Small Molecule Inhibitor Of MET Research Articles

Targeting MET: Discovery of Small Molecule Inhibitors as Non-Small Cell Lung Cancer Therapy.

MET has been considered as a promising drug target for the treatment of MET-dependent diseases, particularly non-small cell lung cancer (NSCLC). Small molecule MET inhibitors with mainly three types of binding modes (Ia/Ib, II, and III) have been developed. In this Review, we provide an overview of the structural features, activation mechanism, and dysregulation pathway of MET and summarize progress on the development and discovery strategies utilized for MET inhibitors as well as mechanisms of acquired resistance to current approved inhibitors. The insights will accelerate discovery of new generation MET inhibitors to overcome clinical acquired resistance.

Discovery and Therapeutic Exploitation of Mechanisms of Resistance to MET Inhibitors in Glioblastoma.

Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or overactivated in GBM. Although clinically applicable MET inhibitors have been developed, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. We aimed to determine the mechanisms of MET inhibitor resistance in GBM and use the acquired information to develop novel therapeutic approaches to overcome resistance.Experimental Design: We investigated two clinically applicable MET inhibitors: crizotinib, an ATP-competitive small molecule inhibitor of MET, and onartuzumab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We developed new MET inhibitor-resistant cells lines and animal models and used reverse phase protein arrays (RPPA) and functional assays to uncover the compensatory pathways in MET inhibitor-resistant GBM. We identified critical proteins that were altered in MET inhibitor-resistant GBM including mTOR, FGFR1, EGFR, STAT3, and COX-2. Simultaneous inhibition of MET and one of these upregulated proteins led to increased cell death and inhibition of cell proliferation in resistant cells compared with either agent alone. In addition, in vivo treatment of mice bearing MET-resistant orthotopic xenografts with COX-2 or FGFR pharmacological inhibitors in combination with MET inhibitor restored sensitivity to MET inhibition and significantly inhibited tumor growth. These data uncover the molecular basis of adaptive resistance to MET inhibitors and identify new FDA-approved multidrug therapeutic combinations that can overcome resistance.

Abstract 4263: A semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model of savolitinib (AZD6094/HMPL-504), a novel MET inhibitor, to explore extent and duration of target inhibition required for optimal efficacy in the EBC-1 mouse xenograft model

Abstract MET is a transmembrane tyrosine kinase receptor that is deregulated across multiple cancer types. Savolitinib is a selective small molecule inhibitor of MET being co-developed with Hutchison MediPharma for papillary renal cell carcinoma (PRCC) and non-small cell lung cancer (NSCLC). In preclinical xenograft models, savolitinib demonstrates rapid and extensive inhibition of phosphorylated-MET (pMET) with an EC50 of 0.35 ng/ml (CI 95% - 0.25 to 0.5), and anti-tumor activity in MET amplified models (RCC-43b & RCC-47 for PRCC; MKN-45, SNU-5 & Hs746T for gastric; EBC-1 for NSCLC). To evaluate the anti-tumor activity of savolitinib under a range of schedules, the EBC-1 xenografts were treated with savolitinib at 30 mg/kg daily (76% tumor growth inhibition (TGI)), every other day (43% TGI), or 4 on/3 off (39% TGI). Intermittent dosing was also explored at 100 mg/kg every other day (80% TGI), 4 on/3 off (67% TGI) and 2 on/5 off (46% TGI). To assess the effect of prolonged pMET inhibition, twice daily dosing at 15 mg/kg (83 % TGI) and 30 mg/kg (94 % TGI) and co-dosing savolitinib at 15 mg/kg (61 % regression) with the cytochrome P450 inhibitor, 1-aminobenzotriazole (ABT) were also tested. A population PK-pMET-TGI model was developed where savolitinib plasma concentration drives pMET inhibition, which in turn drives inhibition of tumor growth. The modelling objectives were to (1) determine whether a generalizable model can be applied to EBC-1, and data from other cell-lines investigated and (2) Determine the extent and duration of pMET inhibition that delivers optimal efficacy. An Emax model of pMET inhibition driving TGI was applied and an Emax was estimated as a factor of intrinsic tumor growth rate, where a value > 1 results in a shrinking tumor. It was found that a single Emax estimate of 3.2 (CI 95% - 2.8 to 3.6) could be applied to EBC-1 and models previously investigated. The pMET inhibition needed for 50% of maximum effect was estimated to be > 90 % confirming that tumor regression is achieved through continuous high levels of pMET inhibition. Estimating drug effect relative to tumor growth rate offers a novel way in which to apply a mathematical model with parameters of drug effect that are shared across different xenograft models. The developed model offers the potential to be translated to predict the expected tumor growth inhibition in humans under varying dose regimens by accounting for mouse-to-man differences in PK and tumor growth dynamics. Citation Format: Rhys D. Jones, Michael Grondine, Alexandra Borodovsky, Maryann San Martin, Michelle DuPont, Celina D'Cruz, Alwin Schuller, Ryan Henry, Evan Barry, Klas Petersson, Tarjinder Sahota, Ghada F. Ahmed. A semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model of savolitinib (AZD6094/HMPL-504), a novel MET inhibitor, to explore extent and duration of target inhibition required for optimal efficacy in the EBC-1 mouse xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4263.

