Wild-type P53 Tumor Research Articles

P53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer

BackgroundDespite advances in early diagnosis and treatment of cancer patients, metastasis remains the major cause of mortality. TP53 is one of the most frequently mutated genes in human cancer, and these alterations can occur during the early stages of oncogenesis or as later events as tumors progress to more aggressive forms. Previous studies have suggested that p53 plays a role in cellular pathways that govern metastasis. To investigate how p53 deficiency contributes to late-stage tumor growth and metastasis, we developed paired isogenic patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC) differing only in p53 status for longitudinal analysis.MethodsPatient-derived isogenic human tumor lines differing only in p53 status were implanted into mouse mammary glands. Tumor growth and metastasis were monitored with bioluminescence imaging, and circulating tumor cells (CTCs) were quantified by flow cytometry. RNA-Seq was performed on p53-deficient and p53 wild-type tumors, and functional validation of a lead candidate gene was performed in vivo.ResultsIsogenic p53 wild-type and p53-deficient tumors metastasized out of mammary glands and colonized distant sites with similar frequency. However, p53-deficient tumors metastasized earlier than p53 wild-type tumors and grew faster in both primary and metastatic sites as a result of increased proliferation and decreased apoptosis. In addition, greater numbers of CTCs were detected in the blood of mice engrafted with p53-deficient tumors. However, when normalized to tumor mass, the number of CTCs isolated from mice bearing parental and p53-deficient tumors was not significantly different. Gene expression profiling followed by functional validation identified B cell translocation gene 2 (BTG2), a downstream effector of p53, as a negative regulator of tumor growth both at primary and metastatic sites. BTG2 expression status correlated with survival of TNBC patients.ConclusionsUsing paired isogenic PDX-derived metastatic TNBC cells, loss of p53 promoted tumor growth and consequently increased tumor cell shedding into the blood, thus enhancing metastasis. Loss of BTG2 expression in p53-deficient tumors contributed to this metastatic potential by enhancing tumor growth in primary and metastatic sites. Furthermore, clinical data support conclusions generated from PDX models and indicate that BTG2 expression is a candidate prognostic biomarker for TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0673-9) contains supplementary material, which is available to authorized users.

Genomic Instability: The Pivotal Role of Mutant P53 in Human Cancers

The tumor suppressor p53 plays a critical role to preserve DNA fidelity from diverse insults through the regulation of cell-cycle checkpoints, DNA repair, senescence and apoptosis. The TP53 is altered in more than half of human cancers. This leads to the production of mutant p53 proteins that loose wild type p53 tumor suppression functions and concomitantly acquire new oncogenic deleterious features implicated in: increased cell proliferation, increased chemoresistance, disruption of tissue architecture, promotion of migration, invasion and metastasis and several other pro-oncogenic properties. Accumulating evidences suggest that mutant p53 proteins drastically perturb the residual genome-stabilizing mechanisms during cancer progression, thereby increasing genomic instability of mutant p53 carrying human cancers. In this commentary we briefly summarize the most important evidences suggesting that mutant p53 plays a relevant role in promoting genomic instability in human cancers.

Abstract A083: Modulation of the immune system as a strategy to treat EGFR mutant lung adenocarcinoma

