Small Molecule Inhibitor Of c-Met Research Articles

Abstract 3558: In vitro and in vivo profiling of class I and class II ATP-competitive c-Met kinase inhibitors defines potential c-Met-specific sensitivity biomarkers

Abstract c-Met is a receptor tyrosine kinase with oncogenic potential for which several small molecule inhibitors are currently being tested in clinical trials. There are suggestions in the literature that c-Met inhibitors could have anti-tumor effects under a variety of contexts, including in tumors in which the MET gene is amplified, contains a gain-of-function mutation or in tumors with receptor overexpression. We have developed ATP-competitive c-Met inhibitors that possess different selectivity profiles and fall into two categories. Class I molecules are highly selective for c-Met while class II molecules are multi-kinase inhibitors. These molecules were profiled in vitro and in vivo to identify biomarkers of c-Met dependence. We show that both classes of molecules inhibited c-Met kinase activity in vitro and in vivo. However, class I and class II molecules differed extensively in their spectrum of anti-tumor activity. The anti-proliferative effects of the different c-Met inhibitors were tested in 359 cancer cell lines in vitro. The activity of class I molecules was exclusively restricted to cell lines that harbor MET amplification. Western blotting in sensitive or resistant cell lines showed that c-Met inhibitors effectively block signaling downstream of c-Met only in MET amplified cell lines. In vivo, class I molecules inhibited only the growth of tumor models that are highly dependent on c-Met activity due to MET amplification or due to a hepatocyte growth factor (HGF)-driven autocrine loop. The same models that were sensitive to class I inhibitors were also sensitive to class II inhibitors. However, class II inhibitors had additional anti-tumor activities in vitro and in vivo in models that did not respond to class I inhibitors. The majority of cell lines that responded only to a class II molecule in vitro required much higher drug concentrations than those required to inhibit the growth of MET amplified cell lines. Finally, the growth inhibitory effects observed in vitro and in vivo were consistent with the selectivity profiles of the different c-Met inhibitors. Together our data demonstrate that MET amplification is a clinically identifiable, potential sensitivity biomarker for selective c-Met kinase inhibitors. c-Met expression alone is not a sensitivity biomarker in preclinical models. The spectrum of c-Met inhibitor sensitive models defined in this work could be used to understand whether other small molecule c-Met inhibitors are exquisitely selective for c-Met or harbor additional activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2011-3558

Abstract 4385: Effect on retinal function as a mechanism for vision disorders with crizotinib (PF-02341066)

Abstract Crizotinib is a small-molecule receptor tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) and c-Met. Crizotinib has been shown to be effective in patients with ALK-positive non-small cell lung cancer. One common adverse event that has been observed is a grade 1 vision disorder (∼45% of patients) characterized as seeing shadows or streaks during changes in lighting conditions. Onset occurs during the first weeks of treatment, with most cases improving with continued treatment and not requiring treatment discontinuation. Such vision disorders were not predicted from preclinical toxicological evaluation of crizotinib. To further understand the possible mechanism of this vision disorder, a rat electroretinography (ERG) study was conducted to determine if retinal function is altered following repeat-dose administration of crizotinib. Male Long-Evans rats (8/group) were administered crizotinib by oral gavage at a dose of 100 mg/kg/day for up to 29 days, during which an evaluation of in-life parameters (clinical signs, body weight, and food consumption), ophthalmic examinations, and ERG were performed. As part of the ERG assessment, five light-stimulation protocols were applied to test scotopic (dark-adapted) and photopic (light-adapted) luminance responses, photopic and scotopic adaptometry, and photopic flicker responses. Terminal plasma and vitreous fluid were examined for crizotinib concentrations in main study animals, and a toxicokinetic profile was obtained from a satellite group of 6 animals. Results of the ERG measurements identified a statistically significant (∼30%) reduction in mean b-wave amplitude relative to control during the initial 16 and 32 minutes of dark adaptation on Days 15 (p<0.05) and 29 (p<0.01), respectively. The magnitude of the reduction did not worsen between Days 15 and 29. Following 2 hours of dark adaptation, b-wave amplitudes were comparable between control and crizotinib-treated animals on both Days 15 and 29. There were no crizotinib-related effects observed for any other ERG parameters or during ophthalmologic examinations. Plasma exposures were ∼6-fold those associated with vision disorders in patients, and vitreous humor concentrations were 0.66-fold those in plasma at an equivalent time post-dose. Results from this preclinical model show that crizotinib causes significant reductions in the rate of retinal dark adaptation, but not the ability to achieve full dark adaptation. Vision disorders reported in the clinical setting may thus be due to direct effects on the retina rather than a central CNS effect. Whether this is a pharmacologically mediated effect at ALK or c-Met receptors expressed in retina or an off-target effect is not clear. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4385. doi:10.1158/1538-7445.AM2011-4385

Targeting of Both the c-Met and EGFR Pathways Results in Additive Inhibition of Lung Tumorigenesis in Transgenic Mice

EGFR and c-Met are both overexpressed in lung cancer and initiate similar downstream signaling, which may be redundant. To determine how frequently ligands that initiate signaling of both pathways are found in lung cancer, we analyzed serum for hepatocyte growth factor (HGF), transforming growth factor-alpha, and amphiregulin (AREG) in lung cancer cases and tobacco-exposed controls. HGF and AREG were both significantly elevated in cases compared to controls, suggesting that both HGF/c-Met and AREG/EGFR pathways are frequently active. When both HGF and AREG are present in vitro, downstream signaling to MAPK and Akt in non-small cell lung cancer (NSCLC) cells can only be completely inhibited by targeting both pathways. To test if dual blockade of the pathways could better suppress lung tumorigenesis in an animal model than single blockade, mice transgenic for airway expression of human HGF were treated with inhibitors of both pathways alone and in combination after exposure to a tobacco carcinogen. Mean tumor number in the group using both the HGF neutralizing antibody L2G7 and the EGFR inhibitor gefitinib was significantly lower than with single agents. A higher tumor K-ras mutation rate was observed with L2G7 alone compared to controls, suggesting that agents targeting HGF may be less effective against mutated K-ras lung tumors. This was not observed with combination treatment. A small molecule c-Met inhibitor decreased formation of both K-ras wild-type and mutant tumors and showed additive anti-tumor effects when combined with gefitinib. Dual targeting of c-Met/EGFR may have clinical benefit for lung cancer.

Efficacy of c-Met inhibitor for advanced prostate cancer

BackgroundAberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR) signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC). Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer.MethodsWe tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression.ResultsWe demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration.ConclusionsThe c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer.

Abstract 3611: LY2801653, an orally available small molecule inhibitor of c-Met, demonstrated broad antitumor efficacy in patient derived xenograft models

Abstract The receptor tyrosine kinase c-Met and its ligand hepatocyte growth factor (HGF) play important roles in cell growth, migration, and survival. Dysregulation of the c-Met/HGF signaling pathway has been found in many human malignancies. LY2801653 was identified and developed as a novel, potent, and orally active small molecule inhibitor of human c-Met. It demonstrated dose dependent inhibition of c-Met phosphorylation in xenograft tumors with a long lasting PD effect. In the current study, the anti-tumor activity of LY2801653 was evaluated in a panel of human patient derived xenograft models. LY2801653 displayed potent anti-tumor efficacy in a number of non small cell lung, renal, pancreatic, and breast tumor models. Examination of c-Met expression in these tumors by immunohistochemistry (IHC) revealed a good correlation between response and c-Met expression in the tumor tissue. Interestingly, the two non small cell lung adenocarcinoma models LXFA526 and LXFA1647 that were resistant to EGFR inhibitors Cetuximab and Erlotinib, displayed high sensitivity towards LY2801653. High level of genomic amplification of c-Met locus was found in these models, consistent with the hypothesis that c-Met activation represents one of the resistant mechanisms to EGFR inhibition. LY2801653 treatment led to increase in functional vessel areas, and decrease in tumor hypoxia. Enhanced anti-tumor efficacy was achieved when Erlotinib was combined with LY2801653. In summary, these results demonstrated the promising therapeutic potential of LY2801653 in mono and combination therapy of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3611.

