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Supplemental Figure 2. AMG 232 treatment causes p53 stabilization, and increased p21 and MDM2 proteins in p53 wild-type cells.

Supplemental Figure 3. Body weights of mice in xenograft efficacy studies

Supplemental Figure 1. AMG 232 chemical structure, and 3-D model of AMG 232 bound to MDM2 protein.

Supplementary Figure 3 from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51. ©2010 AACR.

Supplementary Methods, Figure Legends, and References from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

Supplementary Figure 1 from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

Supplementary Figure 2 from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

Supplementary Figure 4 from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

Supplementary Figure Legend from Complementary Actions of Inhibitors of Angiopoietin-2 and VEGF on Tumor Angiogenesis and Growth

Supplementary Figure 1 from Complementary Actions of Inhibitors of Angiopoietin-2 and VEGF on Tumor Angiogenesis and Growth

Supplementary Table 1 from Complementary Actions of Inhibitors of Angiopoietin-2 and VEGF on Tumor Angiogenesis and Growth

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Mouse double minute 2 (MDM2) is a critical negative regulator of the tumor suppressor p53, playing a key role in controlling its transcriptional activity, protein stability, and nuclear localization. MDM2 expression is up-regulated in numerous cancers, resulting in a loss of p53-dependent activities, such as apoptosis and cell-cycle arrest. Genetic amplification and inheritance of MDM2 promoter single-nucleotide polymorphisms (SNPs) are the two best-studied mechanisms for up-regulating MDM2 activity. This article provides an overview of these events in human cancer, highlighting the frequent occurrence of MDM2 amplification in sarcoma and the role of SNP309 and SNP285 in regulating MDM2 expression and cancer risk. The availability of large-scale genomic profiling datasets, like those from The Cancer Genome Atlas Research Network, have provided the opportunity to evaluate the consequences of MDM2 amplification and SNP inheritance across high-quality tumor samples from diverse cancer indications.

Human murine double minute 2 (MDM2) is a negative regulator of p53, which plays an important role in cell cycle and apoptosis. We report several optimizations to the synthesis of the chromenotriazolopyrimidine series of MDM2-p53 protein-protein interaction inhibitors. Additionally, the in vitro and in vivo stability, pharmacokinetic properties and solubility were improved through N-substitution.

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51. ©2010 AACR.

// Anne Y. Saiki 1 , Sean Caenepeel 1 , Elissa Cosgrove 2, 5 , Cheng Su 3 , Michael Boedigheimer 4 , Jonathan D. Oliner 1 1 Oncology Research, Amgen, Inc., Thousand Oaks, California, USA 2 Genome Analysis Unit, Amgen, Inc., South San Francisco, California, USA 3 Biostatistics, Amgen, Inc., Seattle, Washington, USA 4 Molecular Sciences, Amgen, Inc., Thousand Oaks, California, USA 5 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Correspondence to: Jonathan D. Oliner, e-mail: jonoliner@hotmail.com Keywords: TP53 , MDM2 , amplification Received: October 28, 2014 Accepted: January 08, 2015 Published: February 03, 2015 ABSTRACT Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53 Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53 WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2 -amplified subset of p53 WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

EWS-WT1 is a chimeric transcription factor resulting from fusion of the N-terminal domain of the Ewing sarcoma gene EWS to the three C-terminal zinc fingers of the Wilms tumor suppressor WT1. This translocation underlies desmoplastic small round cell tumor (DSRCT), which is noted for the abundance of reactive stroma surrounding islets of tumor cells, suggestive of paracrine signals contributing to tumor cell proliferation. Hybridization to high-density oligonucleotide microarrays can be used to identify targets of EWS-WT1. Expression of EWS-WT1 from a tetracycline-regulated promoter leads to the induction of growth-associated genes, of which the most remarkable is the beta-chain of the interleukin-2/15 receptor (IL-2/15Rbeta). Potent transcriptional activation by the chimeric protein maps to two bindings sites within the IL-2/15Rbeta promoter. Analysis of primary DSRCT tumor specimens demonstrates high levels of IL-2/15Rbeta within the tumor cells, along with expression of IL-2 and IL-15 by the abundant hyperplastic endothelial cells within the reactive stroma. Activation of this cytokine signaling pathway is consistent with the nuclear localization of its downstream effectors, phosphorylated STAT3 and STAT5. These observations suggest that the transcriptional induction of a cytokine receptor by a tumor-associated translocation product enables a proliferative response of epithelial cancer cells to ligands secreted by the surrounding stroma.

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. Mol Cancer Ther; 14(3); 649–58. ©2015 AACR.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage–responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

AMG 232 is a potent inhibitor of the MDM2-p53 interaction and is a promising clinical candidate for treating tumors, in particular those harboring wild-type p53. AML represents a compelling indication for AMG 232 given the low rate of p53 mutation, frequent MDM2 overexpression, and unmet medical need. We evaluated the effect of AMG 232 treatment on AML tumors in vitro and in vivo, elucidated the mechanism of anti-tumor efficacy, and tested the effect of combining AMG 232 with targeted agents and chemotherapeutics. Combinations were identified based on evidence of in vitro synergy from cell based screens, or based on biological rationale and clinical opportunity with standard of care agents. Combinations evaluated included MEK inhibitors, and p53-inducing, DNA-damaging cytotoxics cytarabine, doxorubicin, and decitabine. In vitro assays demonstrated that AMG 232 as a single agent was effective at inducing cell death across a panel of p53 wild-type AML cell lines. The anti-tumor efficacy involved activation of the p53 pathway, robust inhibition of the cell cycle and induction of apoptosis. AMG 232 treatment caused AML tumor regression in vivo which was related to dose- and time-dependent induction of the p53 targets p21 and PUMA. The combination of AMG 232 and MEK inhibition resulted in synergistic tumor cell killing in vitro, and enhanced in vivo anti-tumor activity which was significantly better than either single agent. Combinations of AMG 232 with chemotherapies which induce DNA damage resulted in synergistic in vitro cell killing, and superior anti-tumor efficacy in vivo with increased induction of p53 signaling in tumors. These data support a clinical strategy for evaluating AMG 232 as a monotherapy and in combination with targeted and cytotoxic agents to treat AML patients. Citation Format: Jude R. Canon, Tao Osgood, Anne Y. Saiki, Jonathan D. Oliner. The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3761.

Structure-based rational design led to the discovery of novel inhibitors of the MDM2–p53 protein–protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

Purpose To determine whether systemic treatment with AMG 386, a selective angiopoietin 1/2-neutralizing peptibody, inhibits neovascular processes in animal models of ocular disease. Methods AMG 386 was tested in a laser-induced choroidal neovascularization (CNV) model in monkeys using fluorescein angiography. The biodistribution of (125)I-AMG 386 was determined in cynomolgus monkeys by whole-body autoradiography and radioanalysis of ocular tissues. A murine retinopathy of prematurity (ROP) model was used to examine the effect of AMG 386 on established and newly formed retinal vessels, either as a single agent or when combined with VEGF inhibition.AMG 386 pharmacokinetics were evaluated in each model. Results In the CNV model, AMG 386 significantly decreased fluorescent angiographic leakage and reduced fibroplasia, indicating an impaired healing response consistent with angiogenesis blockade. Radiolabeled AMG 386 was widely distributed across ocular tissues, with highest concentrations in the choroid, cornea, retinal pigmented epithelium, iris/ciliary body, and sclera. In the ROP model, AMG 386 prevented pathologic retinal angiogenesis when administered from P8 to P16 but transiently impeded regression of these abnormal vessels when administered from P17 to P23. Combining AMG 386 with VEGF inhibition led to cooperative prevention of retinal angiogenesis in this model. No AMG 386-related ocular toxicities occurred, and no treatment-related clinical observations were made in any of the studies. Conclusions In this study, AMG 386 inhibited angiogenesis in animal models of CNV and ROP, supporting investigation of AMG 386 for the treatment of ocular neovascular diseases in the clinical setting.