Abstract Fifteen percent of lung adenocarcinomas harbor mutations in the Epidermal Growth Factor Receptor (EGFR). Although EGFR mutant tumors show sensitivity to specific tyrosine kinase inhibitors (TKIs) like erlotinib, resistance develops after an average of 12 months and there are currently no approved therapeutic strategies to overcome resistance. Therefore, novel approaches to delay resistance and improve patient survival are crucial. Recent discoveries in tumor immunology have revealed the potential of immune-based therapies to treat lung malignancies. In particular, immune checkpoint inhibitors such as anti-PD-1 have shown efficacy in a subset of patients with metastatic lung adenocarcinoma with minimal toxicity. These promising data led us to investigate the benefit of immune modulatory agents in EGFR mutant lung cancer. As a first step, we set out to explore the role of the immune system in this lung adenocarcinoma subset using a tetracycline-inducible mouse model of EGFR mutant lung cancer. We found, in these models, that lung-specific expression of mutant EGFR and consequent tumor formation leads to the establishment of an immunosuppressive tumor microenvironment characterized by an overall decrease in CD4 T cells, infiltration of Tregs, up-regulation of PD-1 expression on both helper and cytotoxic T cells and a diminished cytokine production by these T cells. Additionally, by flow cytometry and quantitative PCR of FACS-sorted cells, we have observed increased expression of PD-L1 on alveolar macrophages, which represent over 60% of the CD45+ cells in these tumors, suggesting an engagement of the adaptive and innate arms of the immune system to mediate immunosuppression via the PD-1/PD-L1 axis. Furthermore, when the tumor suppressor gene p53 was deleted, we observed a marked decrease in tumor infiltrating CD8 T cells when compared to p53 wild-type tumors. Currently, we are evaluating the functional role of elements of the immune system in these models to delineate their effects on tumor progression and therapeutic response. Moreover, we are investigating whether the observed immunosuppression in these tumors can be altered by immunomodulatory agents such as the anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors alone or in combination with EGFR TKIs. Citation Format: Deborah Ayeni, Ping-Chih Ho, Curtis Perry, Susan Kaech, Katerina Politi. Modulation of the immune system as a strategy to treat EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A083.

AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway

The tumor suppressor p53 plays a key role in malignant transformation and tumor development. However, the frequency of p53 mutations within individual types of cancer is different, suggesting the existence of other mechanisms attenuating p53 tumor suppressor activity. Changes in upstream regulators of p53 such as MDM2 amplification and overexpression, expression of viral oncoproteins, estrogen receptor signaling, or changes in p53 transcriptional target genes were previously described in wild-type p53 tumors. We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ER+ breast cancers with wild-type p53 but not ER- or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy.

Regulation of endoplasmic reticulum functions.

The endoplasmic reticulum (ER) is a multifunctional organelle that is specialized for lipid synthesis, Ca2+ homeostasis, and protein synthesis. Protein synthesis is a multistep process involving folding and manipulation of proteins as well as the exportation of proteins to an appropriate location. Several stress responses, such as metabolic and anaerobic stress, induce ER dysfunction and protein unfolding, which accelerates the disruption of ER homeostasis. To prevent the production of unfolded proteins, cells induce the unfolded protein response (UPR), which is a highly regulated protein quality surveillance system. UPR maintains and restores ER homeostasis to increase the secretory function of the ER via the induction of the ER chaperone protein. This stimulates re-folding or degradation of unfolded proteins. However, irreparable ER stress induces a cell death response to eliminate these damaged cells [1]. Three types of ER transmembrane proteins, a protein-kinase and site-specific endoribonuclease, inositol-requiring enzyme 1 α (IRE1α), protein kinase R-like ER kinase/pancreatic eukaryotic initiation factor 2 kinase, and activating transcription factor 6 are essential for the mammalian UPR system. In particular, IRE1α is an essential signal transducer involved in the maintenance of ER function. The IRE1α pathway induces X-box binding protein 1(S) [XBP1(S)], which is the active form of XBP1 generated by IRE1α-dependent splicing of the XBP1 mRNA. XBP1(S) is a transcription factor that increases the expression of the ER chaperone, thus enhancing the secretory function of the ER and suppressing ER stress-mediated cell death [1]. Cancer cells alternate between glycolysis and protein synthesis pathways to survive and proliferate during stress associated with tumor growth and prolonged metabolic and anaerobic stress [2]. Cancer cells produce a lot of new proteins to satisfy their rapid growth using an energy source, such as the cancer-specific aerobic glycolysis pathway. Thus, the augmented function of the ER and resistance of ER stress-mediated cell death is essential for tumor proliferation and survival. Many reports have shown the enhancement of ER function in a variety of human cancers, but the mechanism for the cancer cell-specific regulation of ER function remains unresolved. Our recent study reveals that the tumor suppression gene, p53, is a key regulator of ER function [3]. To the best of our knowledge, this is the first report to connect the cancer-related gene to ER function. p53 is mutated in at least half of human cancers, and defects in the p53 response lead to tumor development. The functions of p53 influence many types of cell signaling transduction pathways, but the role of p53 in the ER is largely unknown [4]. We found that the loss of p53 function increased IRE1α, XBP1(S), and ER chaperone expression in non-stressed and ER-stressed conditions and also increased ER function. Interestingly, IRE1α was abundantly expressed in mutant p53 cell lines compared with wild-type p53 cell lines. The upregulation of IRE1α expression triggered the activation of the IRE1α/XBP1 pathway. Further, our experiments identified the mechanism for increased IRE1 expression with p53 deficiency, and our results revealed that suppressed p53-dependent IRE1α-synoviolin-1 (SYVN1) association induced IRE1α proteasomal degradation. Therefore, wild-type p53 suppresses ER function and adapts to ER stress by keeping IRE1α expression levels low, which highlights the anti-cancer effects of p53. However, loss of p53 function activates the IRE1α/XBP1 pathway and stimulates resistance to ER stress-mediated cell death. Thus, up-regulation of IRE1α expression via the loss of p53 function is one of the mechanisms by which a cancer-specific increase in ER function and activation of the IRE1α/XBP1 pathway is related to some parts of the aggressive cancer phenotype found in p53-mutant cancers. Another important finding of our current study is that an inhibitor of IRE1 selectively prevents the growth of p53-null tumors in vivo compared with that of wild type p53 tumors [3]. Several reports suggested that the inhibition of the IRE1α/XBP1 pathway is a new anti-cancer target. Cubillos-Ruiz et al. reported that the inhibition of activated XBP1 stimulates tumor-infiltrating dendritic cell-induced anti-tumor immunity [5]. The IRE1α inhibitor has an anti-cancer effect on human multiple myeloma and pancreatic cancers [6, 7]. Furthermore, DNA-damaging agents are widely used as treatments for various forms of cancer in the clinic, and its anti-cancer effects depend on the appropriate function of wild-type p53. Thus, mutant p53 cancer cells have been reported to show chemoresistance to many anti-cancer agents, and there is a need for novel therapies against mutant p53 cancers. Therefore, preventing the activation of the IRE1α/XBP1 pathway is a promising target for anti-tumor agents of mutant p53 cancers. In summary, cancer cells need to increase ER function to survive stressful environments. Loss of p53 function augments ER function via the activation of the IRE1α/XBP1 pathway by the disruption of the p53-SYVN1-IRE1α complex. Thus, targeting the IRE1α/XBP1 pathway has potential as a novel therapeutic strategy for malignancy and chemo-resistance of mutant p53 cancers.

PDL1 Regulation by p53 via miR-34.

Background:Although clinical studies have shown promise for targeting PD1/PDL1 signaling in non–small cell lung cancer (NSCLC), the regulation of PDL1 expression is poorly understood. Here, we show that PDL1 is regulated by p53 via miR-34.Methods:p53 wild-type and p53-deficient cell lines (p53–/– and p53+/+ HCT116, p53-inducible H1299, and p53-knockdown H460) were used to determine if p53 regulates PDL1 via miR-34. PDL1 and miR-34a expression were analyzed in samples from patients with NSCLC and mutated p53 vs wild-type p53 tumors from The Cancer Genome Atlas for Lung Adenocarcinoma (TCGA LUAD). We confirmed that PDL1 is a direct target of miR-34 with western blotting and luciferase assays and used a p53R172HΔg/+K-rasLA1/+ syngeneic mouse model (n = 12) to deliver miR-34a–loaded liposomes (MRX34) plus radiotherapy (XRT) and assessed PDL1 expression and tumor-infiltrating lymphocytes (TILs). A two-sided t test was applied to compare the mean between different treatments.Results:We found that p53 regulates PDL1 via miR-34, which directly binds to the PDL1 3’ untranslated region in models of NSCLC (fold-change luciferase activity to control group, mean for miR-34a = 0.50, SD = 0.2, P < .001; mean for miR-34b = 0.52, SD = 0.2, P = .006; and mean for miR-34c = 0.59, SD = 0.14, and P = .006). Therapeutic delivery of MRX34, currently the subject of a phase I clinical trial, promoted TILs (mean of CD8 expression percentage of control group = 22.5%, SD = 1.9%; mean of CD8 expression percentage of MRX34 = 30.1%, SD = 3.7%, P = .016, n = 4) and reduced CD8+PD1+ cells in vivo (mean of CD8/PD1 expression percentage of control group = 40.2%, SD = 6.2%; mean of CD8/PD1 expression percentage of MRX34 = 20.3%, SD = 5.1%, P = .001, n = 4). Further, MRX34 plus XRT increased CD8+ cell numbers more than either therapy alone (mean of CD8 expression percentage of MRX34 plus XRT to control group = 44.2%, SD = 8.7%, P = .004, n = 4). Finally, miR-34a delivery reduced the numbers of radiation-induced macrophages (mean of F4-80 expression percentage of control group = 52.4%, SD = 1.7%; mean of F4-80 expression percentage of MRX34 = 40.1%, SD = 3.5%, P = .008, n = 4) and T-regulatory cells.Conclusions:We identified a novel mechanism by which tumor immune evasion is regulated by p53/miR-34/PDL1 axis. Our results suggest that delivery of miRNAs with standard therapies, such as XRT, may represent a novel therapeutic approach for lung cancer.

Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy.

Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.

Reactivation of p53 via MDM2 inhibition.

Reactivation of p53 via MDM2 inhibition.

The Expanded p53 Interactome as a Predictive Model for Cancer Therapy

The tumor suppressor gene TP53 is implicated in the majority of all human cancers, thus pivotal to genomic integrity. There are more than 72,000 scientific publications describing p53’s function, yet, due to the complexity of its interactions we are still far from fully elucidating p53’s role in tumorigenesis. Computational methodologies are novel tools to depict and dissect complex disease networks. The Boolean PKT206 p53 – DNA damage model has previously demonstrated good predictive capability for p53 wild-type and null tumors in various in silico knockouts. Here, we have expanded PKT206 to generate a more clinically robust representation of p53 dynamics. The new PMH260 model incorporates 260 nodes representing genes, with 980 interactions between them representing inhibitions and activations. Additional biological outputs, including angiogenesis, cell cycle arrest and DNA repair were also amalgamated into the model. Three in silico knockouts of highly connected nodes (p53, MDM2 and FGF2) were generated and logical steady state analysis and dependency relationships applied. 71 % of predictions were considered true from superimposition of human osteosarcoma and HCT116 microarray profiles. In silico knockout analysis revealed 98 potential novel predictions, of which 13 were validated by literature; 83 % of them were overlapping with PKT206. Thus the expanded Boolean PMH260 model offers a promising platform for clinical potential in targeted cancer therapeutics.

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 3531: Companion diagnostics-based telomerase-specific oncolytic virotherapy: preclinical evaluation in human colorectal cancer cell lines differentially affected in the RAS/RAF/MEK signaling pathway

Abstract Colorectal cancers harboring KRAS or BRAF mutations often show resistance to anti-cancer drugs, and are poor prognostic disease that causes metastasis and recurrence compared to those with wild type KRAS and BRAF. KRAS/BRAF-wild type colorectal cancers are sensitive to both cetuximab and panitumumab that are epidermal growth factor receptor (EGFR)-targeting agents. However, KRAS/BRAF-mutant colorectal cancers are resistant to EGFR-targeting agents because of constitutive activation of the EGFR-downstream RAS/RAF/MEK signaling pathway. Therefore, the development of novel therapeutic strategy is required to improve the clinical outcome in patients with KRAS/BRAF-mutant colorectal cancers. We developed two types of telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702 armed with the wild-type p53 tumor suppressor gene. In this study, we established the companion diagnostics-based selection algorithm for our oncolytic viruses. We compared the antitumor effects of OBP-301 and OBP-702 in human colorectal cancer cells with wild type KRAS/BRAF (SW48, Colo320DM), mutated KRAS (DLD-1, SW620) and mutated BRAF (RKO, HT29). We evaluated the antitumor effect of OBP-301 and OBP-702 in human colorectal cancer cells using XTT assay. Oncolytic adenovirus-mediated induction of apoptosis- and autophagy-related cell death was analyzed by Western blot analysis. KRAS-mutant colorectal cancer cells were sensitive to both OBP-301 and OBP-702 as well as KRAS/BRAF-wild type cells. In contrast, BRAF-mutant colorectal cancer cells were sensitive to OBP-702 rather than OBP-301. Western blot analysis showed that OBP-301 induced autophagy, whereas OBP-702 induced both autophagy and apoptosis. Taken together, our data suggest that oncolytic viruses should be used according to the genetic background of the RAS/RAF/MEK signaling pathway, especially in patients with colorectal cancer. Citation Format: Shuta Tamura, Hiroshi Tazawa, Naoto Hori, Takeshi Koujima, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Companion diagnostics-based telomerase-specific oncolytic virotherapy: preclinical evaluation in human colorectal cancer cell lines differentially affected in the RAS/RAF/MEK signaling pathway. [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 3531. doi:10.1158/1538-7445.AM2015-3531

Reactivating p53 functions by suppressing its novel inhibitor iASPP: a potential therapeutic opportunity in p53 wild-type tumors.