Abstract 107: Genome-wide analysis reveals that cMET is a driver of RAS signaling and implicates RAS activation status as a predictor for response to cMET inhibitors

Abstract In order to facilitate the development of HGF/ c-MET inhibitors, we undertook a genome-wide analysis to identify a “c-MET activation signature” that could be used to identify tumors that are potentially dependent on HGF / c-MET signaling and therefore more responsive to HGF/ c-MET inhibition. We first developed a c-MET co-expression signature by identifying genes that correlate with the mRNA expression of c-MET across internal panels of kidney, lung, and colorectal tumors. Many genes correlated with the expression of c-MET, including the epidermal growth factor receptor (EGFR). In order to assess the biology of genes co-expressed with cMET, we performed pathway enrichment analysis to identify pathways or pre-existing signatures that were enriched in this cMET co-expression signature more than we would expect by chance. Our previously reported RAS signature (Loboda et al, 2009 AACR annual meeting) was the top pathway enrichment, indicating a link between genes involved in RAS signaling and cMET expression across tumors. To further assess this relationship, we tested the correlation between c-MET and our RAS signature across independent cohorts of breast and lung tumors, including primary and metastatic samples. A strong correlation between c-MET expression and our RAS signature was present in all cases and was conserved in metastatic tumors. We then assessed the relationship between c-MET and RAS activation using reverse-phase protein arrays measuring approximately 70 proteins across a panel of 89 lung cancer cell lines. Four proteins were identified that significantly correlated positively with the RAS signature: total ERBB4, pMEK, pERK, and pMET. The finding that pMET correlated with RAS signature in addition to pMEK and pERK supports the connection between c-MET signaling and RAS activation, as this relationship is observed on the phospho-protein as well as mRNA levels. Taken together, these data suggest that cMET can serve as a driver of RAS signaling. To test this, we performed a xenograft study in which GTL-16 xenografts (MET amplified gastric cancer cell line) were treated with a novel small molecule inhibitor of c-MET (MK-8033). Animals were treated with vehicle or with a single dose of 10, 33, 66, 100, or 200 mpk MK-8033. At each dose, we collected samples for profiling at 2, 8, and 12 hours post-dose. MK-8033 treatment caused a dose-and time-dependent inhibition of the RAS signature at all three time points, and the level of RAS signature inhibition was related to efficacy. These data support the hypothesis that cMET is a driver of RAS signaling in some tumor contexts and suggest that basal levels of RAS pathway activation may be an important determinant of response to HGF / c-MET inhibition in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 107.

Abstract 4511: LY2801653 treatment increases survival of mice bearing H441 orthotopic lung tumors

Abstract Lung cancer is the leading cause of cancer-related death for both men and women in the world. c-Met receptor tyrosine kinase (RTK) signaling pathway has been implicated in tumor growth, angiogenesis, and metastasis. Sustained activation, over-expression, or mutation of c-Met is associated with poor prognosis in several tumors, including NSCLC. Therefore, c-Met is emerging as a novel target of therapeutic potential for lung cancer. We have discovered and developed a novel, ATP competitive, and orally active small molecule inhibitor of c-Met, LY 2801653. It is a potent inhibitor of c-Met phosphorylation in vivo, and demonstrated anti-tumor activity in several xenograft tumor models. In the current study, we evaluated the efficacy of LY2801653 in both c-Met dependant and c-Met independent NSCLC cells in vitro and in vivo orthotopic lung models that recapitulated the local and regional growth pattern seen in lung cancer patients. In c-Met dependant H441 model, LY2801653 demonstrated the significant inhibition of both primary tumor (90% inhibition) and metastasis (65% inhibition of lymph node and 70% inhibition of chest wall); whereas in c-Met independent H1299 model, LY281653 only inhibited primary tumor growth (70% inhibition). The benefit of LY2801653 treatment was further demonstrated in the survival of mice bearing H441 lung tumors. Treatment with LY2801653 significantly increased the survival of mice to 192% compared to that in the vehicle treated mice. The anti-tumor and/or anti-metastasis mechanism of LY2801653 was also investigated. These results demonstrated therapeutic potential of LY2801653 in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4511.

Abstract 2430: Gastric cancer cell lines with c-Met gene amplification are sensitive to growth inhibition by the bivalent c-Met antibody LA480

Abstract Gastric cancer is common in Asia, and advanced disease is typically incurable. Development of effective treatments for gastric cancer is thus a high priority to address this highly unmet clinical need. c-Met gene amplification resulting in c-Met overexpression and pathway activation has been highly linked to tumor progression and metastasis. It has been reported that approximately 10-20% of gastric tumors have increased copy numbers of the MET gene. c-Met- amplified gastric cancer cell lines that are dependent on an activated c-Met pathway for cell growth have been reported to be highly sensitive to growth inhibition by small molecule inhibitors of c-Met. We report that LA480, a humanized monoclonal antibody specific for c-Met, can directly inhibit HGF-independent in vitro cell proliferation of three c-Met- amplified Asian gastric cell lines (MKN45, SNU-5, and NUGC-4), in a dose-dependent manner. After 48 hours of treatment with LA480, 60-70% percent inhibition of 3H-thymidine incorporation was observed. For one of these gastric lines (NUGC-4), sensitivity to LA480 increases with continuous passage of the culture. This increased sensitivity to LA480 over time appears to be associated with increased expression of cell surface c-Met and increased constitutive phospho-Met expression. In addition to inhibiting proliferation of c-Met- amplified Asian gastric lines, LA480 also directly inhibited proliferation of a c-Met- amplified gastric cell line (Hs 746T) from a Caucasian patient. These data suggest that targeting c-Met amplification with LA480 may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2430.

Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway

Abstract Background c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line. Materials and Method We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome. Results ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells. Conclusion These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1639.

Abstract 5456: Preferential targeting of N-Ras mutant and other wild type B-Raf human melanoma cells with c-Met inhibitor: a preclinical promise

Abstract Melanoma is the deadliest skin cancer known and the incidence continues to rise. The 2009 report from American Cancer Society predicts there would be 68,720 new cases of melanoma this year and deaths amounting to 8650. Melanoma detected early responds well to surgery but metastatic melanoma is highly resistant to conventional radiation and chemotherapy, and most immunotherapy, although this area is improving. Targeted therapies are most attractive as several mutations and altered growth pathways have recently been identified. Approximately 60% of melanoma are known to express mutated B-Raf, about 20% express mutated N-Ras, and the rest a variety of other mutations. The B-Raf mutated tumors are exclusive of the N-Ras, suggesting at least two biologically distinct groups. The c-Met/HGF growth promoting pathway has recently been identified as active in human cutaneous as well as uveal melanoma, with reports showing that HGF may regulate melanoma cell proliferation, migration and differentiation. However, the activation and functional activities of the HGF receptor, c-Met, has been studied only minimally in melanoma. Our studies report that as high as 90% of melanoma cell lines express c-Met and most of these secrete a detectable level of the ligand HGF. Melanoma cells with different somatic mutations, including the mutually exclusive mutated B-Raf or N-Ras genes and other cells with wild type copies of both genes were directly compared for sensitivity to c-Met inhibition using the small molecule c-Met inhibitor PHA665752 from Pfizer. We have previously reported that the N-Ras mutant melanoma cells (SB2, SK-Mel-2 cell lines) are more sensitive compared to the B-Raf mutated lines (A375, WM35, WM793). Subsequently we observed that the majority of cell lines in the non B-Raf mutated populations (including mutant N-Ras) were positive for phospho-c-Met indicating activation of c-Met pathway. After analyses of patient tumors by IHC, we report that 12/19 B-Raf wild type melanoma including N-Ras mutated, show activation of the c-Met pathway by detection of phospho-c-met in situ. Using formalin-fixed paraffin embedded primary cutaneous melanoma tumor tissues that had DNA sequencing after laser capture micro-dissection, we found that most B-Raf wild type tumors have activated c-Met. 5 out of 6 tumors with wild type B-Raf and N-Ras show positive staining for phospho-c-Met and about 83% cells are stained. 7 out of 13 tumors with wild type B-Raf and mutant N-Ras show phospho-c-Met with 54% cells staining positive, but in the group of B-Raf mutant tumors, only 1 out of 5 tumors stained positive for phospho-c-Met with staining in 20% cells. Thus most human melanoma with wild type B-Raf show c-Met activation, as indicated by c-Met phosphorylation, and could be the ideal subgroup for therapeutically targeting c-Met in melanoma. This work was supported by a Melanoma SPORE grant from the NCI (P50 CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5456.

Scatter factor protects tumor cells against apoptosis caused by TRAIL

Scatter factor (SF) and its receptor c-Met are overexpressed in various tumor types, and their expression often correlates with a poor prognosis. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a proposed tumor-specific chemotherapy agent, but its clinical usage is limited by acquisition of TRAIL resistance by tumors. The goals of this study were to determine whether and how SF protects tumor cells against TRAIL and whether SF-induced TRAIL resistance could be reversed. We used MTT assays, trypan blue dye exclusion assays, apoptosis assays, RNA interference, luciferase reporter assays, immunoprecipitation/western blotting, and other cell biological techniques to study SF protection of cultured human tumor cells against TRAIL. SF conferred resistance to TRAIL in various human prostate carcinoma and breast carcinoma cell lines. SF inhibited TRAIL-induced caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and cell death. SF protection against TRAIL required c-Akt; but unlike protection against adriamycin, it did not require Src signaling or the classical pathway of nuclear factor-kappaB activation. Protection against TRAIL was blocked by knockdown of X-linked inhibitor of apoptosis or FLICE-inhibitor protein (FLIP) (a component of the death-inducing signaling complex). We found that c-Met physically associates with several TRAIL receptors and SF regulates their protein stability. Protection against TRAIL was blocked by a novel small molecule inhibitor of c-Met (PHA665752) and by an inhibitor of cyclooxygenase 2. In conclusion, these findings elucidate potential mechanisms of TRAIL resistance in tumors that overexpress the SF/c-Met and identify possible means of reversing this resistance.

Discovery of Small Molecule c-Met Inhibitors: Evolution and Profiles of Clinical Candidates

The scatter factor/hepatocyte growth factor (HGF)-c-Met axis is involved in the malignant phenotype of various tumor types via activation of a wide range of autocrine and paracrine processes. Autocrine activation of tumor cell c-Met receptors enhances tumor cell proliferation, angiogenesis, invasion/metastasis and resistance to apoptosis and cytotoxic therapies. In addition, tumor and stroma cell-derived HGF functions as a potent angiogenic factor. Therefore, the HGF-c-Met axis is critically involved in multiple facets of normal cellular growth and homeostasis and activated in a dysregulated manner in a variety of cancers. Consequently, inhibiting the HGF-c-Met axis would be anticipated to have potent anti-tumor effects in many cancers through multiple complimentary mechanisms including increased sensitivity to current cytotoxic chemo-and radiotherapies. The acceptance of c-Met as a tractable target for cancer therapy has fostered intensive drug discovery efforts across the pharmaceutical industry. This research has led to 20 published crystal structures (with and without ligands) that revealed two distinct binding modes for ATP-competitive inhibitors: Type I ligands which assumes a U shape geometry through interactions with both hinge and activation loop residue Y1230, and Type II ligands which adopt a more extended orientation, either binding a conventional DFG-out conformation or protein conformations with varying degrees of 'DFG-out' character. Nearly a dozen small molecule c-Met inhibitors have entered human clinical trials ranging from Type I inhibitors solely selective for c-Met to Type I inhibitors with broader kinase activities to Type II inhibitors with "spectrum selective" kinase activity. The identification, profiles and properties of these clinical candidates are summarized in this review.

Abstract C190: BMS-777607, a potent small molecule c-MET inhibitor, inhibits prostate cancer progression in vitro

Abstract Overexpression of c-MET is highly associated with prostate cancer progression and therapy resistance. BMS-777607 is a selective and potent c-MET inhibitor that was advanced to clinical evaluation. Herein we report the effects of BMS-777607 in androgen-independent prostate cancer cells (PC-3, DU145). When assessed using an MTT assay, BMS-777607 treatment was found to inhibit cell growth in the absence of hepatocyte growth factor (HGF), with an IC50 >5.0 uM for both cell lines. In a wound-healing assay, BMS-777607 suppressed PC-3 and HGF-induced DU145 cell motility in a dose-dependent fashion (IC50 values of 3.0 and 0.5 uM, respectively). Moreover, BMS-777607 efficiently blocked HGF-triggered DU145 cell scattering in a cell scatter assay; with 40% inhibition at 0.2 uM. Using a Matrigel-based Transwell assay, BMS-777607 also inhibited PC-3 cell invasion in a dose-dependent manner, with 60% inhibition occurring at 1 uM. Western blot analysis demonstrated that BMS-777607 blocked c-MET autophosphorylation, and downregulated multiple downstream kinases involved in c-MET signaling pathway, including Src, Fak and Akt. These data indicate that BMS-777607 inhibited prostate cancer progression in vitro including proliferation, motility, scattering and invasion. Furthermore, the finding of more potent effects on cell migration and invasion than cell proliferation suggests that this small molecule c-MET inhibitor could have utility as an anti-metastatic agent for the treatment of advanced prostate cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C190.

The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

PurposeGlioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy.Materials and methodsEight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies.ResultsMP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time.ConclusionsGBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

Backgroundc-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated.MethodsExpression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migrationResultsHigh c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation.Conclusionc-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted

N3-Arylmalonamides: A new series of thieno[3,2-b]pyridine based inhibitors of c-Met and VEGFR2 tyrosine kinases

A family of thieno[3,2-b]pyridine based small molecule inhibitors of c-Met and VEGFR2 were designed based on lead structure 2. These compounds were shown to have IC50 values in the low nanomolar range in vitro and were efficacious in human tumor xenograft models in mice in vivo.

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c‐Met/PI3K pathway

We previously showed cell-cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.

Assessment of two dosing schedules of GSK1363089 (GSK089), a dual MET/VEGFR2 inhibitor, in metastatic gastric cancer (GC): Interim results of a multicenter phase II study

4502 Background: GSK089 is an oral, small-molecule inhibitor of cMET and VEGFR2/KDR, and has potent antitumor activity in cMET-amplified cell lines and xenografts. cMET is reportedly amplified, overexpressed and/or activated in GC, making it an attractive therapeutic target. This phase II study examines the safety and efficacy of 2 dosing schedules of GSK089 as a single agent in patients (pts) with metastatic GC. Methods: Pts with distal esophagus, GE junction or stomach adenocarcinoma, 0–2 prior chemotherapy regimens, adequate organ function, measurable disease, and ECOG PS 0–2 are sequentially enrolled in 2 cohorts: 1) Intermittent 5 days on/9 off - GSK089 240 mg/d on D1–5 repeated every 14 days, and 2) Daily - 80 mg/d. Primary study endpoint (response) is assessed every 8 weeks. cMET amplification by FISH (≥2 copies of cMET per copy of chromosome 7) is not an entry criterion, but is determined on archival tissue for all pts. Pre- and on-treatment tumor biopsies in select pts and plasma samples in all pts are analyzed for GSK089 effects on direct and downstream drug targets. GSK089 pharmacokinetics (PK) are also evaluated. Results: As of 12/19/08, enrollment to the 5 on/9 off cohort is complete with 41 evaluable pts, and 14 evaluable pts have enrolled on the daily cohort. cMET amplification was seen in 3/43 (7%) pts tested; all 3 were in 5 on/9 off cohort. Best response of stable disease (SD), was noted in 6/41 (15%) pts in the 5 on/9 off cohort (none were cMET-amplified), with 2 pts SD >6 months, 1 ongoing; and in 3/14 (21%) pts in the daily cohort. Among all-enrolled pts (48 pts in 5 on/9 off, 16 pts in daily cohort), LFT abnormalities (9%), fatigue (5%), and venous thromboembolism (3%) were the most common GSK089 related grade 3- 4 adverse events (AE). Only 1 possibly related grade 5 AE, death due to unknown cause, was noted. Conclusions: GSK089 is well tolerated on both dosing schedules. cMET amplification in metastatic GC is rare. Single agent GSK089 demonstrates minimal antitumor activity in a cMET-unselected gastric population on the 5 on/9 off schedule. Enrollment on the daily dosing schedule continues, now with mandatory pre- and on-treatment biopsies to better define cMET pathway and target inhibition with GSK089. No significant financial relationships to disclose.

QS301. A Small Molecule Inhibitor of C-MET Sensitizes Esophageal Adenocarcinoma to Chemotherapy

QS301. A Small Molecule Inhibitor of C-MET Sensitizes Esophageal Adenocarcinoma to Chemotherapy

First in human phase I study of MK-2461, a small molecule inhibitor of c-Met, for patients with advanced solid tumors

14657 Background: MK-2461 is a potent small molecule inhibitor of c-Met, a receptor tyrosine kinase involved in tumor cell proliferation and motility. Over-expression of c-Met or Met gene amplification has been associated with poorer prognosis in several tumor types. Methods: Multicenter, open-label, phase I dose escalation study in patients with advanced solid tumors refractory to standard therapy. Drug was administered by mouth daily or twice daily (BID) for 28 days followed by a 14 day rest period during cycle 1. Patients received continuous dosing subsequently for 28-day cycles. Maximum tolerated dose (MTD) was determined using a standard 3+3 dosing design. PK analyses were performed on days 1 and 28 of cycle 1. Results: Fourteen patients (10 M/ 4 F), mean age 54 (range 19–76), have received 31 cycles (range 1–6). Dose levels tested include 60mg daily, 60mg BID, 120mg BID, and 180 mg BID. Toxicity data are available for 11 patients treated at the 60mg daily-120mg BID dosing cohorts. Ten patients (91%) have not experienced > Grade 1 drug-related toxicity. Dose limiting toxicity has not been reached and no objective antitumor responses have been observed. One patient with mucinous carcinoma of the appendix had stable disease for 6 cycles. Common drug related toxicities are outlined in the table below. Four patients experienced serious adverse experiences that were considered not related to MK-2461. PK analysis revealed a rapid Tmax (1–3 hr) across all dosing cohorts with a terminal half life of 6.3 hr following the final day of dosing for the QD dosing cohort. Conclusions: Twice daily administration of MK-2461 at the doses tested is well tolerated. Terminal t1/2 suggests acceptable drug plasma concentrations expected at BID dosing. Dose escalation continues. Toxicity Grade 1 (%) Grade 2 (%) Anorexia 2(18%) 0 Fatigue 1(9%) 1(9%) Nausea 1(9%) 0 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Merck Merck Merck Exelixis, Merck