Abstract PR09: Clonal dynamics during breast cancer dormancy and recurrence

Abstract Tumor progression is the process by which cancer cells acquire increasingly aggressive characteristics, including resistance to therapies and an increased propensity to metastasize. The combination of these two processes results in recurrence of tumors in distant sites, and is chiefly responsible for cancer mortality. For instance, tumor recurrence is the leading cause of death from breast cancer. Recurrent breast cancers are thought to arise from a population of residual cells that survive treatment and persist, often in a dormant state, for years or decades before resuming proliferation. Primary breast tumors are heterogeneous, harboring different subclones of (epi)genetically distinct cells, and tumor recurrence may result from the progressive outgrowth of aggressive subclones. As a consequence, understanding the clonal dynamics of dormancy and recurrence is essential for developing strategies to prevent or treat recurrence. However, studying these processes in patients is challenging, given the difficulty in identifying residual disease and obtaining recurrent tumors. To overcome these obstacles, we have used DNA barcoding to study the clonal dynamics of breast cancer recurrence in inducible genetically engineered mouse models. These models exhibit key features of breast cancer progression as it occurs in women, including the survival of cancer cells following therapy and their eventual spontaneous recurrence. In these models doxycycline (dox) administration induces expression of an oncogene (e.g., HER2/neu, Wnt1, or Myc), leading to mammary tumor formation. Subsequent withdrawal of dox induces oncogene downregulation and complete tumor regression, mimicking targeted therapies. However, a cluster of residual cells survives oncogene downregulation and resides in a dormant, nonproliferative state in the mammary gland, and these cells eventually reinitiate proliferation to form a recurrent tumor. Using these models, we find that breast tumors can follow two distinct evolutionary routes to recurrence. Approximately half of the recurrent tumors exhibit a striking clonal dominance in which one or two subclones comprise the vast majority of the tumor. These clonal recurrent tumors exhibit evidence of de novo acquisition of Met amplification, and are sensitive to small-molecule Met inhibitors. The other half of recurrent tumors exhibit marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors are not sensitive to Met inhibitors, but have acquired dependence upon an epigenetic pathway for their growth and survival and are sensitive to drugs targeting this pathway. These results suggest that tumor recurrence can proceed via multiple independent routes, producing recurrent tumors with distinct clonal dynamics, genetic alterations, and drug sensitivities. This abstract is also being presented as Poster B45. Citation Format: Jeffrey Damrauer, James V. Alvarez. Clonal dynamics during breast cancer dormancy and recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR09.

Identification of Phytochemicals Targeting c-Met Kinase Domain using Consensus Docking and Molecular Dynamics Simulation Studies.

c-Met receptor tyrosine kinase is a proto-oncogene whose aberrant activation is attributed to a lower rate of survival in most cancers. Natural product-derived inhibitors known as “fourth generation inhibitors” constitute more than 60% of anticancer drugs. Furthermore, consensus docking approach has recently been introduced to augment docking accuracy and reduce false positives during a virtual screening. In order to obtain novel small-molecule Met inhibitors, consensus docking approach was performed using Autodock Vina and Autodock 4.2 to virtual screen Naturally Occurring Plant-based Anti-cancer Compound–Activity–Target database against active and inactive conformation of c-Met kinase domain structure. Two hit molecules that were in line with drug-likeness criteria, desired docking score, and binding pose were subjected to molecular dynamics simulations to elucidate intermolecular contacts in protein–ligand complexes. Analysis of molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area studies showed that ZINC08234189 is a plausible inhibitor for the active state of c-Met, whereas ZINC03871891 may be more effective toward active c-Met kinase domain compared to the inactive form due to higher binding energy. Our analysis showed that both the hit molecules formed hydrogen bonds with key residues of the hinge region (P1158, M1160) in the active form, which is a hallmark of kinase domain inhibitors. Considering the pivotal role of HGF/c-Met signaling in carcinogenesis, our results propose ZINC08234189 and ZINC03871891 as the therapeutic options to surmount Met-dependent cancers.

Abstract 102: Overcoming MET inhibitor resistance in GBM therapy

Abstract Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or over activated in GBM. Clinically applicable MET inhibitors have been developed and tested in the lab and in clinical trials. However, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. This study aimed to determine the mechanisms of MET inhibitor resistance in GBM and to use the acquired information to develop novel therapeutic approaches to overcome resistance. We investigated two clinically applicable MET inhibitors: PF-02341066, an ATP-competitive small molecule inhibitor of MET, and MetMab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We generated PF-02341066 and MetMab resistant GBM cell lines and primary cells by subjecting them to increasing concentrations of drug over a period of time. We utilized RNA sequencing (RNA-seq) and reverse phase protein arrays (RPPA) in addition to death and proliferation assays to identify the pathways altered in the resistant GBM cells compared to wild type GBM cells. We discovered many critical proteins that were altered in the resistant cells lines compared to wild type cells. These included FAK, COX-2, p-FGFR1, Vimentin, mTOR and p-STAT3. There was substantial but not complete overlap between the molecules that were altered in cells resistant to the small molecule as compared to molecules that were changed in cells resistant to the antibody. The protein changes in resistant GBM cells were verified by western blotting. Notably, we discovered that both COX-2 and p-FGFR1 were upregulated in GBM resistant cells and thus investigated whether inhibition of these targets could restore MET inhibitor sensitivity. Celecoxib, an FDA-approved drug, acts to inhibit COX-2 and Debio-1347 acts to inhibit the FGFR family, with a higher affinity for FGFR1. Combining Celecoxib or Debio-1347 with PF-02341066 or MetMab led to increased cell death in resistant cells compared to either agent alone. In addition, the drug combination decreased resistance to cell proliferation inhibition compared to either agent alone, indicating restored sensitivity to both MET inhibitors. We are currently undertaking in vivo experiments to establish whether the combination of celecoxib and MET inhibitors can restore sensitivity to MET inhibition in resistant cells as effectively in vivo as seen with in vitro experiments. These data indicate that MET inhibitor resistance can be overcome by targeting the resulting upregulated pathways using FDA-approved drugs and that multi-drug combinations may revert resistance during treatment. Citation Format: Nichola A. Cruickshanks, Ying Zhang, Sarah Hatef, Julia Wulfkuhle, Isela Gallagher, Alexander Koeppel, David Schiff, See Phan, Stephen Turner, Emanuel Petricoin, Roger Abounader. Overcoming MET inhibitor resistance in GBM therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 102. doi:10.1158/1538-7445.AM2017-102

Abstract 2642: Evaluation of the mechanism of MET-dependent cellular transformation and potent cytoreductive activity of MGCD265 in novel MET exon 14 mutation positive cancer models

Abstract MET splice site mutations that result in the deletion of exon 14 (METex14del) are implicated as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). MET exon 14 contains the Y1003 CBL ubiquitin ligase regulatory binding site that normally mediates CBL-dependent MET degradation and signal attenuation. Deletion of this exon results in sustained activation of MET and its downstream signaling pathways. The diverse splice site mutations leading to exon 14 skipping comprise a unique and unprecedented class of RTK activating mutations and the molecular mechanism by which these genetic alterations transform cancer cells is not fully understood. One major challenge in understanding the utility of MET inhibition of the METex14del class has been the lack of available pre-clinical models. In the present study, we generated and characterized multiple METex14del-driven cancer models to study the mechanism of MET-dependent cellular transformation as well as the response to MGCD265, a small molecule inhibitor of MET and AXL. METex14del models were identified via mining patient-derived xenograft (PDX) databases or were engineered using genome editing techniques to generate isogenic pairs of METex14del and WT cell lines. The METex14del cell lines formed increased size and number of colonies in anchorage independent growth assays compared to their WT counterparts. The transformation of METex14del cells was associated with an increase in durable HGF-dependent activation of MET and downstream signaling pathways potentially due to dysregulated MET processing and signaling attenuation. MGCD265 was shown to effectively inhibit this growth and MET-dependent signal transduction in a concentration-dependent manner. When evaluated in the amplified METex14del-driven gastric xenograft model Hs746T, significant tumor regression was observed following MGCD265 treatment. In addition, MGCD265 demonstrated substantial regression of large established tumors, in two novel NSCLC METex14del-positive PDX models. Together, these data confirm METex14del mutations are bona fide oncogenic drivers and sensitive to targeted therapeutics. Moreover, the models described in this study represent a relevant pre-clinical platform to further study receptor hyper-activation and drug action that is clinically actionable. Identification, development, and understanding of METex14del models will likely help further guide precision medicine strategies to treat NSCLC patients harboring these mutations. Citation Format: Lars D. Engstrom, Ruth W. Tang, David M. Briere, Harrah Chiang, Peter Olson, James G. Christensen. Evaluation of the mechanism of MET-dependent cellular transformation and potent cytoreductive activity of MGCD265 in novel MET exon 14 mutation positive cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2642.

Abstract 413: Validation of an antibody independent tool for patient selection: RNA in situ hybridization detects Met expression levels predictive for response to Met inhibition by Bay 853474

Abstract This study was performed in order to validate RNA in situ hybridization (RNA ISH) as tool for patient selection. Met expression was analyzed in a matched sample set of FFPE versus fresh frozen tumor samples comprising 20 cases of gastric cancer. Classical immunohistochemistry using the antibody SP44 and RNA ISH (RNAscope by ACD) were used to detect c-met expression in FFPE material. The results were confirmed by sandwich-immunoassays on Met and its phosphorylation on tyrosine 1349 (MSD) as well as mass spectrometry. The level of functional relevance was determined by testing a set of cell lines comprising some with genomic amplification of the met gene as well as some non-amplified lines showing different expression levels. Among gastric cancer cell lines only those with met amplification respond to treatment with the small molecule Met inhibitor Bay 853474. The cell line result generates a responder hypothesis that can be used to define a cutoff for clinical samples. 2 of the 20 investigated clinical samples were shown to have high level Met expression by RNA ISH and IHC that could be confirmed by sandwich-immunoassays also showing high level of functional activity by phosphotyrosine 1349. Met expression in these cases was also confirmed by mass spectrometry. Expression levels and functional activity in these 2 cases were in the range that predicts response to treatment as established with gastric cancer cell lines. Determination of predictive biomarkers by immunohistochemistry can be limited due to lack of high quality antibodies of sufficient specificity. Due to its high specificity, RNA in situ hybridization is a technique that can be used to confirm the findings obtained by immunohistochemistry and may potentially even replace immunohistochemistry it if no suitable antibodies are available or not specific enough e.g. to discriminate between closely related protein isoforms. We show the biological relevance of RNA in situ hybridization on FFPE samples by correlation with immunohistochemistry, ELISA based approaches and mass spectrometry. RNA ISH is shown to be specific and sensitive enough to identify cases of functionally relevant MET overexpression levels in gastric cancers and can be used to select patients for treatment. Citation Format: Oliver von Ahsen, Thomas Krahn, Christoph Schatz. Validation of an antibody independent tool for patient selection: RNA in situ hybridization detects Met expression levels predictive for response to Met inhibition by Bay 853474. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 413.