Although mutational inactivation of p53 is found in 50% of all human tumors, a subset of tumors display defective p53 function, but retain wild-type (WT) p53. Here, direct and indirect mechanisms leading to the loss of WT p53 activities are discussed. We summarize the oncogenic roles of iASPP, an inhibitor of WT p53, in promoting proliferation, invasion, drug or radiation-resistance and metastasis. From the therapeutic view, we highlight promising perspectives of microRNA-124, peptide and small molecules that reduce or block iASPP for the treatment of cancer. High iASPP expression enhances proliferation, aggressive behavior, the resistance to radiation/chemotherapy and correlates with poor prognosis in a range of human tumors. Overexpression of iASPP accelerates tumorigenesis and invasion through p53-dependent and p53-independent mechanisms. MicroRNA-124 directly targets iASPP and represses the growth and invasiveness of cancer cells. The disruption of iASPP-p53 interaction by a p53-derived peptide A34 restores p53 function in cancer cells. The inhibition of iASPP phosphorylation with small molecules induces p53-dependent apoptosis and growth suppression. The mechanisms underlying aberrant expression of iASPP in human tumors should be further investigated. Reactivating WT p53 functions by targeting its novel inhibitor iASPP holds promise for potential therapeutic interventions in the treatment of WT p53-containing tumors.

A distinct p53 target gene set predicts for response to the selective p53-HDM2 inhibitor NVP-CGM097.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.

Small-molecule MDM2-p53 inhibitors: recent advances.

Potent and selective small-molecule inhibitors of the p53-MDM2 interaction intended for the treatment of p53 wild-type tumors have been designed and optimized in a number of chemical series. This review details recent disclosures of compounds in advanced optimization and features key series that have given rise to clinical trial candidates. The structure-activity relationships for inhibitor classes are discussed with reference to x-ray structures, and common structural features are identified.

The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-Inducing Cytotoxic Agents.

p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2-p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics.

Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors.

Abstract PR06: Lymphocyte invasion of basal breast tumors is associated with wild-type p53

Abstract The tumor suppressor p53 is mutated in approximately 30% of breast adenocarcinomas, but the clinical implications of p53 mutation depend on the subtype of breast tumor. Although p53 mutation is associated with worse outcome for patients with Luminal B and Her2 subtypes, the same is not true in Basal tumors, despite the fact that Basal tumors have the highest frequency of p53 mutation of any subtype. Lymphocytic infiltration is associated with better prognosis in several epithelial malignancies including triple-negative breast tumors. In this study we identify an association in Basal breast cancer between lymphocytic infiltration and the number of copies of wild-type p53 by combining gene expression, p53 sequencing, DNA copy number analysis, and semi-quantitative immune cell histology in 1,281 tumors. We use the Nanodissect algorithm to define signatures for T cell types and show that the infiltration consists primarily of cytotoxic T lymphocytes. Elevated expression of this gene signature is associated with better outcome in Basal tumors, and differential pathway activity analysis identifies significant activation of caveolin 1 signaling to TGF-beta in p53 wild-type Basal tumors. These findings identify a new link between the p53 pathway and beneficial adaptive immune response in breast tumors not responsive to the estrogen receptor, supporting a connection between a lack of p53 function and the failure of tumor immunosurveillance. This abstract is also presented as Poster A35. Citation Format: David A. Quigley, Charlie Vaske, Laxmi Silwa-Pandit, Ruth Dannenfelser, Anita Langerød, Hans Kristian M. Vollan, Olga Troyanskaya, Suet-Feung Chin, Carlos Caldas, Allan Balmain, Anne-Lise Børresen-Dale, Vessela Kristensen. Lymphocyte invasion of basal breast tumors is associated with wild-type p53. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR06. doi:10.1158/1538-7445.CHTME14-PR06

Targeting Melanoma with Small Molecules: Inhibitors of the Calcium-Binding Protein S100B

Long used as a prognostic indicator, the calcium binding protein, S100B, has been directly linked to Malignant Melanoma (MM). S100B binds the typically wild-type p53 tumor suppressor in MM and promotes its degradation. In order to restore p53 levels within MM along with its tumor suppressor and apoptotic activities, we have implemented a program for the discovery and optimization of S100B inhibitors (often referred to as SBiXs). In the process, we have uncovered and probed the three persistent binding sites within S100B. Liganding within these sites was characterized using structural biology techniques (NMR and X-ray crystallography) and inhibitor efficacy was evaluated using Fluorescent Polarization Competition Assays and cellular assays. Efforts to discover/synthesize and/or to improve existing inhibitors of S100B to restore p53 activity in human malignant melanoma are ongoing and SBiXs occupying the persistent binding Sites 1, 2, and 3 simultaneously are desired. Such compounds can then be examined for in vivo efficacy. The compounds presented offer potential bridging scaffolds between Sites 1, 2, and 3; and will act as the basis for the design of improved SBiXs. SBiXs may also have therapeutic value for treating other cancers with elevated S100B and wt p53 such as astrocytoma, renal tumors, and some forms of leukemia.

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&amp;lt;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 805: Disruption of the MDM2-p53 interaction synergizes with MEK inhibition to induce cell death and promote tumor regression in p53 wild-type, Ras or Raf-mutant tumor models

Abstract Disruption of the interaction between p53 and MDM2 with small molecules, and subsequent reactivation of p53, is an attractive treatment strategy for p53 wild-type tumors that has shown striking pre-clinical activity in models exhibiting genomic amplification of the MDM2 gene. However, MDM2 amplified tumors represent only a small proportion of the p53 wild-type tumor population, and single agent regressions may be limited outside of the MDM2 amplified context. In order to increase the potential clinical benefit of MDM2-p53 protein-protein interaction (PPI) inhibitors beyond tumors exhibiting amplification of MDM2, we performed an enhancer library combination screen to identify optimal combination partners for MDM2-p53 PPI inhibitors in p53 wild-type, MDM2 non-amplified tumor cells. The MDM2-p53 PPI inhibitor SAR405838 and a related molecule were examined in combination with an enhancer library of 200 oncology drugs representing a broad molecular diversity of targets across a panel of twenty p53 wild-type cancer cell lines. Analysis results showed that MEK inhibitors were the most statistically significant synergistic combination partners in this screen, with synergy observed in cell lines harboring mutations that activated MAP kinase pathway signaling. These synergy results were validated in independent experiments using the ray design methodology, and extended to multiple K-ras, N-ras and B-raf mutant contexts in vitro. In K-ras, N-ras or B-raf mutant cell lines, the combination of SAR405838 with the MEK inhibitor pimasertib resulted in p53 pathway activation, inhibition of pERK levels, induction of the apoptotic mediators PUMA and BIM, induction of phosphatidyl serine exposure, and induction of caspase activity. In the Ras/Raf wild-type cell line MCF7, no synergy was observed. In cell line and patient-derived xenografts in vivo, the combination of SAR405838 and pimasertib resulted in greater anti-tumor activity than single agents and induced regression in K-ras, N-ras and B-raf mutant tumor models derived from different tissue types, including complete regressions in multiple models. These data indicate that MEK inhibitors synergize with MDM2-p53 PPI inhibitors in tumor models that exhibit activated MAP kinase pathway signaling and p53 wild-type status. Given the relative lack of overlapping clinically significant toxicities of MDM2-p53 PPI inhibitors and MEK inhibitors, such a combination merits further investigation for the treatment of K-ras, N-ras, or B-raf mutant, p53 wild-type tumors, representing a large patient population with significant unmet medical need. Citation Format: Isabelle Meaux, Jean-Paul Nicolas, Steve Rowley, Sukhvinder Sidhu, Francoise Herve, Laurent Dassencourt, Fanny Windenberger, Dimitri Gorge-Bernat, Pascal Pannier, Donald Bergstrom, Laurent Debussche, James Watters. Disruption of the MDM2-p53 interaction synergizes with MEK inhibition to induce cell death and promote tumor regression in p53 wild-type, Ras or Raf-mutant tumor models. [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 805. doi:10.1158/1538-7445.AM2014-805