Inhibition of angiopoietin-2 (Ang2) can slow tumor growth, but the underlying mechanism is not fully understood. Because Ang2 is expressed in growing blood vessels and promotes angiogenesis driven by vascular endothelial growth factor (VEGF), we asked whether the antitumor effect of Ang2 inhibition results from reduced sprouting angiogenesis and whether the effect is augmented by inhibition of VEGF from tumor cells. Using Colo205 human colon carcinomas in nude mice as a model, we found that selective inhibition of Ang2 by the peptide-Fc fusion protein L1-7(N) reduced the number of vascular sprouts by 46% and tumor growth by 62% over 26 days. Strikingly, when the Ang2 inhibitor was combined with a function-blocking anti-VEGF antibody, the number of sprouts was reduced by 82%, tumor vascularity was reduced by 67%, and tumor growth slowed by 91% compared with controls. The reduction in tumor growth was accompanied by decreased cell proliferation and increased apoptosis. We conclude that inhibition of Ang2 slows tumor growth by limiting the expansion of the tumor vasculature by sprouting angiogenesis, in a manner that is complemented by concurrent inhibition of VEGF and leads to reduced proliferation and increased apoptosis of tumor cells. Cancer Res; 70(6); 2213–23

Despite extensive data linking mutations in the p53 gene to human tumorigenesis, little is known about the cellular regulators and mediators of p53 function. MDM2 is a strong candidate for one such cellular protein; the MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells and the MDM2 protein subsequently shown to bind to p53 in rat cells transfected with p53 genes. To determine whether MDM2 plays a role in human cancer, we have cloned the human MDM2 gene. Here we show that recombinant-derived human MDM2 protein binds human p53 in vitro, and we use MDM2 clones to localize the human MDM2 gene to chromosome 12q13-14. Because this chromosomal position appears to be altered in many sarcomas, we looked for changes in human MDM2 in such cancers. The gene was amplified in over a third of 47 sarcomas, including common bone and soft tissue forms. These results are consistent with the hypothesis that MDM2 binds to p53, and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virally-induced tumours, in which viral oncogene products bind to and functionally inactivate p53.

Tumor protein 53 (p53) is a critical regulator of cell cycle and apoptosis that is frequently disabled in human tumors. In many tumor types, p53 is deleted or mutated, but in others p53 is inactivated by overexpression or amplification of its negative regulator mouse double minute 2 (MDM2). A high-throughput screening effort identified 6,7-bis(4-bromophenyl)-7,12-dihydro-6H-chromeno[4,3-d][1,2,4]triazolo[1,5-a]pyrimidine as a potent inhibitor of the MDM2-p53 protein-protein interaction. This screening hit was found to be chemically unstable and difficult to handle due to poor DMSO solubility. Co-crystallization with the target protein helped to direct further optimization and provided a tractable lead series of novel MDM2-p53 inhibitors. In cellular assays, these compounds were shown to upregulate p53 protein levels and p53 signaling and to cause p53-dependent inhibition of proliferation and apoptosis.

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have complex actions in angiogenesis and vascular remodeling due to their effects on Tie2 receptor signaling. Ang2 blocks Ang1-mediated activation of Tie2 in endothelial cells under certain conditions but is a Tie2 receptor agonist in others. We examined the effects of selective inhibitors of Ang1 (mL4-3) or Ang2 (L1-7[N]), alone or in combination, on the vasculature of human Colo205 tumors in mice. The Ang2 inhibitor decreased the overall abundance of tumor blood vessels by reducing tumor growth and keeping vascular density constant. After inhibition of Ang2, tumor vessels had many features of normal blood vessels (normalization), as evidenced by junctional accumulation of vascular endothelial-cadherin, junctional adhesion molecule-A, and platelet/endothelial cell adhesion molecule-1 in endothelial cells, increased pericyte coverage, reduced endothelial sprouting, and remodeling into smaller, more uniform vessels. The Ang1 inhibitor by itself had little noticeable effect on the tumor vasculature. However, when administered with the Ang2 inhibitor, the Ang1 inhibitor prevented tumor vessel normalization, but not the reduction in tumor vascularity produced by the Ang2 inhibitor. These findings are consistent with a model whereby inhibition of Ang2 leads to normalization of tumor blood vessels by permitting the unopposed action of Ang1, but decreases tumor vascularity primarily by blocking Ang2 actions.