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.

Abstract 4427: Apoptosis, autophagy and ATP: The dynamic interaction of cellular ATP and drug resistance in MET amplified gastric cancer

Abstract Background: MET amplification occurs in 20% of gastric tumors, MET protein overexpression is present in approximately 50% of advanced gastric cancers and activating MET mutations are exceedingly rare in gastric cancer. Several ongoing trials have reported responses in gastric and esophageal patients with MET amplification .Methods: To investigate the effects, of MET inhibition on proliferation and cell death in gastric cancer I used two complementary approaches 1) siRNA mediated knockdown of MET; 2) incubation with crizotinib, a clinically approved small molecule inhibitor of MET. Both resulted in decreased proliferation and increased cell death only in the MET amplified gastric cancer cell lines. Results: The effects on growth arrest were stronger than the effects on cell death. To look for possible resistance mechanisms we performed whole genome mRNA expression profiling. Molecular changes related to autophagy were among the top alterations observed. Consistent with these findings, autophagy levels increased in a concentration dependent manner when exposed to crizotinib. In order to investigate the role of autophagy on cell death, I inhibited autophagy using two approaches 1) siRNA mediated knockdown of ATG5 and ATG7; 2) exposure to chloroquine, a clinically approved agent that inhibits autophagy by raising lysosomal pH. I then measured apoptosis levels with the autophagy inhibitor alone or in combination with MET knockdown or crizotinib. Autophagy inhibition caused a dramatic decrease in apoptosis in one of the MET amplified cell lines (MKN45) but not in the other (SNU-5). Because autophagy provides energy for cells, we explored the effects of MET or autophagy inhibition on cellular ATP levels using the Cell Titer-glo assay. The MKN45 cells had the lowest basal levels of ATP and these levels were further diminished in combination with autophagy inhibition. Conclusions: Overall, our data reveal a novel relationship between low ATP levels and resistance to MET inhibitor-induced cell death. Clinically this is significant because it may discourage the use of combination therapies with autophagy inhibitors in gastric cancer patients whose tumors contain MET amplification. Citation Format: Rebecca Dunbar Schroeder, David Hong, David J. McConkey. Apoptosis, autophagy and ATP: The dynamic interaction of cellular ATP and drug resistance in MET amplified gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4427. doi:10.1158/1538-7445.AM2015-4427

Abstract 5391: Preclinical evaluation of AMG 337, a highly selective small molecule MET inhibitor, in hepatocellular carcinoma

Abstract Purpose: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Aberrant hepatocyte growth factor / MET signaling has been implicated in hepatocarcinogenesis, suggesting that MET may serve as an attractive therapeutic target in HCC. The purpose of this study was to investigate the anti-tumor activity in vitro and in vivo of AMG 337, a potent and highly selective small molecule MET kinase inhibitor, in preclinical models of HCC. Experimental Design: The anti-proliferative activity of AMG 337 was evaluated across a panel of 40 HCC cell lines in a 72-hour viability assay. Daily oral administration of AMG 337 at doses of 3, 10 and 30 mg/kg was used to evaluate the in vivo anti-tumor activity of AMG 337 in two patient-derived mouse xenograft (PDX) models of HCC, LI0612 and LI1078. Effects on MET signaling were assessed by western blot analysis of downstream effector proteins including p-GAB1, p-AKT and p-ERK. MET protein levels and gene copy number for both PDX models and a subset of HCC cell lines were assessed by immunohistochemistry (IHC; Dako) and fluorescence in situ hybridization (FISH; Dako), respectively. High MET expression was defined as the presence of any tumor cells with membrane staining at 3+ intensity. MET amplification was defined as MET:CEN-7 ratio >2.0 or MET SNP array log2 copy number >2.5. Results: AMG 337 displayed potent anti-proliferative activity in two of 40 HCC cell lines (MHCC97H and HCCLM3) (IC50 0.015 μM and 0.025 μM, respectively); both cell lines were amplified for MET (MET/CEN-7 >2.0) and showed high MET expression (3+ IHC). AMG 337 potently inhibited p-MET in all cell lines with detectable levels of total MET. However, dose-dependent inhibition of p-GAB1, p-AKT and p-ERK was limited to those cell lines that were sensitive to AMG 337 in viability assays. AMG 337 significantly inhibited tumor growth at all doses tested (p < 0.001) in the MET-amplified and MET-high expressing HCC PDX model LI0612. However, AMG 337 had no effect on tumor growth in the non-MET-amplified and MET-low expressing HCC PDX model LI1078. Analysis of The Cancer Genome Atlas (TCGA) data identified four of 164 (2.4%) HCC tumor samples with MET amplification (https://tcga-data.nci.nih.gov/tcga), representing a patient population with potential to derive clinical benefit from AMG 337. Conclusions: These preclinical studies show that the efficacy of AMG 337 in HCC was highly associated with MET amplification. AMG 337 represents a promising, novel clinical therapeutic strategy for targeting HCC with a dependence on the MET pathway. Citation Format: Zhiqiang Du, Sean Caenepeel, Yuqing Shen, Karen Rex, Yanni Zhang, En-Tzu Tang, Ouhong Wang, Wenge Zhong, Hui Zhou, Jacqueline Huang, Eric Huang, Liaoyuan Hu, Angela Coxon, Mingqiang Zhang. Preclinical evaluation of AMG 337, a highly selective small molecule MET inhibitor, in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2015-5391

Abstract LB-241: HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells

Abstract The MET oncoprotein is a tyrosine kinase that is the only known receptor for its ligand, HGF (Hepatocyte Growth Factor). In approximately 5% of gastric cancers, MET is activated by focal amplification, rendering it constitutively active and ligand independent. MET-amplified cancer cell lines and xenografts are typically dependent on MET signaling for proliferation or survival. To test this dependency in patients, a number of small molecule MET inhibitors, including AMG337, are currently undergoing clinical evaluation for the treatment of MET-amplified tumors.Here we characterize resistance mechanisms to MET inhibitor AMG337 in vitro. We show that under standard culture conditions, resistance does not readily arise in MET-amplified gastric cell lines SNU620 and MKN45 treated with a therapeutic dose (>IC90) of AMG337. Surprisingly however, exogenous recombinant human HGF enables resistance to readily appear, with emergent cultures possessing resistance mutations in the MET activation loop at positions D1228 and Y1230. Phosphoprotein analysis shows that HGF increases autophosphorylation and activation of mutant MET sufficiently to drive cell proliferation even in the presence of AMG337.Notably, deep sequencing and clonal analysis of individually barcoded cells show that pre-existing mutant clones are likely present in cultures prior to drug treatment. However, such clones are unable to robustly proliferate in drug-containing media unless HGF is present. Based on copy number analysis, we propose that a single or small number of pre-existing mutant MET alleles in an individual cell is sufficient to drive resistance in the presence of exogenous HGF. In contrast, in HGF-negative media, a single mutant MET allele is insufficient and thus resistance does not readily arise unless gradual drug dose escalation protocols that select for increased mutant MET copy number are employed.Our results identify a key mechanism by which HGF can promote resistance to MET inhibitors in MET-amplified cancer cell lines. Because HGF is present in tumor microenvironments, combining anti-HGF antibodies with maximally-tolerated doses of small molecule MET inhibitors such as AMG337 may be beneficial in preventing resistance and in promoting durable responses. Citation Format: Marcia Gordon, Alexandra Croft, Chi-Ming Li, Sean Taylor, Sean R. Caenepeel, Kim Quon. HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-241. doi:10.1158/1538-7445.AM2015-LB-241

Targeting the MET receptor tyrosine kinase in non-small cell lung cancer: emerging role of tivantinib.

MET receptor tyrosine kinase and its natural ligand, hepatocyte growth factor, have been implicated in a variety of cancers, including non-small cell lung cancer (NSCLC). Mechanisms by which cellular deregulation of MET occurs include overexpression, genomic amplification, mutation, or alternative splicing. MET overexpression or activation is a known cause of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in NSCLC. Inhibition of MET signaling in these EGFR tyrosine kinase inhibitor-resistant cells may potentially restore sensitivity to EGFR inhibitors. Tivantinib (ARQ 197), reported as a small-molecule MET inhibitor, has demonstrated antitumor activity in early clinical studies. This review focuses on MET and lung cancer, the clinical development of tivantinib, the clinical trials of tivantinib in NSCLC to date, its current/emerging role in the management of NSCLC, and future directions.

Abstract 3114: Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC

Abstract MET is a transmembrane tyrosine kinase receptor that is deregulated (gene amplification, mutation and over-expression) across multiple cancer types. Signaling through MET is normally activated through interactions with its specific ligand, hepatocyte growth factor (HGF). Aberrant MET/HGF activation can stimulate tumor growth, promote angiogenesis, induce metastasis and may contribute to resistance mechanisms in several tumor types. Several non-selective MET inhibitors have entered clinical development; results have been mixed based on potency, selectivity, and/or patient selection. Volitinib is a potent (IC50 4 nM) and selective (>650 fold selectivity over 265 kinases), small molecule inhibitor of MET. Recent evaluation of Volitinib across a panel of cancer cell lines demonstrated selectivity for MET-driven disease, with MET amplified cell lines being most sensitive (IC50s of 1nM) and also suggesting limited off target activity. Volitinib resistant cell lines with MET amplification were identified and used to better understand the relationship of concurrent mutations with response. In addition to cancer cell line selectivity, we are analyzing preclinical models of NSCLC (non small cell lung cancer) that are representative of key patient segments, namely MET amplification and over expression. In newly diagnosed NSCLC adenocarcinomas, focal MET amplification events represent ∼3% of the population while over expression of MET (without gene amplification) is observed in the majority of patients. In preclinical models, focal Met amplification in EBC-1 and NCI-H1993 caused significant tumor growth inhibition, confirming sensitivity to Volitinib. Given the prevalence of over expression in NSCLC, however, we sought to build a platform of evidence for the therapeutic use of Volitinib in preclinical models lacking amplification of the MET gene. Using a patient-derived xenograft model (PDX) of EGFR WT, KRAS WT and metastatic NSCLC disease (HLXF-036LN), we demonstrate that Volitinib induces tumor regression as monotherapy and has added therapeutic benefit when used in combination with taxotere. In addition, we show robust efficacy effects for additional preclinical models, LG0567, LG0645 and Calu-3, for either Volitinib alone or in combination with taxotere. In parallel pharmacodynamic studies, we demonstrate that Volitinib inhibits p-MET and downstream signaling in each model. Together, using an integrated platform of molecular characterization and MET FISH and IHC scores, corresponding antitumor responses to Volitinib are being evaluated for several patient segments. We are using these studies to inform the design of patient selection criteria for upcoming clinical trials in NSCLC. Citation Format: Celina D'Cruz, Melanie Frigault, Ammar Adam, Minhui Shen, Garry Beran, Evan Barry, Paul Gavine, Yongxin Ren, Shiming Fan, Feng Zhou, