838, as well as the pathways that these modulated genes enriched. SAR405838 effectively stabilized p53 and activated the p53 pathway, resulting in abrogated cellular proliferation, cell-cycle arrest, and apoptosis. Similar results were observed with Nutlin-3a and MI-219; however, significantly higher concentrations were required. In vitro effectiveness of SAR405838 activity was recapitulated in DDLPS xenograft models where significant decreases in tumorigenicity were observed. Microarray analyses revealed genes enriching the p53 signaling pathway as well as genomic stability and DNA damage following SAR405838 treatment. SAR405838 is currently in early-phase clinical trials for a number of malignancies, including sarcoma, and our in vitro and in vivo results support its use as a potential therapeutic strategy for the treatment of DDLPS.

Reactivation of p53 via MDM2 inhibition.

Reactivation of p53 via MDM2 inhibition.

Highlights of This Issue

Tumor adaptive resistance to therapeutic radiation remains a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of protein deacetylases, prevents cell aging by promoting mitochondrial metabolic homeostasis. Liu and colleagues discovered that SIRT3 was transcriptionally regulated by NF-κB upon radiation, and its enzymatic activity was further enhanced via phosphorylation by mitochondria-localized Cyclin B1–CDK1 complex. Phosphorylation of SIRT3 by CDK1 was required for mitochondrial functions as well as cell survival upon therapeutic radiation shown by in vitro and in vivo radiation tests. Thus, targeting CDK1-mediated SIRT3 phosphorylation is a potential effective approach to enhance tumor response to radiotherapy.Cholangiocarcinoma is a highly malignant carcinoma with poor prognosis. The molecular chaperone, heat shock protein 90 (HSP90), plays an important role in the posttranslational maturation and activation of many critical oncogenic proteins. Here, Shirota and colleagues discovered that the expression of HSP90 was significantly associated with 5-year survival for intrahepatic and extrahepatic cholangiocarcinoma. Furthermore, HSP90 inhibitor NVP-AUY922 showed potent anti- cholangiocarcinoma effect in vitro and in vivo. These data indicate that HSP90 expression is an independent prognostic factor, and that NVP-AUY922 may be an effective treatment option for a subset of patients with cholangiocarcinoma.There is compelling clinical rationale to identify molecular targeting agents that enhance tumor response to radiation. Agents that target MDM2 and p53 are excellent candidates to modulate radiosensitivity. Werner, Huang, and colleauges demonstrate that AMG 232, a potent small molecule MDM2 inhibitor, augments radiation response across a spectrum of human tumors using in vitro and in vivo model systems. Combined AMG 232 and radiation treatment resulted in inhibition of DNA damage repair and induction of senescence, apoptosis, and autophagy. This work previews a promising combination treatment approach for tumors harboring functional p53.Increasing evidence implicates EGFR and HER3 in the modulation of tumor cell response to radiation. In this study, Li, Huang, and colleagues investigate the effect of a dual-specific antibody (MEHD7945A) that simultaneously targets EGFR and HER3 to radiation response in head and neck and lung cancers. The authors found that MEHD7945A enhanced radiation response in both in vitro and in vivo model systems, possibly through an increase of γ-H2AX associated with DNA double-strand breaks and a significant reduction of markers related to tumor proliferation and vasculature. These data suggest the potential value of MEHD7945A as a radiation sensitizer in future clinical trial designs.

The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma.

Neuroblastoma is an aggressive pediatric malignancy which is >98% p53 wild-type at diagnosis. As a primary repressor of p53 activity and part of a p53-activated negative feedback loop, targeting of mouse double minute 2 homolog (MDM2) is an attractive therapeutic approach to reactivation of p53. Since development of the first selective MDM2 inhibitor, Nutlin-3a, newer compounds have been developed for increased potency and improved bioavailability. Herein, we sought to determine the efficacy and specificity of a second-generation MDM2 inhibitor, RG7388, in neuroblastoma cell lines and xenografts and examine its effect on the p53-independent pathway of hypoxia-inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF). Cell viability and apoptosis studies were performed on the neuroblastoma cell lines, NGP, SH-SY5Y, LAN-5, LAN-5 si-p53 (p53 silenced), and SK-N-AS (p53 null). RG7388 potently decreased cell proliferation and activated p53-dependent apoptosis. Tumor-bearing mice treated with RG7388 demonstrated significant tumor inhibition by 59% in NGP (P=0.003), 67% in SH-SY5Y (P=0.006), and 75% in LAN-5 (P=0.0019) p53 wild-type xenograft tumors, but no inhibitory effect on LAN-5 si-p53 or SK-N-AS p53-silenced/null xenograft tumors. Moreover, RG7388 was found to inhibit the p53-independent pathway of HIF-1α/VEGF with decreased gene expression and alteration of angiogenesis. Our study supports the further evaluation of RG7388 as a novel treatment option in p53 wild-type neuroblastoma at diagnosis and relapse.

Abstract 1271: MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion

Abstract An attractive therapeutic approach for treatment of cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63. Both compounds rapidly increase intracellular wt p53 levels and induce apoptosis or a senescence growth arrest, depending on the target cell. A critical question in the clinical development of MDM2 inhibitors is their potential for normal tissue toxicity owing to their ability to cause p53-dependent apoptosis or cellular senescence. Recent work suggest senescent cells exhibit a senescence-associated secretory phenotype (SASP) that can stimulate cancer progression. We critically examined the effect of MDM2 small molecule inhibitors Nutlin-3a and MI-63 on non-senescent and senescent cells. Both compounds caused normal cells to undergo a transient growth arrest. Continuous incubation with MDM2 small molecule inhibitors caused normal cells to exhibit some hallmarks of senescence (arrested growth, SA-β-Gal expression and secretion of High Mobility Group Box 1 protein), but the cells resumed growth upon removal of the MDM2 inhibitors. Senescent cells cultured with MDM2 inhibitors failed to resume growth upon removal of the compounds, as expected. However, their SASP was markedly dampened, as determined by multiplex ELISA assays. Since NF-κB and IL-1α are major drivers of the SASP, we examined their status in senescent cells cultured with MDMD2 inhibitors. The small molecule MDM2 inhibitors significantly reduced NF-κB activity and IL-1α mRNA expression in senescent cells. Our data suggest using combinatorial therapy of chemotherapeutics or irradiation and MDMD2 small molecule inhibitors may provide a potent therapeutic strategy to promote apoptosis of p53 positive tumor cells after radio- or chemo-therapy and attenuate the potentially deleterious effects of the SASP. Citation Format: Nicholas Schaum, Fatouma Almirah, Gary Scott, Christopher Benz, Judith Campisi, Albert R. Davalos. MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1271. doi:10.1158/1538-7445.AM2015-1271

Inhibiting MDM2-p53 Interaction Suppresses Tumor Growth in Patient-Derived Non–Small Cell Lung Cancer Xenograft Models

The tumor suppressor p53 is frequently inactivated in non-small cell lung cancer (NSCLC). Activation of the p53 pathway by inhibition of its negative regulator MDM2 may offer an attractive approach for NSCLC therapy. We evaluated the antitumor activity of the small-molecule MDM2 inhibitor RG7388 in patient-derived xenograft (PDX) models of NSCLC. We investigated the effect of RG7388 treatment on cell proliferation, cell cycle arrest, and apoptosis using a panel of human NSCLC cell lines (A549, H157, H1650, H1395, and H358) and PDX cell lines (human lung cell lines 12, 137, 277, and 196). PDX-bearing mice were used to test the therapeutic efficacy and pharmacodynamic effects of RG7388 treatment. We demonstrated that RG7388 promotes low nanomolar antiproliferative activity selectively in cell lines with wild-type p53 and p53 pathway activation, resulting in cell cycle arrest and apoptosis. In PDX models, oral administration of RG7388 led to potent dose-dependent and time-dependent activation of p53 and had a significant impact on p53 downstream targets. Daily treatment of RG7388 in mice at 50 and 80 mg/kg/day inhibited tumor growth in three wild-type p53 PDX models. Activation of the p53 pathway inhibited cell proliferation as observed by reduced Ki-67-positive cells in xenograft tumors. However, induction of apoptotic caspase activity was not observed in these tumors. Notably, RG7388 treatment remains effective in tumors lacking MDM2 amplification but expressing wild-type p53. MDM2 small-molecule inhibitor is effective in treating NSCLC tumors with wild-type p53, supporting further clinical investigation as a potential NSCLC therapy.

Abstract B58: The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo

Abstract Introduction: Novel less toxic therapeutic approaches to neuroblastoma therapy are urgently needed to improve survival and reduce treatment related side effects. As neuroblastoma is nearly uniformly p53 wild-type, research efforts have focused on activating innate p53-mediated apoptotic mechanisms, specifically through the inhibition of MDM2, the primary inhibitor of p53. The compound RG7388 has proven effective against adult tumors and is under consideration for clinical trials in pediatrics. Here we demonstrate the efficacy of this new class of MDM2 inhibitors against p53 wild-type neuroblastoma in vitro and in vivo orthotopic models of neuroblastoma. Methods: The human neuroblastoma cell lines, NGP, SH-SY5Y, LAN5 and LAN5 si-p53 (p53 silenced by shRNA), were utilized for in vitro and in vivo studies. Using graded concentrations of RG7388 (0 nM - 1500 nM), cell viability was measured by MTS assay. Apoptosis with RG7388 (0 nM, 100 nM, 500 nM) was measured using a flow cytometric annexin assay. In vivo effect of RG7388 was assessed in an orthotopic mouse model of neuroblastoma, in which neuroblastoma cells were intrarenally implanted into 80 female NCr nude mice (NGP n=22, SH-SY5Y n=18, LAN5 n=20, LAN5 si-p53 n=20). The mice were randomly divided into a control group which received vehicle (hydroxypropylcellulose/Tween 80) and a treatment group which received RG7388. RG7388 was initiated three weeks post-implantation by intraperitoneal injection once daily for 14 days at a concentration of 25 mg/kg for mice implanted with NGP and SH-SY5Y, and 35 mg/kg for mice implanted with LAN5 and LAN5 si-p53. At five weeks post-implantation, all mice were sacrificed, xenograft tumors resected and weighed. Xenograft tumor specimens underwent immunohistochemistry for apoptosis. Results: Using MTS assay and annexin apoptosis assay to measure cell viability and apoptosis in several cell lines of neuroblastoma (NGP, SH-SY5Y, LAN5) treated with RG7388, we found robust decrease in cell proliferation and increase in apoptosis compared to vehicle control in all p53 wild-type cell lines. The p53 silenced cell line, LAN5 si-p53, was found to be resistant to RG7388 with no decrease in cell proliferation and no increase in apoptosis. In vivo, all mice tolerated RG7388 well without morbidity. RG7388 significantly inhibited tumor growth by 59% in NGP (p=0.003), 67% in SH-SY5Y (p=0.006), and 75% in LAN5 (p=0.0019) xenograft tumors. However, RG7388 had no inhibitory effect on LAN5 si-p53 xenograft tumors compared to vehicle treatment (p=0.57). Tumor apoptosis was analyzed by cleaved caspase-3 staining, which showed xenografts treated with RG7388 had significantly increased apoptotic cells per high power field in NGP xenografts (p<0.0001) and SH-SY5Y xenografts (p=0.0014). Conclusion: The MDM2 small molecule inhibitor RG7388 significantly inhibits p53 wild-type neuroblastoma tumor growth but has no effect on p53-silenced neuroblastoma xenografts in an orthotopic mouse model. Our studies suggest that RG7388 inhibits tumor growth by p53-mediated apoptosis. Ongoing studies include assessment of the effect of RG7388 on tumor angiogenesis and metastasis. In vitro studies are ongoing to verify the molecular mechanism of RG7388 and confirm increase in p53 levels as well as downstream p53 pathways. Further studies are also being performed in several other p53 mutant neuroblastoma cell lines to elucidate the role of p53 for RG7388 treatment. Citation Format: Anna Lakoma, Eveline Barbieri, Matthew C. McVay, Zaowen Chen, Charitra Adhikari, Jason M. Shohet, Eugene S. Kim. The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo. [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 B58.

Abstract 1797: Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial

Abstract Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors is being pursued as a promising cancer therapeutic strategy. In the present study, we show the identification of NVP-CGM097, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-CGM097 binds to human Mdm2 protein with a Ki value of 1.3 nM, activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. Importantly, NVP-CGM097 displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability, which triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancer. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-CGM097 in pre-selected patients with p53 wild-type tumors. Citation Format: Sebastien Jeay, Joerg Berghausen, Nicole Buschmann, Patrick Chène, Robert Cozens, Dirk Erdmann, Stéphane Ferretti, Pascal Furet, Tobias Gabriel, François Gessier, Diana Graus-Porta, Francesco Hofmann, Philipp Holzer, Moriko Ito, Edgar Jacoby, Michael Jensen, Joerg Kallen, Marc Lang, Joanna Lisztwan, Masato Murakami, Carole Pissot-Soldermann, Stephan Ruetz, Caroline Rynn, Dario Sterker, Stefan Stutz, Thérèse Valat, Marion Wiesmann, Keiichi Masuya. Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2014-1797

Discovery of a small molecule MDM2 inhibitor (AMG 232) for treating cancer.

We recently reported the discovery of AMG 232 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 protein-protein interaction. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer. This article provides an overview of its discovery from the de novo design of the piperidinone series to the structure-activity studies leading to the identification of 1. In addition, this article also describes the preclinical pharmacology and pharmacokinetics of 1, along with its drug metabolism and safety assessment.

Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy

The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

Design, synthesis and in vitro and in vivo antitumour activity of 3-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors of the MDM2–p53 interaction

A novel class of small-molecule inhibitors of MDM2–p53 interaction with a (E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual screening strategy that combined both pharmacophore- and structure-based approaches. The hit optimisation identified several compounds with more potent activity than the hit compound and the positive drug nutlin-3a, especially compound 1b, which exhibited both the highest binding affinity to MDM2 (Ki = 0.093 μM) and the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI50 = 13.42 μM). Additionally, 1b dose-dependently inhibited tumour growth in BALB/c mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary, compound 1b represents a novel and promising lead structure for the development of anticancer drugs as MDM2–p53 interaction disruptors.

Discovery of Potent and Orally Active p53-MDM2 Inhibitors RO5353 and RO2468 for Potential Clinical Development.

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.

A p53 Drug Response Signature Identifies Prognostic Genes in High-Risk Neuroblastoma

Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6) whose expression together strongly predicted overall and event-free survival (p<0.0001). The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma.

Abstract B55: Antitumor activity of the MDM2 antagonist RG7388.

Abstract The p53 tumor suppressor is a transcription factor that inhibits tumor development by inducing cell cycle arrest or apoptosis in response to diverse stresses. In normal cells, p53 levels are tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers, thereby impairing p53 function. Antagonists of p53-MDM2 interaction can reactivate p53 and offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the nutlins, provided preclinical proof-of-concept for MDM2 antagonists as therapeutics for patients with tumors expressing wild-type p53. Subsequently, a member of the nutlin family, RG7112, was the first small molecule inhibitor of MDM2 to be tested clinically. Here we describe, RG7388, a highly optimized agent with analogous mechanism of action representing an entirely new branch of the nutlin family of MDM2 antagonists. Like RG7112, RG7388 binds selectively to the p53 site on the surface of the MDM2 molecule, effectively displacing p53 from MDM2, and leading to p53 stabilization and activation of the p53 pathway. However, RG7388 is derived from a distinct chemical series that binds with higher potency and selectivity to the MDM2 protein. RG7388 has substantially improved pharmacological properties, resulting in superior preclinical efficacy at lower doses and exposures as compared with its predecessor, RG7112. RG7388 elicits growth arrest and apoptosis at approximately 10-fold lower concentrations in vitro and in vivo, has an improved CYP inhibition profile, and a 2.5- to 20-fold lower projected human efficacious dose as compared with RG7112. Tumors with functional p53 signaling and MDM2 over-expression or amplification are likely to be the most sensitive to RG7388, however preclinical studies indicate that tumors with normal MDM2 levels may also respond to this novel therapeutic strategy. Results from preclinical safety and toxicology studies support further exploration of this compound in cancer patients. RG7388 is a promising agent that may offer a new therapeutic option and is currently in clinical testing in both solid and hematologic malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B55. Citation Format: Brian Higgins, Christian Tovar, Kelli Glenn, Antje Walz, Zoran Filipovic, Yu-E Zhang, Markus Dangl, Bradford Graves, Lyubomir Vassilev, Kathryn Packman. Antitumor activity of the MDM2 antagonist RG7388. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B55.

Small Molecule Inhibitors of MDM2-p53 and MDMX-p53 Interactions as New Cancer Therapeutics

Inactivation of the function of tumor suppressor p53 is common in human cancers. In approximately half of human cancers, the tumor suppressor function of p53 is inactivated by deletion or mutation of TP53, the gene encoding p53 protein. In the remaining 50% of human cancers, p53 tumor suppressor function can be effectively inhibited by oncoprotein MDM2 or its homolog MDMX. Since inhibition of p53 by MDM2 or MDMX protein is mediated by their direct interaction with p53, small-molecule inhibitors designed to block the MDM2-p53 or MDMX-p53 protein-protein interaction (MDM2 or MDMX inhibitors) can activate p53 in tumor cells retaining wild-type p53. In the last few years, several classes of potent, selective, and efficacious small molecule MDM2 inhibitors have been designed and developed, and six such compounds are being evaluated in clinical trials as new anticancer drugs. Additionally, non-peptide, small-molecule MDMX inhibitors have been reported. We review herein the design and development of potent small-molecule MDM2 and MDMX inhibitors.

Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence

Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated β-glactosidase (SA-β-gal) staining, decreased BrdU incorporation and elevated expression of p16INK4a (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53–p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53–p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.

Diastereomeric Spirooxindoles as Highly Potent and Efficacious MDM2 Inhibitors

Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new class of MDM2 small-molecule inhibitors. We report herein a reversible ring-opening-cyclization reaction for some of these spirooxindoles, which affords four diastereomers from a single compound. Our biochemical binding data showed that the stereochemistry in this class of compounds has a major effect on their binding affinities to MDM2, with >100-fold difference between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an animal model of human cancer.

Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development.

The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.