Inhibition Of c-Met Phosphorylation Research Articles

Design, synthesis, and biological evaluation of thiazole/thiadiazole carboxamide scaffold-based derivatives as potential c-Met kinase inhibitors for cancer treatment

As part of our continuous efforts to discover novel c-Met inhibitors as antitumor agents, four series of thiazole/thiadiazole carboxamide-derived analogues were designed, synthesised, and evaluated for the in vitro activity against c-Met and four human cancer cell lines. After five cycles of optimisation on structure–activity relationship, compound 51am was found to be the most promising inhibitor in both biochemical and cellular assays. Moreover, 51am exhibited potency against several c-Met mutants. Mechanistically, 51am not only induced cell cycle arrest and apoptosis in MKN-45 cells but also inhibited c-Met phosphorylation in the cell and cell-free systems. It also exhibited a good pharmacokinetic profile in BALB/c mice. Furthermore, the binding mode of 51am with both c-Met and VEGFR-2 provided novel insights for the discovery of selective c-Met inhibitors. Taken together, these results indicate that 51am could be an antitumor candidate meriting further development.

In Silico Target Identification of Galangin, as an Herbal Flavonoid against Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies in the bile duct, which proliferates aggressively. CCA is highly prevalent in Northeastern Thailand wherein it is associated with liver fluke infection, or Opisthorchis viverrini (OV). Most patients are diagnosed in advanced stages, when the cancer has metastasized or severely progressed, thereby limiting treatment options. Several studies investigate the effect of traditional Thai medicinal plants that may be potential therapeutic options in combating CCA. Galangin is one such herbal flavonoid that has medicinal properties and exhibits anti-tumor properties in various cancers. In this study, we investigate the role of Galangin in inhibiting cell proliferation, invasion, and migration in OV-infected CCA cell lines. We discovered that Galangin reduced cell viability and colony formation by inducing apoptosis in CCA cell lines in a dose-dependent manner. Further, Galangin also effectively inhibited invasion and migration in OV-infected CCA cells by reduction of MMP2 and MMP9 enzymatic activity. Additionally, using proteomics, we identified proteins affected post-treatment with Galangin. Enrichment analysis revealed that several kinase pathways were affected by Galangin, and the signature corroborated with that of small molecule kinase inhibitors. Hence, we identified putative targets of Galangin using an in silico approach which highlighted c-Met as candidate target. Galangin effectively inhibited c-Met phosphorylation and subsequent signaling in in vitro CCA cells. In addition, Galangin was able to inhibit HGF, a mediator of c-Met signaling, by suppressing HGF-stimulated invasion, as well as migration and MMP9 activity. This shows that Galangin can be a useful anti-metastatic therapeutic strategy in a subtype of CCA patients.

Transforming Type II to Type I c-Met kinase inhibitors via combined scaffold hopping and structure-guided synthesis of new series of 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-one derivatives

Novel 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-one derivatives 3a-e, 4a-f and 5a-f were designed as Type I c-Met kinase inhibitors based on scaffold hopping of our previous Type II c-Met kinase lead. Target compounds were then synthesized under the guidance of molecular docking analysis to identify the potential inhibitors that fit the binding pocket of c-Met kinase in the characteristic manner as the reported Type I c-Met kinase inhibitors. All synthesized derivatives were evaluated for their c-Met kinase inhibitory activity at 10 µM concentration, where 3d, 5d and 5f displayed >80% inhibition. Further IC50 investigation of these compounds identified 5d as the most potent c-Met kinase inhibitor with IC50 value of 1.95 µM. Moreover, 5d showed selective antitumor activity against c-Met over-expressing colon HCT-116 and lung A549 adenocarcinoma cells with IC50 values of 6.18 and 10.6 µg/ml, respectively. More significantly, 5d effectively inhibited c-Met phosphorylation in the Western blot experiment. Also, 5d induced cellular apoptosis in HCT-116 cancer cells as well as cell cycle arrest with accumulation of cells in G2/M phase. Finally, kinase selectivity profiling of 5d against nine oncogenic kinases revealed its selectivity to only Tyro3 kinase (% inhibition = 80%, IC50 = 3 µM). All these experimental findings clearly demonstrate that 5d is a potential dual acting inhibitor against c-Met and Tyro3 kinases, standing out as a viable lead that deserves further investigation and development to new generation of antitumor agents.

Development and biological investigations of hypoxia-sensitive prodrugs of the tyrosine kinase inhibitor crizotinib.

Despite the huge success of tyrosine kinase inhibitors as anticancer agents, severe side effects are a major problem. In order to overcome this drawback, the first hypoxia-activatable 2-nitroimidazole-based prodrugs of the clinically approved ALK and c-MET inhibitor crizotinib were developed. The 2-aminopyridine functionality of crizotinib (essential for target kinase binding) was considered as ideal position for prodrug derivatization. Consequently, two different prodrugs were synthesized with the nitroimidazole unit attached to crizotinib either via carbamoylation (A) or alkylation (B) of the 2-aminopyridine moiety. The successful prodrug design could be proven by docking studies and a dramatically reduced ALK and c-MET kinase-inhibitory potential. Furthermore, the prodrugs showed high stability in serum and release of crizotinib in an enzymatic nitroreductase-based cleavage assay was observed for prodrug A. The in vitro activity of both prodrugs was investigated against ALK- and c-MET-dependent or -overexpressing cells, revealing a distinct hypoxia-dependent activation for prodrug A. Finally, inhibition of c-MET phosphorylation and cell proliferation could also be proven in vivo. In summary of the theoretical, chemical and biological studies, prodrug derivatization of the 2-aminopyridine position can be considered as a promising strategy to reduce the side effects and improve the anticancer activity of crizotinib.

Cabozantinib and dastinib exert anti-tumor activity in alveolar soft part sarcoma.

BackgroundAlveolar soft part sarcoma (ASPS) is an extremely rare metastatic soft tissue tumor with a poor prognosis for which no effective systemic therapies have yet been established. Therefore, the development of novel effective treatment approaches is required. Tyrosine kinases (TKs) are being increasingly used as therapeutic targets in a variety of cancers. The purpose of this study was to identify novel therapeutic target TKs and to clarify the efficacy of TK inhibitors (TKIs) in the treatment of ASPS.Experimental designTo identify novel therapeutic target TKs in ASPS, we evaluated the antitumor effects and kinase activity of three TKIs (pazopanib, dasatinib, and cabozantinib) against ASPS cells using an in vitro assay. Based on these results, we then investigated the phosphorylation activities of the identified targets using western blotting, in addition to examining antitumor activity through in vivo assays of several TKIs to determine both the efficacy of these substances and accurate targets.ResultsIn cell proliferation and invasion assays using pazopanib, cabozantinib, and dasatinib, all three TKIs inhibited the cell growth in ASPS cells. Statistical analyses of the cell proliferation and invasion assays revealed that dasatinib had a significant inhibitory effect in cell proliferation assays, and cabozantinib exhibited marked inhibitory effects on cellular functions in both assays. Through western blotting, we also confirmed that cabozantinib inhibited c-MET phosphorylation and dasatinib inhibited SRC phosphorylation in dose-dependent fashion. Mice that received cabozantinib and dasatinib had significantly smaller tumor volumes than control animals, demonstrating the in vivo antitumor activity of, these substances.ConclusionsOur findings suggest that cabozantinib and dasatinib may be more effective than pazopanib against ASPS cells. These in vitro and in vivo data suggest that c-MET may be a potential therapeutic target in ASPS, and cabozantinib may be a particularly useful therapeutic option for patients with ASPS, including those with pazopanib-resistant ASPS.

Synthesis and evaluation of a series of pyridine and pyrimidine derivatives as type II c-Met inhibitors

In this study, a series of novel pyridine and pyrimidine-containing derivatives were designed, synthesized and biologically evaluated for their c-Met inhibitory activities. In the biological evaluation, half of the target compounds exhibited moderate to potent c-Met inhibitory activities. Among which, it is noteworthy that compounds 13d not only showed most potent c-Met inhibitory potency but also displayed excellent anti-proliferative activity (IC50=127nM against EBC-1 cell line) as well as an acceptable kinase selectivity profile. Moreover, the western blot assay indicated that 13d inhibited c-Met phosphorylation in EBC-1 cells in a dose-dependent manner, with complete abolishment at 0.1mM. All these experimental results suggested that 13d could be served as a promising lead compound for the development of anticancer agents.

Cabozantinib and Tivantinib, but Not INC280, Induce Antiproliferative and Antimigratory Effects in Human Neuroendocrine Tumor Cells in vitro: Evidence for ‘Off-Target' Effects Not Mediated by c-Met Inhibition

Background/Aims: The hepatocyte growth factor/transmembrane tyrosine kinase receptor c-Met has been defined as a potential target in antitumoral treatment of various carcinomas. We aimed to investigate the direct effect of c-Met inhibition on neuroendocrine tumor cells in vitro. Methods: The effects of the multi-tyrosine kinase inhibitors cabozantinib and tivantinib and of the highly specific c-Met inhibitor INC280 were investigated in human pancreatic neuroendocrine BON1, bronchopulmonary NCI-H727 and midgut GOT1 cells in vitro. Results: INC280, cabozantinib and tivantinib inhibited c-Met phosphorylation, respectively. However, while equimolar concentrations (10 μ<smlcap>M</smlcap>) of cabozantinib and tivantinib inhibited cell viability and cell migration, INC280 had no inhibitory effect. Knockdown experiments with c-Met siRNA also did not demonstrate effects on cell viability. Cabozantinib and tivantinib caused a G2 arrest in neuroendocrine tumor cells. Conclusions: Our in vitro data suggest that c-Met inhibition alone is not sufficient to exert direct antitumoral or antimigratory effects in neuroendocrine tumor cells. The multi-tyrosine kinase inhibitors cabozantinib and tivantinib show promising antitumoral and antimigratory effects in neuroendocrine tumor cells, which are most probably ‘off-target' effects, not mediated by c-Met.

Quercetin inhibits HGF/c-Met signaling and HGF-stimulated melanoma cell migration and invasion.

BackgroundMelanoma is notorious for its propensity to metastasize, which makes treatment extremely difficult. Receptor tyrosine kinase c-Met is activated in human melanoma and is involved in melanoma progression and metastasis. Hepatocyte growth factor (HGF)-mediated activation of c-Met signaling has been suggested as a therapeutic target for melanoma metastasis. Quercetin is a dietary flavonoid that exerts anti-metastatic effect in various types of cancer including melanoma. In a previous report, we demonstrated that quercetin inhibited melanoma cell migration and invasion in vitro, and prevented melanoma cell lung metastasis in vivo. In this study, we sought to determine the involvement of HGF/c-Met signaling in the anti-metastatic action of quercetin in melanoma.MethodsTranswell chamber assay was conducted to determine the cell migratory and invasive abilities. Western blotting was performed to determine the expression levels and activities of c-Met and its downstream molecules. And immunoblotting was performed in BS3 cross-linked cells to examine the homo-dimerization of c-Met. Quantitative real-time PCR analysis was carried out to evaluate the mRNA expression level of HGF. Transient transfection was used to overexpress PAK or FAK in cell models. Student’s t-test was used in analyzing differences between two groups.ResultsQuercetin dose-dependently suppressed HGF-stimulated melanoma cell migration and invasion. Further study indicated that quercetin inhibited c-Met phosphorylation, reduced c-Met homo-dimerization and decreased c-Met protein expression. The effect of quercetin on c-Met expression was associated with a reduced expression of fatty acid synthase. In addition, quercetin suppressed the phosphorylation of c-Met downstream molecules including Gab1 (GRB2-associated-binding protein 1), FAK (Focal Adhesion Kinase) and PAK (p21-activated kinases). More importantly, overexpression of FAK or PAK significantly reduced the inhibitory effect of quercetin on the migration of the melanoma cells.ConclusionsOur findings suggest that suppression of the HGF/c-Met signaling pathway contributes to the anti-metastatic action of quercetin in melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0367-4) contains supplementary material, which is available to authorized users.

Design, Synthesis, and Biological Evaluation of Novel Imidazo[1,2-a]pyridine Derivatives as Potent c-Met Inhibitors.

A series of imidazo[1,2-a]pyridine derivatives against c-Met was designed by means of bioisosteric replacement. In this study, a selective, potent c-Met inhibitor, 22e was identified, with IC50 values of 3.9 nM against c-Met kinase and 45.0 nM against c-Met-addicted EBC-1 cell proliferation, respectively. Compound 22e inhibited c-Met phosphorylation and downstream signaling across different oncogenic forms in c-Met overactivated cancer cells and model cells. Compound 22e significantly inhibited tumor growth (TGI = 75%) with good oral bioavailability (F = 29%) and no significant hERG inhibition. On the basis of systematic metabolic study, the pathway of all possible metabolites of 22e in liver microsomes of different species has been proposed, and a major NADPH-dependent metabolite 33 was generated by liver microsomes. To block the metabolic site, 42 was designed and synthesized for further evaluation. Taken together, the imidazo[1,2-a]pyridine scaffold showed promising pharmacological inhibition of c-Met and warrants further investigation.

AI-11 * c-MET-MEDIATED ENDOTHELIAL-MESENCHYMAL TRANSITION DRIVES GLIOBLASTOMA PROGRESSION AND THERAPEUTIC RESISTANCE

Excessive and abnormal vasculature characterizes the microenvironment that fuels the progression of malignant tumors including glioblastoma multiforme (GBM), the grade IV glioma. GBM is among the most lethal human malignancies, distinguished by extensive microvascular hyperplasia due to intravessel endothelial cell (EC) proliferation with unknown etiology. Here, we show that c-Met phosphorylation induces endothelial-mesenchymal transition (Endo-MT), contributing significantly to vascular abnormality, GBM progression, and therapeutic resistance. Utilizing murine orthotropic GBM models induced by transplantation of GL26 glioma cells and RCAS/tv-a-mediated somatic gene transfer of platelet-derived growth factor (PDGF) in Ink-4a/Arf−/-PTEN−/- mice, we reveal robust Endo-MT in tumor vasculature, characterized by expression of the fibroblast-specific marker FSP-1 in 60% of CD31+ or CD105+ ECs, and the co-expression level correlates with the aggressiveness of glioma in humans. Cell fate analysis by lineage tracing Tie2+ ECs shows over half of FSP-1+ fibroblasts are of EC origin in the mouse GBM models. Tumor-conditioned medium induces in vitro EC acquisition of mesenchymal gene signature including FSP-1 and N-cadherin expression, confirming the microenvironment-dependent Endo-MT. Furthermore, proteomic analysis identifies a critical role of c-Met in Endo-MT, which is required for the increased EC proliferation and migration and vessel hyperpermeability in the GBM microenvironment. EC-specific c-Met phosphorylation induces MMP-14 expression, leading to Endo-MT and vascular abnormality. Finally, pharmacological inhibition of c-Met phosphorylation normalizes blood vessels, reduces vascular density and Endo-MT, blocks tumor progression, and sensitizes tumor to temozolomide treatment; all of which improves overall survival in the GBM-bearing mice. These findings reveal a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting c-Met phosphorylation and Endo-MT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors.

Anti-tumor activity of oridonin on SNU-5 subcutaneous xenograft model via regulation of c-Met pathway

Gastric cancer is the leading cause of cancer death worldwide. Oridonin, a diterpenoid isolated from Rabdosia rubescens, has attracted considerable attention as a potential treatment for gastric cancer based on its anti-tumor effects in many tumor cell lines. However, detailed anti-tumor mechanisms of oridonin remain a matter of speculation. In the present study, a gastric carcinoma cell line harboring c-Met gene amplification SNU-5 was used to investigate the underlying mechanisms. The results showed that in vitro, oridonin potently inhibited c-Met phosphorylation and c-Met-dependent cell proliferation (IC50 value, 36.8 μM), meanwhile down-regulated the expression of the downstream signaling molecules including phospho-c-Raf, phospho-Erk, and phospho-Akt. In vivo, oridonin showed efficacy at well-tolerated doses, including marked cytoreductive anti-tumor activity in SNU-5 subcutaneous xenograft model. The anti-tumor efficacy of oridonin was dose-dependent and showed strong inhibition of c-Met phosphorylation. Additional mechanism of action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), and reduction of microvessel density (CD31). These results suggested that the anti-tumor activity of oridonin may be mediated by direct effects on tumor cell growth or survival as well as anti-angiogenic mechanisms. In summary, the results indicated that oridonin exerted anti-tumor growth on human gastric cancer SNU-5 in vitro and in vivo by direct regulation of c-Met signaling pathway and the anti-tumor effects was mainly based on its anti-proliferation and anti-angiogenesis.

Abstract A52: c-Met and β-catenin independently regulate cancer stem cell self-renewal and maintenance in hepatocellular carcinoma cells.

Abstract Background: Physiologic activation of the hepatocyte growth factor receptor, c-Met, or Wnt/β-catenin has been shown to drive a variety of cellular processes including cell proliferation, motility and tissue repair. In hepatocellular carcinoma (HCC) overexpression and/or constitutive activation of c-Met or β-catenin is correlated with increased tumorigenesis and poor patient prognosis. Moreover, interactions between the c-Met or β-catenin signaling pathways have been described. Cancer stem cells (CSCs) are thought to play a key role in malignant tumor growth, as well as tumor recurrence and metastasis. Identification of the mechanisms that regulate CSC growth and survival is, therefore, of crucial importance for the development of HCC therapeutics. In this regard, we have recently shown that ionizing radiation or chemotherapy can induce stemness in HCC cells via upregulation of the pluripotency factors Oct3/4 and Sox2. Aim: The goal of this study was to determine whether c-Met plays a role in the self-renewal and maintenance of HCC CSCs, and whether crosstalk between the c-Met and Wnt signaling pathways participates in this process.Methods: To examine the role of c-Met or β-catenin HCC cells were transfected with asymmetric interfering RNAs (aiRNAs) against c-Met or β-catenin (100nM). Cell viability was assessed by MTT assay. Levels of phospho/total c-Met and phospho/total β-catenin protein were evaluated by Western blot. Levels of the c-Met signaling proteins Akt and Erk1/2 were also investigated. CSC self-renewal was assessed by spherogenesis assay. Single cell proliferation was analyzed by colony formation assay. Results: Huh7 and HepG2 cell viability was significantly inhibited by treatment with aiRNAs against c-Met or β-catenin. Moreover, aiRNA knockdown of c-Met inhibited c-Met phosphorylation and reduced HCC sphere formation by 50% compared to untreated cells, or cells treated with a control GFP aiRNA. Similarly, treatment with β-catenin aiRNA inhibited HCC self-renewal ability by ∼60% in both cell lines. aiRNA-mediated blockade of c-Met or β-catenin also reduced the colony formation ability of both Huh7 and HepG2 cells. Silencing of c-Met, however, had little effect on β-catenin expression suggesting c-Met and β-catenin independently regulate HCC CSC self-renewal. Similar results were obtained when cells were treated with the c-Met inhibitor Tivantinib. Conclusion: Our findings demonstrate that c-Met plays a key role in the self-renewal, growth and maintenance of HCC CSCs. Moreover, this appears to be independent of β-catenin signaling. These findings suggest that combined targeting c-Met and β-catenin may be an effective strategy for HCC therapeutics development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A52. Citation Format: Laura Ghisolfi, Andrew C. Keates, Dong-ki Lee, Chiang J. Li. c-Met and β-catenin independently regulate cancer stem cell self-renewal and maintenance in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A52.

A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human cancer with poor prognosis due to late diagnosis and metastasis. Common genomic alterations in ESCC include p53 mutation, p120ctn inactivation, and overexpression of oncogenes such as cyclin D1, EGFR, and c-Met. Using esophageal epithelial cells transformed by the overexpression of EGFR and p53R175H, we find novel evidence of a functional link between p53R175H and the c-Met receptor tyrosine kinase to mediate tumor cell invasion. Increased c-Met receptor activation was observed upon p53R175H expression and enhanced further upon subsequent EGFR overexpression. We inhibited c-Met phosphorylation, resulting in diminished invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53R175H), suggesting that the mechanism of increased invasiveness upon EGFR and p53R175H expression may be the result of increased c-Met activation. These results suggest that the use of therapeutics directed at c-Met in ESCC and other squamous cell cancers.

EMD 1214063 and EMD 1204831 Constitute a New Class of Potent and Highly Selective c-Met Inhibitors

The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions. The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models. EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment. Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies.

Effects of Dual Targeting of Tumor Cells and Stroma in Human Glioblastoma Xenografts with a Tyrosine Kinase Inhibitor against c-MET and VEGFR2

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.

Abstract 1244: Differential requirements for the receptor tyrosine kinase c-Met in scirrhous gastric carcinoma cell lines

Abstract The prognosis of patients with scirrhous gastric carcinoma (SGC) has been very poor. SGC cells do not form obvious tumor mass, but cause diffuse infiltration and eventually rigid thickening of the gastric wall. Furthermore, SGC cells frequently infiltrate through gastric serosa and cause peritoneal dissemination. However, the mechanisms underlying the progression of SGC have not yet been fully elucidated. In the present study, we performed immunoblotting and proteomic analyses of tyrosine-phosphorylated proteins in differentiated non-SGC cell lines (MKN7, MKN1, and MKN74) and poorly differentiated or SGC cell lines (MKN45, NUGC-4, KATO III, HSC59, 58As9, HSC44-PE, and 44As3) to identify signaling pathways and molecules that are characteristics of SGC. We found that the tyrosine kinase receptor c-Met is overexpressed and highly phosphorylated in SGC cell lines. c-Met is a cell surface receptor for HGF and implicated in the growth, survival, invasion, and metastasis of various human cancers. Amplification of c-Met gene in SGC was also previously reported. We analyzed the downstream signaling pathways of c-Met in SGC cells by using two c-Met inhibitors PHA-665752 and JNJ-38877605. Treatment of 58As9 cells with the c-Met inhibitors markedly inhibited phosphorylation of Erk, Akt, and Stat3, but promoted phosphorylation of Src. In contrast, 44As3 cells treated with the c-Met inhibitors showed no significant changes in phosphorylation of these molecules, in spite of successful inhibition of c-Met phosphorylation. Reflecting these observations, c-Met inhibitors blocked cell proliferation and caused morphological alterations in 58As9 cells but not in 44As3 cells. Similar results were obtained when MKN45 and NUGC-4 cells were analyzed in the same manner: MKN45 cells but not NUGC-4 cells were sensitive to c-Met inhibition. These results suggest that SGC can be classified into two subtypes according to their dependencies on the c-Met signaling pathway. Further studies are now conducted to identify therapeutic targets in c-Met-independent SGC cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1244. doi:1538-7445.AM2012-1244

Sensitivity of Selected Human Tumor Models to PF-04217903, a Novel Selective c-Met Kinase Inhibitor

The c-Met pathway has been implicated in a variety of human cancers for its critical role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule inhibitor of c-Met kinase. PF-04217903 showed more than 1,000-fold selectivity for c-Met compared with more than 150 kinases, making it one of the most selective c-Met inhibitors described to date. PF-04217903 inhibited tumor cell proliferation, survival, migration/invasion in MET-amplified cell lines in vitro, and showed marked antitumor activity in tumor models harboring either MET gene amplification or a hepatocyte growth factor (HGF)/c-Met autocrine loop at well-tolerated dose levels in vivo. Antitumor efficacy of PF-04217903 was dose-dependent and showed a strong correlation with inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft models that express relatively high levels of c-Met, complete inhibition of c-Met activity by PF-04217903 only led to partial tumor growth inhibition (38%-46%) in vivo. The combination of PF-04217903 with Recepteur d'origine nantais (RON) short hairpin RNA (shRNA) knockdown in the HT29 model that also expresses activated RON kinase-induced tumor cell apoptosis and resulted in enhanced antitumor efficacy (77%) compared with either PF-04217903 (38%) or RON shRNA alone (56%). PF-04217903 also showed potent antiangiogenic properties in vitro and in vivo. Furthermore, PF-04217903 strongly induced phospho-PDGFRβ (platelet-derived growth factor receptor) levels in U87MG xenograft tumors, indicating a possible oncogene switching mechanism in tumor cell signaling as a potential resistance mechanism that might compromise tumor responses to c-Met inhibitors. Collectively, these results show the use of highly selective inhibition of c-Met and provide insight toward targeting tumors exhibiting different mechanisms of c-Met dysregulation.

Abstract 2786: Identification and preclinical characterization of EMD 1204831 – A selective c-Met kinase inhibitor in clinical phase 1

Abstract The involvement of the mesenchymal endothelial transition factor (c-Met) in the primary event of oncogenic transformation and the secondary ability to mediate metastatic spread has been convincingly demonstrated in preclinical and early clinical settings. The clinical benefits of c-Met kinase inhibitors with various modes of actions and selectivity profiles are currently under investigation hoping that inhibitors of c-Met might emerge as valuable cancer therapeutics in the future. During an HTS run 3-(diethylamino)propyl N-[3-[[5-(3,4-dimethoxyphenyl)-2-oxo-6H-1,3,4-thiadiazin-3-yl]methyl]phenyl]carbamate was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): &amp;gt;100 µg/ml, IC50 (cMet enzyme): 30 nM, IC50 (cMet A549): 800 nM) providing a valid starting point. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting with the main chain nitrogen atom of Met1160 within the hinge region and with the main chain nitrogen of Asp1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1204831 was identified as development compound. EMD1204831 is currently being investigated in a phase 1 clinical trial. The pyridazinone EMD1204831 inhibits enzymatic and cellular c-Met kinase activity with IC50 values of 12 nM and 15 nM, respectively. EMD1204831 displayed an exquisite selectivity when tested in vitro against a panel of more than 400 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1204831 demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models, including U87-MG glioblastoma cells (autocrine HGF expression), TPR-Met-transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric cancer cells (c-Met gene amplification and HGF-independent activation). Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose- and time-dependent inhibition of c-Met phosphorylation, reduction of IL-8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The overall profile of EMD1204831 including first time structural disclosure, some structure activity relationships, in vitro potency, selectivity profile and in-vivo data will be presented. 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 2786. doi:10.1158/1538-7445.AM2011-2786

Abstract 5372: Target proteins involved in resistance to c-Met inhibitors and their therapeutic role in human melanoma

Abstract c-Met is activated by binding with its ligand, hepatocyte growth factor (HGF), and is an attractive target for molecular therapeutic inhibition. c-Met is over-expressed and mutated in human melanoma and plays a role in tumorigenecity and metastasis. c-Met overexpression could be one of the factors responsible for the severe invasiveness of metastatic melanoma resulting in poor patient prognosis. We found overexpression of c-Met in 6 out of 7 melanoma cell lines studied by immunoblotting. MU, WK, RU, MC, EP and LH over-expressed c-Met, however, in AN we found minimal c-Met expression. Small molecule tyrosine kinase inhibitors (TKI's), such as SU11274, which is an ATP-competitive small molecule inhibitor, can inhibit phosphorylation, activation and tumorigenecity of c-Met. We found that SU11274 inhibited c-Met phosphorylation and cell growth in all cell lines by 85-98% at 96 hrs with an IC50 between 1 and 2.5 μM and caused apoptosis (12-58%) in all cell lines except AN. To test the therapeutic effects of SU11274 in vivo, five million RU melanoma cells were injected subcutaneously into the hind flanks of RAG (−/-) mice. Tumors were allowed to develop for a week after which daily intratumoral injections of 50μg of SU11274 or equal volumes of vehicle control were given. We found that SU11274 decreased the size of MU Melanoma tumor xenografts in RAG (−/-) mice by 6.2 fold compared to mice treated with a vehicle control. While c-Met inhibitors are currently in clinical trials, resistance to TKI's has been observed in cancer patients. To determine the mechanism of c-Met inhibitor resistance, four melanoma lines (MU, RU, WK, and EP) were exposed to increasing concentrations of SU11274 to develop drug resistance. The resistant melanoma cell lines proliferated in the presence of 7-9 fold higher concentration of SU11274. After generating cell lines resistant to SU11274, the expression levels of AKT and ERK were determined by immunoblotting to compare expression in parental vs. resistant cells. It was discovered that MU and EP drug resistant melanoma cells exhibited upregulation of p-AKT (up to 2.8 fold) and ERK (up to1.5 Fold) in the presence of SU11274. This upregulation may be through a c-Met independent pathway, possibly through the PI3K or HER-2/neu pathway, or there may be multiple pathways mediating resistance to c-Met TKI's. Conversely, RU and WK resistant lines showed down regulation of p-AKT (up to 7.3), however these cell lines showed upregulation of p-S6-kinase (up to1.3 fold) indicating that cell survival in these cell lines maybe mediated by p-S6- kinase. Additionally, WK and EP lines showed upregulation of p-4E-BP1 (up to 5.8 fold). Since both p-4E-BP1 and p-S6-Kinase are phosphorylated by mTOR, in human melanoma these results indicate that the mTOR pathway may play a key role in mediating resistance and further studies are being conducted to break SU11274 resistance in melanoma by rapamycin which targets the mTOR pathway. 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 5372. doi:10.1158/1538-7445.AM2011-5372

Abstract 3622: Preclinical characterization of EMD1214063, a potent and highly selective inhibitor of the c-Met kinase in Phase I clinical trials

Abstract c-Met, the receptor for hepatocyte growth factor (HGF), is a well characterized receptor tyrosine kinase, which is crucial for cell functions, such as survival, proliferation, motility and migration. Activating point mutations of c-Met have been identified in human malignancies, including renal carcinoma and lung cancer and constitutive activation of the HGF/c-Met pathway leads to tumor development and progression in cancer animal models. In the light of these findings, c-Met has become a key target for oncology therapeutics. In order to develop c-Met specific inhibitors, we have conducted a biochemical HTS run with the recombinant kinase domain of c-Met. This approach has enabled us to identify a new class of c-Met inhibitors. Our report will focus on pharmacological and pre-clinical data, characterising EMD1214063, a promising therapeutic candidate currently under clinical testing. Inhibition of the c-Met kinase by EMD1214063 was potent and highly selective. In in vitro kinase reactions and binding assays, EMD1214063 at a dose of 1μM, inhibited only 5 additional kinases in a panel of more than 250 different potential off-targets more than 50%. In vitro, EMD1214063 interfered with survival, anchorage-independent growth and HGF-induced migration of tumor cells, by blocking HGF-dependent as well as constitutive phosphorylation of c-Met. In vivo, oral administration of EMD1214063 resulted in a strong inhibition - ranging from delayed growth to complete regression - of HGF-dependent and -independent c-Met driven tumor xenografts. Such anti-tumor activity was observed at a broad dose range, suggesting that EMD1214063 has a wide therapeutic index. The inhibitor was well tolerated, as indicated by the lack of significant weight loss in treated mice. The mechanism of action of EMD1214063 was investigated in vivo in a series of PK/PD studies. Administration of a single dose of EMD1214063 induced a complete and long-lasting inhibition of c-Met phosphorylation in the tumor xenografts. Furthermore, it strongly reduced the plasma levels of tumor-derived IL-8. c-Met phosphorylation and plasma IL-8 levels decreased as a function of the drug concentration in the tumor and plasma. Taken together, the high c-Met selectivity, potency and in vivo efficacy of EMD1214063 render this compound a very promising candidate for the treatment of patients bearing c-Met-driven tumors. 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 3622.