Rilotumumab Resistance Acquired by Intracrine Hepatocyte Growth Factor Signaling.

Drug resistance is a long-standing impediment to effective systemic cancer therapy and acquired drug resistance is a growing problem for molecularly-targeted therapeutics that otherwise have shown unprecedented successes in disease control. The hepatocyte growth factor (HGF)/Met receptor pathway signaling is frequently involved in cancer and has been a subject of targeted drug development for nearly 30 years. To anticipate and study specific resistance mechanisms associated with targeting this pathway, we engineered resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma cells harboring autocrine HGF/Met signaling, a frequent abnormality of this brain cancer in humans. We found that rilotumumab resistance was acquired through an unusual mechanism comprising dramatic HGF overproduction and misfolding, endoplasmic reticulum (ER) stress-response signaling and redirected vesicular trafficking that effectively sequestered rilotumumab and misfolded HGF from native HGF and activated Met. Amplification of MET and HGF genes, with evidence of rapidly acquired intron-less, reverse-transcribed copies in DNA, was also observed. These changes enabled persistent Met pathway activation and improved cell survival under stress conditions. Point mutations in the HGF pathway or other complementary or downstream growth regulatory cascades that are frequently associated with targeted drug resistance in other prevalent cancer types were not observed. Although resistant cells were significantly more malignant, they retained sensitivity to Met kinase inhibition and acquired sensitivity to inhibition of ER stress signaling and cholesterol biosynthesis. Defining this mechanism reveals details of a rapidly acquired yet highly-orchestrated multisystem route of resistance to a selective molecularly-targeted agent and suggests strategies for early detection and effective intervention.

A homogeneous SIRPα-CD47 cell-based, ligand-binding assay: Utility for small molecule drug development in immuno-oncology.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.

CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

In Vitro and In Vivo Activity of AMG 337, a Potent and Selective MET Kinase Inhibitor, in MET-Dependent Cancer Models.

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model.

INTRODUCTION: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using (18)F-FDG and (18)F-FLT PET. METHODS: U-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 µg) were administered, and mice were evaluated with (18)F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 µg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using (18)F-FDG and (18)F-FLT PET. RESULTS: In the dose response study, rilotumumab at doses of 300 and 500 µg was similarly effective against tumor growth. Treatment with 300 and 500 µg rilotumumab inhibited (18)F-FDG accumulation with significant decreases of -37% and -40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 µg rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation with a maximum %ID/g of -41% and -64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed. CONCLUSIONS: Rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate (18)F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.

Molecular imaging of c-Met tyrosine kinase activity.

The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities and can lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/c-Met receptor coupling or c-Met signaling events in real time and in living cells and animals, here we describe a genetically engineered reporter where bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. c-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor-bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter's bioluminescence activity in a dose-dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102.

The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.

Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth.

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.

Biochemical characterization of AMG 102: a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor.

AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC(50) values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH(2)-terminus of the beta-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH(2)-terminus of the beta-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400-9.

Safety, pharmacokinetics, and pharmacodynamics of AMG 102, a fully human hepatocyte growth factor-neutralizing monoclonal antibody, in a first-in-human study of patients with advanced solid tumors.

PURPOSE: The aims were to assess the safety, pharmacokinetics, maximum tolerated dose, and antitumor activity of AMG 102, a fully human hepatocyte growth factor/scatter factor (HGF/SF)-neutralizing monoclonal antibody, in patients with solid tumors. EXPERIMENTAL DESIGN: Patients (N = 40) with refractory advanced solid tumors were enrolled into six sequential dose-escalation cohorts (0.5, 1, 3, 5, 10, or 20 mg/kg AMG 102 i.v. every 2 weeks) and a dose-expansion cohort (20 mg/kg AMG 102 every 2 weeks). Safety, anti-AMG 102 antibody formation, pharmacokinetics, tumor response, and exploratory biomarkers were assessed. RESULTS: AMG 102 was well tolerated up to the planned maximum dose of 20 mg/kg, and the maximum tolerated dose was not reached. Treatment-related adverse events were generally mild and included fatigue (13%), constipation (8%), nausea (8%), vomiting (5%), anorexia (5%), myalgia (5%), and hypertension (5%). Two patients experienced dose-limiting toxicities: one patient (0.5 mg/kg cohort) experienced grade 3 hypoxia and grade 3 dyspnea and one patient (1 mg/kg cohort) experienced grade 3 upper gastrointestinal hemorrhage. No anti-AMG 102 antibodies were detected, and AMG 102 had linear pharmacokinetics within the dose range investigated. Sixteen of 23 (70%) evaluable patients had a best response of stable disease with progression-free survival ranging from 7.9 to 40 weeks. Circulating levels of the biomarker HGF/SF (bound and unbound) increased in a dose-dependent manner, whereas soluble c-Met concentrations were generally similar across doses. CONCLUSIONS: AMG 102 is safe and well tolerated, has a favorable pharmacokinetic profile, and will be further investigated as a monotherapy and in combination with other agents.

Identification of the receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor, as therapeutic targets in clear cell sarcoma.

Clear cell sarcoma (CCS), a childhood tumor of the tendons and aponeuroses, is uniformly fatal once it has metastasized because of its profound therapeutic resistance. CCS is characterized by production of a chimeric transcription factor, EWS-ATF1, which is formed as the result of a disease-specific chromosomal translocation. EWS-ATF1 activates the melanocyte transcription factor MITF, which in turn activates transcription of c-Met, an oncogenic receptor tyrosine kinase recently shown to be activated in CCS. Based on this connection, we hypothesized that c-Met inhibition may offer a strategy to treat CCS, as an indirect tactic to defeat a transforming pathway downstream of EWS-ATF1. Here, we show that primary CCS and CCS-derived cell lines express c-Met, which is activated in an autocrine fashion by its ligand hepatocyte growth factor (HGF)/scatter factor in some CCS cell lines. c-Met expression is critical for CCS invasion, chemotaxis, and survival. Blocking c-Met activity with a small-molecule inhibitor (SU11274) or a neutralizing antibody to its ligand HGF (AMG 102) significantly reduced CCS cell growth in culture. Similarly, AMG 102 significantly suppressed in vivo tumor growth in an autocrine xenograft model of CCS. Collectively, these findings suggest the HGF:c-Met signaling axis as a candidate therapeutic target to improve clinical management of CCS.

Therapeutic potential of hepatocyte growth factor/scatter factor neutralizing antibodies: inhibition of tumor growth in both autocrine and paracrine hepatocyte growth factor/scatter factor:c-Met-driven models of leiomyosarcoma.

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, c-Met, have been implicated in the growth and progression of a variety of solid human tumors. Thus, inhibiting HGF/SF:c-Met signaling may provide a novel therapeutic approach for treating human tumors. We have generated and characterized fully human monoclonal antibodies that bind to and neutralize human HGF/SF. In this study, we tested the effects of the investigational, human anti-human HGF/SF monoclonal antibody, AMG 102, and a mixture of mouse anti-human HGF/SF monoclonal antibodies (Amix) on HGF/SF-mediated cell migration, proliferation, and invasion in vitro. Both agents had high HGF/SF-neutralizing activity in these cell-based assays. The HGF/SF:c-Met pathway has been implicated in the growth of sarcomas; thus, we also investigated the effect of AMG 102 on the growth of human leiomyosarcoma (SK-LMS-1) in HGF/SF transgenic C3H severe combined immunodeficient mice engineered to express high levels of human HGF/SF, as well as tumor growth of an autocrine variant of the SK-LMS-1 cell line (SK-LMS-1TO) in nude mice. The results indicate that interrupting autocrine and/or paracrine HGF/SF:c-Met signaling with AMG 102 has profound antitumor effects. These findings suggest that blocking HGF/SF:c-Met signaling may provide a potent intervention strategy to treat patients with HGF/SF:c-Met-dependent tumors.

Soluble c-Met receptors inhibit phosphorylation of c-Met and growth of hepatocyte growth factor: c-Met-dependent tumors in animal models.

c-Met is a receptor tyrosine kinase frequently overexpressed or amplified in many types of human cancers. Hepatocyte growth factor (HGF, also known as scatter factor) is the only known ligand for c-Met. In this study, soluble human and murine c-Met receptor-Fc fusion proteins were generated and were shown to bind to human and murine HGF as measured by fluorescence-activated cell sorting and surface plasmon resonance (Biacore) assays. Also, both human and murine c-Met-Fc showed activity in functional cell assays, inhibiting HGF-induced c-Met phosphorylation in PC3 and 4T1 cells, respectively, and inhibiting HGF-driven cellular invasion in a dose-dependent manner. Pharmacokinetic analysis showed that both reagents were suitable for in vivo testing. Systemic administration of human c-Met-Fc significantly inhibited tumor growth in the human HGF-dependent U-87 MG xenograft model at daily doses of 30 or 100 µg (P < 0.0001). Similarly, murine c-Met-Fc, at 100 µg daily, significantly inhibited tumor growth in the murine HGF-dependent CT-26 syngeneic tumor model (P < 0.002). Human and murine c-Met-Fc seemed to be well-tolerated in animals. In conclusion, both mouse and human versions of c-Met-Fc effectively block HGF-induced activation of c-Met and inhibit growth of tumor xenografts, providing further evidence that c-Met is an important target for oncology therapeutics.

Design, synthesis, and biological evaluation of potent c-Met inhibitors.

c-Met is a receptor tyrosine kinase that plays a key role in several cellular processes but has also been found to be overexpressed and mutated in different human cancers. Consequently, targeting this enzyme has become an area of intense research in drug discovery. Our studies began with the design and synthesis of novel pyrimidone 7, which was found to be a potent c-Met inhibitor. Subsequent SAR studies identified 22 as a more potent analog, whereas an X-ray crystal structure of 7 bound to c-Met revealed an unexpected binding conformation. This latter finding led to the development of a new series that featured compounds that were more potent both in vitro and in vivo than 22 and also exhibited different binding conformations to c-Met. Novel c-Met inhibitors have been designed, developed, and found to be potent in vitro and in vivo.

Identification of a novel recepteur d'origine nantais/c-met small-molecule kinase inhibitor with antitumor activity in vivo.

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC(50)s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor-mediated and macrophage-stimulating protein-mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONDelta160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met-dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated.

c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.

PURPOSE: Hepatocyte growth factor (HGF/SF) and its receptor c-Met have previously been shown to be up-regulated in multiple human cancers, including glioblastoma multiforme. To better understand if AMG 102, a fully human, anti-HGF/SF-neutralizing antibody, could be incorporated into current clinical practice, AMG 102 was tested preclinically in combination with temozolomide or docetaxel to determine if enhanced efficacy was observed compared with AMG 102 alone. EXPERIMENTAL DESIGN: The effects of AMG 102 were tested for antiproliferative activity in combination with temozolomide or docetaxel on U-87 MG cells in vitro and for antitumor activity in a U-87 MG xenograft model in vivo. Apoptotic activity was also measured for AMG 102 and docetaxel combined in vitro. RESULTS: Treatment with temozolomide combined with AMG 102 resulted in increased inhibition of cell growth in vitro compared with treatment with either single agent alone. In U-87 MG xenografts in vivo, AMG 102 combined with temozolomide or docetaxel significantly increased the inhibitory effect on tumor growth when compared with treatment with either agent alone (P < 0.0001 and P < 0.015, respectively). In vitro, docetaxel alone induced both caspase-3/7 activity as well as poly(ADP)ribose polymerase and caspase-7 cleavage in U-87 MG cells; these events were enhanced when used in combination with AMG 102. Importantly, there was no evidence of interference between AMG 102 and either temozolomide or docetaxel in vitro or in vivo. CONCLUSION: These studies support testing of AMG 102 in combination with temozolomide or docetaxel. Such combinations may represent promising, novel clinical therapeutic strategies for cancers that are dependent on the HGF/SF/SF:c-Met pathway in the oncology setting.

c-Met ectodomain shedding rate correlates with malignant potential.

PURPOSE: Many proteins are proteolytically released from the cell surface by a process known as ectodomain shedding. Shedding occurs under normal physiologic conditions and can be increased in certain pathologies. Among the many receptors for which ectodomain shedding has been shown is c-Met, the hepatocyte growth factor (HGF) receptor tyrosine kinase. HGF stimulates mitogenesis, motogenesis, and morphogenesis in a variety of cellular targets during development, homeostasis, and tissue regeneration. Inappropriate HGF signaling resulting in unregulated cell proliferation, motility, and invasion occurs in several human malignancies. This can occur through paracrine signaling, autocrine loop formation, receptor mutation, gene amplification, or gene rearrangement, accompanied frequently with overexpression of ligand and/or receptor proteins. We hypothesized that c-Met overexpression in cancer might result in increased ectodomain shedding, and that its measure could be a useful biomarker of tumor progression. EXPERIMENTAL DESIGN: We developed a sensitive electrochemiluminescent immunoassay to quantitate c-Met protein in cell lysates, culture supernatants, and biological samples. RESULTS: A survey of cultured cell models of oncogenic transformation revealed significant direct correlations (P < 0.001, t test or ANOVA) between malignant potential and the rate of c-Met ectodomain shedding that was independent of steady-state receptor expression level. Moreover, weekly plasma and urine samples from mice harboring s.c. human tumor xenografts (n = 4 per group) displayed soluble human c-Met levels that were measurable before tumors became palpable and that correlated directly with tumor volume (R2 > 0.92, linear regression). CONCLUSIONS: For a variety of human cancers, c-Met ectodomain shedding may provide a reliable and practical indicator of malignant potential and overall tumor burden.

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors.

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.