Murine Double Minute 2 Inhibitor Research Articles

Degree of MDM2 Amplification Affects Clinical Outcomes in Dedifferentiated Liposarcoma.

Dedifferentiated liposarcomas (DDLPS) are mesenchymal tumors associated with universally poor response to treatment. Genomic amplification of murine double minute 2 (MDM2) is used as a diagnostic biomarker; however, no established biomarkers exist to guide DDLPS treatment. In the largest study of its kind, we report that the extent of MDM2 amplification, not simply the presence of MDM2 amplification, may be biologically important to the actions of DDLPS. The distribution of MDM2 amplification in DDLPS was assessed using data from a commercial sequencing laboratory (n = 642) and The Cancer Genome Atlas (n = 57). Data from two retrospective clinical trials (n = 15, n = 16) and one prospective clinical trial (n = 25) were used to test MDM2's utility as a clinical biomarker. in vitro and in vivo assessments were conducted in DDLPS cell lines. Genomic MDM2 amplification follows a highly reproducible log-normal distribution. In patients with DDLPS treated with complete tumor resection, elevated MDM2 was associated with shortened time to recurrence as measured by genomic amplification (p = .003) and mRNA expression (p = .04). In patients requiring systemic therapy, higher MDM2 amplification was associated with reduced overall survival (p = .04). Doxorubicin treatment of DDLPS cells in vitro demonstrated variable sensitivity based on baseline MDM2 levels, and doxorubicin treatment elevated MDM2 expression. In vivo, treatment with doxorubicin followed by an MDM2 inhibitor improved doxorubicin sensitivity. MDM2 amplification levels in DDLPS follow a reproducible distribution and are associated with clinical outcomes and drug sensitivity. These results suggest that a prospective study of MDM2 as a predictive biomarker in DDLPS is warranted. No validated biomarkers exist for treatment selection in dedifferentiated liposarcoma (DDLPS). Although murine double minute 2 (MDM2) is currently used for diagnosis, the clinical relevance of MDM2 amplification has yet to be fully assessed. This study found that MDM2 amplification follows a predictable distribution in DDLPS and correlates with clinical and biological outcomes. These data suggests that MDM2 amplification may be a useful biomarker in DDLPS.

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.

Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via “computational conformer selection” for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 μM) > bepridil (23 μM) > azelastine (25 μM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

MDM2 inhibitor ameliorates cisplatin-induced nephropathy via NFκΒ signal inhibition.

Cisplatin is a platinum‐containing chemotherapeutic drug, which is widely used and highly effective. While effective against tumors, its use is limited by severe side effects such as nephrotoxicity and bone marrow suppression. Murine double minute 2 (MDM2) is the E3 ubiquitin ligase of the tumor suppressor gene, p53, and inhibition of MDM2 can suppress tumor cell growth. However, independent of p53, MDM2 acts as a co‐transcription factor for nuclear factor‐κB (NFκB), whose signaling can be involved in cisplatin‐induced tubular injury. We therefore examined the effects of MDM2 inhibitor on cisplatin cytotoxicity. In order to induce acute kidney injury and to investigate MDM2 inhibitory effects, we injected cisplatin into rats with or without the MDM2 inhibitor, DS‐5272, and analyzed kidney physiology/histology and NFκB signaling. Serum creatinine was significantly lower in the DS‐5272 group than in the vehicle group on day 3 (0.55 ± 0.069 vs 0.70 ± 0.072 mg/dL, P < 0.05). DS‐5272 also significantly decreased kidney injury molecule‐1 (KIM‐1) expression, improved tubular injury, and decreased apoptotic cells. Western blotting showed that cisplatin increased NFκB phosphorylation in kidneys, which was significantly suppressed by DS‐5272. In vitro, we treated HEK 293 cells with cisplatin, in the absence or presence of DS‐5272, and examined cytotoxicity and NFκB transcriptional activity. DS‐5272 co‐treatment reduced both cisplatin‐induced cell death and NFκB transcriptional activity. Collectively, these findings suggest that DS‐5272 can ameliorate cisplatin nephrotoxicity via NFκB signal inhibition.

Discovery and Characterization of Dual Inhibitors of MDM2 and NFAT1 for Pancreatic Cancer Therapy.

Overexpression and activation of the murine double minute 2 (MDM2) or nuclear factor of activated T cells 1 (NFAT1) oncoproteins frequently occur in pancreatic cancer. Most MDM2 inhibitors under development target MDM2-p53 binding and have little or no effect on cancers without functional p53, including pancreatic cancer. Some available compounds indirectly inhibit NFAT1 activity by interfering with calcineurin activity, but there are currently no specific inhibitors against NFAT1. Here we performed a high-throughput virtual and cell-based screening to yield a lead compound (MA242) that can directly bind both MDM2 and NFAT1 with high affinity, induce their protein degradation, and inhibit NFAT1-mediated transcription of MDM2 As a result of this binding, MA242 decreased cell proliferation and induced apoptosis in pancreatic cancer cell lines regardless of p53 status. MA242 alone or in combination with gemcitabine inhibited pancreatic tumor growth and metastasis without any host toxicity. Our data indicate that targeting both MDM2 and NFAT1 represents a novel and effective strategy to treat pancreatic cancer.Significance: These findings suggest that pharmacological inhibition of both MDM2 and NFAT1 is a promising strategy for the treatment of pancreatic cancer, even in tumors lacking functional p53. Cancer Res; 78(19); 5656-67. ©2018 AACR.

Efficacy of the MDM2 Inhibitor SAR405838 in Glioblastoma Is Limited by Poor Distribution Across the Blood-Brain Barrier.

Controversy exists surrounding whether heterogeneous disruption of the blood-brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893-901. ©2018 AACR.

Chlamydia trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis via the PI3K-AKT-mediated MDM2-p53 axis

Chlamydia trachomatis, the most common human pathogen that causes trachoma and sexually transmitted disease, has developed various strategies for inhibiting host cell apoptosis. Activation of the PI3K (phosphoinositide 3-kinase)/AKT-mediated MDM2 (murine double minute 2)-p53 pathway plays a prominent role in the apoptosis resistance arising from C. trachomatis infection. However, the precise upstream mechanisms by which C. trachomatis activates this pathway have not been adequately investigated. Here, we reveal that the secreted C. trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis in HeLa cells. This process requires the activation of the PI3K/AKT signaling pathway, thereby leading to phosphorylation and nuclear entry of MDM2, and p53 degradation. PI3K inhibitor LY294002 and MDM2 inhibitor Nutlin-3a block Pgp3-induced inhibition of HeLa cell apoptosis, suggesting a critical role for the PI3K/AKT pathway and its effect on the MDM2-p53 axis in Pgp3 anti-apoptotic activity.

In silico Activity Analysis of Saponins and 2, 5-Piperazinedione from Marine Organism against Murine Double Minute-2 Inhibitor and Procaspase-3 Activator

Objective: Cancer is a disease that can occur because of apoptosis failure. One of the causes of apoptosis failure is the presence of MDM2 inhibiting the activity of p53 so procaspase-3 could not be activated to caspase-3. Currently, the treatment of cancer has been applied widely, but more effective treatment always needed because cancer has been mutated. One of the treatments is the search for natural resources that come from the sea. The pursuit of the bioactive compound from marine organisms is not straightforward because it takes a long time and costly. Methods: Therefore, in silico method is used. This study conducted a docking of bioactive compounds from saponin and 2, 5-Piperazinedione as the MDM2 inhibitor and procaspase-3 activator by using Auto Dock and Vina. Results: The results showed that 18-Oxotryprostatin A and I ntercedenside A were the best bioactive compounds to serve as MDM2 Inhibitor and 6-Methoxyspirotryprostatin B and Frondoside A as a procaspase-3 activator due to their low binding energy. Conclusion: Most recommended bioactive compounds are those who have low binding energy, which is 18-Oxotryprostatin A with the value of -8,6 kcal/mol and Intercedenside A with the value of -7,0 kcal/mol. Key words: 2,5-Piperazinedione, Activator procaspase-3, Inhibitor MDM2, Cancer, Molecular docking, Saponin.

Inhibition of Noncanonical Murine Double Minute 2 Homolog Abrogates Ocular Inflammation through NF-κB Suppression

Uveitis is estimated to account for 10% of all cases of blindness in the United States, including 30,000 new cases of legal blindness each year. Intraocular and oral corticosteroids are the effective mainstay treatment, but they carry the risk of serious long-term ocular and systemic morbidity. New noncorticosteroid therapies with a favorable side effect profile are necessary for the treatment of chronic uveitis, given the paucity of existing treatment choices. We have previously demonstrated thatNutlin-3, a small-molecule inhibitor of murine double minute 2 (MDM2) homolog, suppresses pathologic retinal angiogenesis through a p53-dependent mechanism, but the noncanonical p53-independent functions have not been adequately elucidated. Herein, we demonstrate an unanticipated function of MDM2 inhibition, where Nutlin-3 potently abrogates lipopolysaccharide-induced ocular inflammation. Furthermore, we identified a mechanism by which transcription and translation of NF-κB is mediated byMDM2, independent of p53, in ocular inflammation. Small-molecule MDM2 inhibition is a novel noncorticosteroid strategy for inhibiting ocular inflammation, which may potentially benefit patients with chronic uveitis.

Abstract 4870: Targeted MDM2 degradation as a novel and efficacious cancer therapy

Abstract The E3 ubiquitin ligase MDM2 (Murine Double Minute 2) is the most important negative regulator of p53 tumor suppressor primarily by targeting it for ubiquitination and proteasomal degradation. Amplification or overexpression of the MDM2 gene occurs in many human cancers contributing to tumor development, progression and metastasis. Targeting MDM2 to restore p53 function has become an attractive therapeutic strategy for cancers harboring wild-type TP53. Currently, numerous drugs inhibiting the MDM2-p53 interaction, such as RG7112 (Hoffmann-La Roche), MI-773 (Sanofi) and DS-3032b (Daiichi Sankyo), have entered different stages of clinical trials. However, even in p53 wild-type tumors, MDM2 inhibitors can exert limited efficacy as monotherapy in some models, probably due to inadequate p53 induction and side effects. Therefore novel and improved strategies to effectively target the MDM2-p53 pathway are needed. Recently, we have designed a series of MDM2 degraders by conjugating a small molecule MDM2 inhibitor to phthalimide. The phthalimide moiety interacts with its target protein Cereblon (CRBN) and recruits the CUL4-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase complex to promote ubiquitination and proteasome degradation of MDM2. Our initial evaluation focused on acute leukemias that are mostly p53 wild-type and express high levels of MDM2. The MDM2 degraders exert significantly improved growth inhibitory activity compared to the inhibitor in human acute leukemia cell lines with IC50s &amp;lt; 5 nM. We performed Western Blots for MDM2 and p53 protein levels and qRT-PCR analysis for mRNA levels of p53 target genes to demonstrate MDM2 protein degradation and activation of p53 downstream pathways. Flow cytometry analysis confirmed the induction of apoptosis by MDM2 degraders in leukemia cells. In agreement with our design, the action of our MDM2 degraders depends on binding to Cereblon, as demonstrated by competition with free phthalimide, and on proteasome function, as demonstrated by proteasome inhibition. siRNA knockdown of p53 significantly abolished the activity of the degraders demonstrating the dependence upon p53 activation. Our studies demonstrate that the MDM2 degraders robustly activate wild-type p53 by inducing rapid degradation of MDM2 and activation of p53 pathways leading to strong apoptosis in leukemia cell lines in a CRBN-binding, proteasome and p53 dependent manner. More importantly, the MDM2 degraders showed great efficacy in inducing complete tumor regression or strong tumor growth inhibition in human leukemia xenograft models and significantly improved survival in disseminated human leukemia models in mice at well-tolerated dose-schedules. Our data provide strong preclinical rationale to further develop MDM2 degraders as a new class of therapy for the treatment of human acute leukemia and potentially other types of human cancer. Citation Format: Jiuling Yang, Yangbing Li, Angelo Aguilar, Donna McEachern, Sally Przybranowski, Ester Fernandez-Salas, Jianfeng Lu. Targeted MDM2 degradation as a novel and efficacious cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4870.

Predictive Gene Signatures Determine Tumor Sensitivity to MDM2 Inhibition.

Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type TP53 tumors are sensitive to MDM2 inhibition. Therefore, more potent inhibitors and biomarkers predictive of tumor sensitivity are needed. The novel MDM2 inhibitor DS-3032b is 10-fold more potent than the first-generation inhibitor nutlin-3a. TP53 mutations were predictive of resistance to DS-3032b, and allele frequencies of TP53 mutations were negatively correlated with sensitivity to DS-3032b. However, sensitivity to DS-3032b of TP53 wild-type tumors varied greatly. We thus used two methods to create predictive gene signatures. First, by comparing sensitivity to MDM2 inhibition with basal mRNA expression profiles in 240 cancer cell lines, a 175-gene signature was defined and validated in patient-derived tumor xenograft models and ex vivo human acute myeloid leukemia (AML) cells. Second, an AML-specific 1,532-gene signature was defined by performing random forest analysis with cross-validation using gene expression profiles of 41 primary AML samples. The combination of TP53 mutation status with the two gene signatures provided the best positive predictive values (81% and 82%, compared with 62% for TP53 mutation status alone). In addition, the top-ranked 50 genes selected from the AML-specific 1,532-gene signature conserved high predictive performance, suggesting that a more feasible size of gene signature can be generated through this method for clinical implementation. Our model is being tested in ongoing clinical trials of MDM2 inhibitors.Significance: This study demonstrates that gene expression profiling combined with TP53 mutational status predicts antitumor effects of MDM2 inhibitors in vitro and in vivoCancer Res; 78(10); 2721-31. ©2018 AACR.

Synergistic targeting of malignant pleural mesothelioma cells by MDM2 inhibitors and TRAIL agonists.

Malignant Pleural Mesothelioma (MPM) is a chemoresistant tumor characterized by low rate of p53 mutation and upregulation of Murine Double Minute 2 (MDM2), suggesting that it may be effectively targeted using MDM2 inhibitors. In the present study, we investigated the anticancer activity of the MDM2 inhibitors Nutlin 3a (in vitro) and RG7112 (in vivo), as single agents or in combination with rhTRAIL.In vitro studies were performed using MPM cell lines derived from epithelioid (ZL55, M14K), biphasic (MSTO211H) and sarcomatoid (ZL34) MPMs. In vivo studies were conducted on a sarcomatoid MPM mouse model.In all the cell lines tested (with the exception of ZL55, which carries a biallelic loss-of-function mutation of p53), Nutlin 3a enhanced p21, MDM2 and DR5 expression, and decreased survivin expression. These changes were associated to cell cycle arrest but not to a significant induction of apoptosis. A synergistic pro-apoptotic effect was obtained through the association of rhTRAIL in all the cell lines harboring functional p53. This synergistic interaction of MDM2 inhibitor and TRAIL agonist was confirmed using a mouse preclinical model. Our results suggest that the combined targeting of MDM2 and TRAIL might provide a novel therapeutic option for treatment of MPM patients, particularly in the case of sarcomatoid MPM with MDM2 overexpression and functional inactivation of wild-type p53.

Oral delivery of anti-MDM2 inhibitor SP141-loaded FcRn-targeted nanoparticles to treat breast cancer and metastasis

We have recently discovered a specific Murine Double Minute 2 (MDM2) oncogene inhibitor, called SP141, which exerts potent anticancer activity in various breast cancer models. However, its low oral bioavailability is the major hurdle for moving this drug to clinical trial. The present study was designed to discover and validate a novel nano-oral delivery system for this promising anticancer agent. Herein, we report the preparation, characterization, and evaluation of the efficacy and safety of the SP141-loaded IgG Fc-conjugated maleimidyl-poly(ethylene glycol)-co-poly(ε-caprolactone) (Mal-PEG-PCL) nanoparticles (SP141FcNP) as an orally cancer therapeutic agent. Our results indicated that SP141FcNP showed a biphasic release pattern and increased transepithelial transport in vitro and in vivo with the involvement of FcRn-mediated transcytosis. SP141FcNP also exhibited increased intestinal epithelial permeability, cellular uptake, and oral bioavailability, with extended blood circulation time, increased tumor accumulation, enhanced MDM2 inhibition, and stronger responses in anti-tumor growth and metastasis effects in vitro and in vivo, without apparent host toxicity. Collectively, this newly developed nanoparticle oral delivery system provides a basis for evaluation of SP141 as a potential clinical candidate for cancer therapy.

MDM2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Ablation and Radiotherapy in a p53-Dependent Manner

PURPOSE: Increased murine double minute 2 (MDM2) expression, independent of p53 status, is associated with increased cancer-specific mortality for men with prostate cancer treated with radiotherapy. We assessed MI-219, a small molecule inhibitor of MDM2 with improved pharmacokinetics over nutlin-3, for sensitization of prostate cancer cells to radiotherapy and androgen deprivation therapy, a standard treatment option for men with high-risk prostate cancer. EXPERIMENTAL DESIGN: The effect of MDM2 inhibition by MI-219 was assessed in vitro and in vivo with mouse xenograft models across multiple prostate cancer cell lines containing varying p53 functional status. RESULTS: MDM2 inhibition by MI-219 resulted in dose- and time-dependent p53 activation and decreased clonogenic cell survival after radiation in a p53-dependent manner. Mechanistically, radiosensitization following inhibition of MDM2 was largely the result of p53-dependent increases in apoptosis and DNA damage as evidenced by Annexin V flow cytometry and γ-H2AX foci immunofluorescence. Similarly, treatment with MI-219 enhanced response to antiandrogen therapy via a p53-dependent increase in apoptotic cell death. Lastly, triple therapy with radiation, androgen deprivation therapy, and MI-219 decreased xenograft tumor growth compared with any single- or double-agent treatment. CONCLUSION: MDM2 inhibition with MI-219 results in p53-dependent sensitization of prostate cancer cells to radiation, antiandrogen therapy, and the combination. These findings support MDM2 small molecule inhibitor therapy as a therapy intensification strategy to improve clinical outcomes in high-risk localized prostate cancer. TRANSLATIONAL RELEVANCE: The combination of radiotherapy and androgen deprivation therapy is a standard treatment option for men with high-risk prostate cancer. Despite improvements in outcomes when androgen deprivation therapy is added to radiation, men with high-risk prostate cancer have significant risk for disease recurrence, progression, and even death within the first 10 years following treatment. We demonstrate that treatment with MI-219 (an inhibitor of MDM2) results in prostate cancer cell sensitization to radiation and androgen deprivation therapy in vitro and in vivo. Triple therapy with MI-219, radiation, and androgen deprivation therapy dramatically decreased tumor growth compared with any single- or double-agent therapy. These findings provide evidence that inhibition of MDM2 is a viable means by which to enhance the efficacy of both radiation and androgen deprivation therapy and thereby improve outcomes in the treatment of prostate cancer. As such, further investigation is warranted to translate these findings to the clinical setting.

MDM2 promotes rheumatoid arthritis via activation of MAPK and NF-κB

Murine double minute-2 (MDM2) has pleiotropic roles in immune activation and regulation. However, the role of MDM2 in rheumatoid arthritis (RA) remains unknown. We undertook this study to investigate the role of MDM2 in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were isolated from 25 patients with active RA and 25 patients with osteoarthritis (OA). FLS were stimulated in the presence or absence of IL-1β in vitro. Mice with collagen-induced arthritis (CIA) were treated with Nutlin-3a (100mg/kg) or vehicle twice daily for 2weeks. MDM2 expression was determined by Western blot. MDM2 was down-regulated by specific gene silencing. The concentrations of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) were analyzed using enzyme-linked immunosorbent assay (ELISA). The pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were investigated by Western blot. Arthritis scoring and histological analysis were conducted. MDM2 expression was significantly higher in RA-FLS than in OA-FLS. MDM2 protein expression was positively correlated with disease activity of RA. MDM2 promoted the production of TNF-α, IL-6, MMP1 and MMP13 through MAPK and NF-κB pathways in RA-FLS. Nutlin-3a treatment decreased the arthritis severity and joint damage in CIA. Nutlin-3a also inhibited the activation of MAPK and NF-κB in arthritic joints. In conclusion, MDM2 inhibition exhibits anti-inflammatory activity and MDM2 might be a new therapeutic target for RA.

Molecular Modeling Investigation of Folic Acid Conjugation to MDM2 Inhibitors for Enhanced Cellular Uptake and Target Binding.

The activation of tumor suppressor p53 protein through inhibition of its interaction with the oncogenic Murine Double Minute 2 (MDM2) protein presents a novel therapeutic strategy against cancer. Accordingly, several small-molecule inhibitors have been developed that mimic three hydrophobic groups of p53 involved in p53-MDM2 binding and thus block the p53-binding pocket on MDM2. Interestingly, presence of a fourth, solvent-exposed hydrophilic moiety in these MDM2 inhibitors is shown to enhance their binding to MDM2 by protecting the inhibitor-MDM2 binding interface from surrounding solvent. In this context, we hypothesized that vitamin folic acid (FA) may prove to be suitable as the hydrophilic cover for enhancing activity of present MDM2 inhibitors. The proposed conjugation of FA to MDM2 inhibitors may also lead to their enhanced and selective uptake by cancer cells, owing to significantly higher expression of the FA receptors on cancer cells compared to normal cells. Therefore, based on our novel hypothesis we designed FA-conjugated MDM2 inhibitors and investigated their binding with MDM2 protein as well as the FA receptor. Specifically, a molecular modeling approach combining flexible receptor docking and molecular mechanics energy minimization calculations revealed highly favorable interactions of FA-conjugated MDM2 inhibitors with both MDM2 protein and the FA receptor as compared to native crystal ligands. Furthermore, these binding interactions were found to be stable using 50,000 ps molecular dynamics simulations. In summary, the newly-designed molecules of this kind, with better MDM2 target binding and enhanced cellular uptake potential, may prove highly useful against cancer and thus warrant further experimental investigations.

Triptolide induces apoptosis of gastric cancer cells via inhibiting the overexpression of MDM2.

Triptolide has been reported to exhibit antitumor effects in several cancers. This study investigates the mechanism by which triptolide induces apoptosis of gastric cancer cells. Gastric biopsies were collected for histological evaluation and detection of murine double minute 2 (MDM2) expression. Gastric cancer cells were cultured and treated with different concentrations of triptolide at indicated time points. The expression of MDM2, p53 protein, and target proteins including p21, PUMA, and X-linked inhibitor of apoptosis protein (XIAP) was detected. Apoptosis of cells treated with or without triptolide was evaluated. Our results showed that MDM2 protein was overexpressed in gastric cancer (p < 0.01, resp.). Triptolide induced significant apoptosis of gastric cancer cells in a dose- and time-dependent manner (p < 0.05). In addition, treatment with triptolide strongly inhibited the overexpression of MDM2 in gastric cancer cells, and this MDM2 inhibition led to increased levels of p53 protein and inhibition of XIAP (p < 0.05). However, triptolide failed to increase the expression of p53 target protein p21 and PUMA (p > 0.05). In conclusion, triptolide may induce apoptosis of gastric cancer cells via the inhibition of MDM2 overexpression in a p53-independent manner.

Abstract 1799: Targeted murine double minute 2 (MDM2) inhibition results in dramatic tumor regression in an MDM2-amplified glioblastoma multiforme (GBM) xenograft model

Abstract Disruption of the MDM2/p53 pathway is an oncogenic driver alteration observed in the vast majority of GBM. This study evaluated the efficacy of the small molecule, human-specific, MDM2 inhibitor SAR405838 across a panel of GBM xenografts with distinct alterations within the MDM2/p53 pathway. Through screening a panel of 56 GBM xenografts with a combination of array CGH and MDM2-specific PCR amplification, five xenograft models were identified with high-level MDM2 amplification (GBM 5, 102, 108, 122, and 143). MDM2 transcript expression was elevated in all of these lines except GBM102, which had expression levels similar to non-amplified lines. The effects of SAR405838 were studied in 2 of the MDM2 amplified lines (GBM102, GBM108), and non-amplified lines with wild-type p53 (GBM10) or mutant p53 (GBM12). Using a combination of neurosphere and Cyquant assays to assess in vitro sensitivity, 100 nM SAR405838 completely inhibited growth of GBM108 (6% +/- 1% absorbance relative to control), partially inhibited GBM102 neurosphere formation (34 +/- 17% relative to control), and had nominal impact on relative neurosphere formation in GBM10 (51% +/- 4% relative to control) or GBM12 (96% +/- 4% relative to control). Twenty-four hours after treatment with 100 nM SAR405838, transcript expression of p53 target genes p21 and PUMA were elevated by 14 and 12 fold relative to control in GBM108, while more limited changes in expression were observed in GBM102 (4 and 2.5 fold increase, respectively). Similarly, analysis of apoptosis after 72h by Annexin-V staining demonstrated a 5-fold increase in apoptotic fraction from control in GBM108 compared to no increase in GBM102. A more striking difference in efficacy was observed in flank tumor xenograft studies where the time to reach a median tumor volume of 1000 mm3 was marginally extended in GBM102 (36 days for placebo vs. 44 days for SAR405838). In contrast, GBM108 placebo-treated mice grew to a median volume of 1000 mm3 in 53 days while treatment with SAR405838 resulted in complete tumor regression at 70 days. SAR405838 is a known substrate for the P-glycoprotein efflux pump that functions in the blood brain barrier to exclude xenobiotics from the central nervous system. Consistent with limited penetration of SAR405838 into the brain, a parallel study in an intracranial xenograft model for GBM108 demonstrated no significant survival benefit with SAR405838 treatment compared to placebo. Thus, although SAR405838 provides marked efficacy in vitro and in a flank tumor model of an MDM2 amplified tumor line, the lack of efficacy in an orthotopic model of this study suggests that more prudent investigation is needed before advancing the development of this specific drug in human tumors growing in the brain. Citation Format: Ann C. Mladek, Isabelle Meaux, Katrina Bakken, Pascal Pannier, Cedric Barriere, James W. Watters, Laurent R.J. Debussche, Jann N. Sarkaria. Targeted murine double minute 2 (MDM2) inhibition results in dramatic tumor regression in an MDM2-amplified glioblastoma multiforme (GBM) xenograft model. [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 1799. doi:10.1158/1538-7445.AM2014-1799

Abstract 741: Combined inhibition of MDM2 and MEK for KRAS mutant non-small cell lung cancer

Abstract There are currently no effective targeted therapies for KRAS mutant NSCLC. Combination drug strategies that target downstream pathways activated by mutant KRAS are currently being investigated, however efficacy may be limited by inability to induce a sufficient apoptotic response. MDM-2 (murine double minute 2) is a ubiquitin ligase that negatively regulates p53 function by promoting its degradation. Inhibitors of the MDM2-p53 interaction lead to p53 activation and transcription of genes promoting cell cycle arrest and apoptosis. We investigated the efficacy of combined MEK and MDM2 inhibition for KRAS mutant NSCLC. In KRAS mutant NSCLC cell lines with wild-type p53, pimasertib (MEKi) and SAR405838 (MDM-2i) led to upregulation of BIM and PUMA respectively. Induction of apoptosis and cell cycle arrest correlated with in vitro efficacy, and the combination exhibited marked in vivo activity against KRAS mutant p53 wild-type NSCLC xenograft tumors. These studies provide rationale for future clinical investigation of combined MEK and MDM-2 inhibition for KRAS mutant NSCLC. Citation Format: Aaron N. Hata, Hannah L. Archibald, Maria D. Gomez-Caraballo, Laurent R. DeBussche, Sukhvinder Sidhu, James Watters, Jeffrey A. Engelman. Combined inhibition of MDM2 and MEK for KRAS mutant non-small cell lung cancer. [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 741. doi:10.1158/1538-7445.AM2014-741

MDM2 inhibitors in the search for an optimized neovascular age-related macular degeneration treatment

Murine double minute-2 inhibitors offer a durable treatment for neovascular age-related macular degeneration by inducing apoptosis in pathologically proliferating cells, a similar effect as radiation therapy, but with potentially a more favorable side-effect profile since it does not induce DNA damage. Murine double minute-2 inhibitors may be complementary to current anti-VEGF treatments and potentially reduce the cumulative number of intravitreal injections.