Purpose AMG 386 is an investigational peptide-Fc fusion protein (ie, peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 and angiopoietin-2 with their receptor, Tie2. This first-in-human study evaluated the safety, pharmacokinetics (PK), pharmacodynamics, and antitumor activity of AMG 386 in adults with advanced solid tumors. Patients and Methods Patients in sequential cohorts received weekly intravenous AMG 386 doses of 0.3, 1, 3, 10, or 30 mg/kg. Results Thirty-two patients were enrolled on the study and received AMG 386. One occurrence of dose-limiting toxicity was seen at 30 mg/kg: respiratory arrest, which likely was caused by tumor burden that was possibly related to AMG 386. The most common toxicities were fatigue and peripheral edema. Proteinuria (n = 11) was observed without clinical sequelae. Only four patients (12%) experienced treatment-related toxicities greater than grade 1. A maximum-tolerated dose was not reached. PK was dose-linear and the mean terminal-phase elimination half-life values ranged from 3.1 to 6.3 days. Serum AMG 386 levels appeared to reach steady-state after four weekly doses, and there was minimal accumulation. No anti–AMG 386 neutralizing antibodies were detected. Reductions in volume transfer constant (Ktrans; measured by dynamic contrast-enhanced magnetic resonance imaging) were observed in 10 patients (13 lesions) 48 hours to 8 weeks after treatment. One patient with refractory ovarian cancer achieved a confirmed partial response (ie, 32.5% reduction by Response Evaluation Criteria in Solid Tumors) and withdrew from the study with a partial response after 156 weeks of treatment; four patients experienced stable disease for at least 16 weeks. Conclusion Weekly AMG 386 appeared well tolerated, and its safety profile appeared distinct from that of vascular endothelial growth factor–axis inhibitors. AMG 386 also appeared to impact tumor vascularity and showed antitumor activity in this patient population.

Interactions between Tie2, an endothelial cell-specific receptor tyrosine kinase, and its ligands the angiopoietins have been shown to play critical roles in multiple steps of angiogenesis and vascular remodeling. These include the stabilization of immature vessels, the destabilization of mature vessels, and endothelial cell migration and survival. Angiopoietin 1 has been shown to be a Tie2 agonist, stimulating autophosphorylation and the activation of downstream signaling pathways responsible for the maturation and stabilization of the developing vasculature. In contrast, angiopoietin 2 is largely thought to function as a contextdependent antagonist of Tie2 signaling, inducing vascular plasticity and sensitivity to pro-angiogenic factors such as VEGF-A. Furthermore, Ang2 exhibits broad expression in the vasculature of human tumors but limited expression in normal tissues, suggesting it could be an attractive target for safe and effective anti-angiogenic therapy. Potent and specific antibodies and peptideFc fusion proteins neutralizing the interaction between Ang2 and Tie2 have now been developed. In a variety of nonclinical studies in rodents, treatment with these agents resulted in an inhibition of the growth of human tumor xenografts and an inhibition of VEGF-stimulated corneal angiogenesis. In addition, mechanism of action studies suggest that Ang2 antagonists achieve efficacy by targeting the tumor vasculature. Data in support of the clinical utility of Ang2 inhibition in the oncology setting is currently being collected, with AMG 386, an Ang2 inhibitor, having been advanced into phase I oncology clinical trials in patients with solid tumors.

The p53 tumor suppressor is controlled by MDM2, which binds p53 and negatively regulates its transcriptional activity and stability. Many tumors overproduce MDM2 to impair p53 function. Therefore, restoration of p53 activity by inhibiting p53-MDM2 binding represents an attractive, novel approach to cancer therapy. We previously reported the discovery of AM-8553, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction (Rew et al. J. Med. Chem. 2012, 55, 4936). We report here continued optimization of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface that led to the discovery of a variety of extremely potent sulfonamides such as 14 with an IC50 of 5.3 nM in the cell proliferation assay. The compound 14 interacts specifically with the p53-binding pocket of MDM2 and releases the p53 protein from negative control. Treatment of cancer cells expressing wild-type p53 with sulfonamide 14 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. The compound 14 showed excellent efficacy and caused tumor regression in the SJSA-1 tumor xenograft model. Citation Format: Zhihong Li, Jiasheng Fu, Yosup Rew, Michael W. Gribble, Jude Canon, Ada Chen, John Eksterowicz, Xin Huang, Lixia Jin, Mei-Chu Lo, Lawrence R. McGee, Tao Osgood, Anne Y. Saiki, Paul Shaffer, Daqing Sun, Sarah Wortman, Qiuping Ye, Dongyin Yu, Xiaoning Zhao, Jing Zhou, Jonathan D. Oliner, Steve H. Olson, Julio C. Medina. Discovery of sulfonamide-piperidinones as potent inhibitors of the MDM2-p53 protein-protein interaction. [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 3663. doi:10.1158/1538-7445.AM2015-3663

Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.

We identified REDD1 as a novel transcriptional target of p53 induced following DNA damage. During embryogenesis, REDD1 expression mirrors the tissue-specific pattern of the p53 family member p63, and TP63 null embryos show virtually no expression of REDD1, which is restored in mouse embryo fibroblasts following p63 expression. In differentiating primary keratinocytes, TP63 and REDD1 expression are coordinately downregulated, and ectopic expression of either gene inhibits in vitro differentiation. REDD1 appears to function in the regulation of reactive oxygen species (ROS); we show that TP63 null fibroblasts have decreased ROS levels and reduced sensitivity to oxidative stress, which are both increased following ectopic expression of either TP63 or REDD1. Thus, REDD1 encodes a shared transcriptional target that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation.

Lymphocyte activation is known to be associated with the induction of genes implicated in cytokine signaling and cellular proliferation. High-density microarrays offer the means to monitor global cellular expression profiles, temporal relationships between classes of transcripts, and alterations associated with human disease or immunosuppression. We sought to determine whether microarray analysis would accurately reflect the normal pattern of gene expression following human T cell activation, and whether the complex expression patterns identified could be analyzed to produce a functional profile of lymphocyte activation. We examined a time course of sequential expression profiles for 6,800 cellular transcripts in human lymphocytes activated with concanavalin A. Expression patterns were grouped using clustering analysis and validated using Northern blotting. Genes known to be induced following T cell activation were accurately identified, and the qualitative patterns of gene expression were well correlated between Northern and microarray analyses. Quantitative differences in gene expression levels were less well correlated between these two techniques. Expression profile analysis revealed the sequential induction of groups of functionally similar genes, whose temporal coregulation underscores known cellular events during T cell activation. This functional "fingerprint" of lymphocyte activation may prove useful for comparisons of lymphocyte responses under experimental conditions and in disease states.

PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G(1) arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2-PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.

The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway.

The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin- and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin.

Introduction: Cancer therapies that combine agents directly targeting tumor cells with those impacting the tumor stroma hold the promise of improving patient outcomes. In this preclinical study, we examined the effects of inhibiting both EGF signaling in human tumor cells and angiopoietin signaling in the murine stroma. Using tumor xenograft models, we tested a treatment combination of panitumumab, a fully human monoclonal antibody against human EGF receptor, and trebananib, an investigational peptide-Fc fusion protein that prevents binding of angiopoietins 1 and 2 to their receptor, Tie2. We assessed the effect of this combination on tumor growth and explored mechanisms of enhanced efficacy. Methods: Athymic nude mice were injected SC in the right flank with either 1x107 A431 (human epidermoid carcinoma) or 5x106 DLD-1 (colon adenocarcinoma) cells. When tumors were ∼ 200 mm3, 20 μg panitumumab IP and/or 70 μg trebananib SC were given twice weekly. Controls were isotype antibody or human Fc. In mechanism of action studies, DLD-1 tumors (∼ 300-400 mm3) received 2 treatments over 5 days before harvest and processing for paraffin sectioning. To examine endothelial cell proliferation, treated tumors were enzyme-digested and stained with anti-CD31, anti-CD45, and anti-BrdU antibodies. The percentage of BrdU-positive tumor-associated endothelial cells (CD31high/CD45neg) was determined by flow cytometry. Results: In both tumor models, combined treatment with panitumumab and trebananib resulted in significantly greater tumor growth inhibition (p≤0.014) than treatment with either single agent alone. All treatments were well tolerated; no significant weight loss was observed. Mechanistic studies revealed that combination treatment did not alter the single-agent activity of either treatment: histological analyses showed a significant reduction in estimated blood vessel area within viable tumor after treatment with either trebananib (p=0.0006) or panitumumab (p=0.012) alone; this reduction was not affected by combination treatment. Similarly, BrdU incorporation assays demonstrated that single-agent panitumumab significantly impaired tumor cell proliferation (p=0.0003), whereas single-agent trebananib significantly reduced endothelial cell proliferation (p Conclusions: This study demonstrates that combined treatment with agents targeting EGF signaling in the tumor and angiopoietin signaling in the stroma results in significantly enhanced antitumor activity. Neither agent appears to impact the biochemical activities of the other, but instead gives rise to an orthogonal enhancement of antitumor activities. These findings support further clinical testing of agents that target the tumor stroma in combination with direct tumor-targeting therapies. Citation Format: James V. Bready, Paula Kaplan-Lefko, Jodi Moriguchi, Marc Payton, Stephen Kaufman, Jonathan Oliner, Robert Radinsky, Angela Coxon. Combined treatment of trebananib (AMG 386) with panitumumab in preclinical tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2013-5089

Normal p53 function is required for optimal arrest of cells in the G1 phase of the cell cycle following certain types of DNA damage. Loss of this cell cycle checkpoint may contribute to tumor development by increasing the number of genetic abnormalities in daughter cells following DNA damage. The MDM2 protein is an endogenous gene product that binds to the p53 protein and is able to block p53-mediated transactivation of cotransfected reporter constructs; thus, interactions between MDM2 and p53 in this checkpoint pathway following ionizing irradiation were examined. Though increases in p53 protein by DNA damage were not abrogated by MDM2 overexpression, increased levels of MDM2, resulting either from endogenous gene amplification or from transfection of an exogenous expression vector, were associated with a reduction in the ability of cells to arrest in G1 following irradiation. In addition, expression of endogenous MDM2 was enhanced by ionizing irradiation at the level of transcription in a p53-dependent fashion. These observations demonstrate that MDM2 overexpression can inhibit p53 function in a known physiologic pathway and are consistent with the hypothesis that MDM2 may function in a "feedback loop" mechanism with p53, possibly acting to limit the length or severity of the p53-mediated arrest following DNA damage.

DCC is a candidate tumor-suppressor gene encoding a protein with sequence similarity to cell adhesion molecules such as N-CAM. A set of overlapping YAC clones that contains the entire DCC coding region was isolated. Studies of this YAC contig showed that the DCC gene spans approximately 1.4 Mb. For elucidation of exon-intron structure, lambda phage clones containing all known coding sequences were isolated from a genomic library. These clones were used to demonstrate the existence of 29 DCC exons, and the sequences of the exon-intron boundaries were determined for each. Twenty-three polymorphic markers from chromosome 18 were then studied in a panel of primary colorectal tumors that had lost some, but not all, of chromosome 18. In most of these tumors, the region that was lost included DCC. Finally, Southern blot and PCR-based approaches were used to search for subtle mutations in several DCC exons. One tumor that had a point mutation in exon 28 was found, resulting in a proline to histidine substitution. A second tumor with a point mutation in intron 13 was also found. The regional map and genomic structure of DCC should provide the means to more extensively study DCC gene alterations and protein function in normal and neoplastic cells.

This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities. PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector. PCR products and vector DNA are then simply co-transfected into E. coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation. The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).

Of the many genes mutated on the road to tumor formation, few have received as much attention as p53. The gene has come to occupy center stage for the simple reason that it is more frequently altered in human tumors than any other known gene, undergoing mutation at a significant rate in almost every tumor type in which it has been studied. This association between p53 mutation and tumorigenesis has spurred a flurry of research attempting to delineate the normal function of p53 and, by extension, the role of p53 mutation in tumor formation. At the cellular level, p53 has been shown to suppress growth. Recent efforts to further discern the function of p53 have centered on the underlying molecular basis for this growth suppression. In particular, research has focused on the identification of cellular molecules (specifically DNA and proteins) with which the p53 protein associates. p53 has now been shown to bind DNA in a sequence-specific manner, and mounting evidence suggests that p53 acts as a transcription factor, perhaps regulating the expression levels of genes involved in the inhibition of cell growth. The logical next step in understanding p53 function involves the resolution of two questions: (1) what are the physiological transcriptional targets of p53, and (2) what cellular proteins regulate or mediate the ability of p53 to modulate transcription? Some initial clues to these puzzles are now emerging, and these form the subject of this review.

THE tumour-suppressor gene p53 is inactivated in most human malignancies1 either by missense mutations1 or by binding to oncogenic proteins2–4. In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification4. MDM2 is an oncogene product5,6 that may function by binding to p53 and inhibiting its ability to activate transcription3. Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain. The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery.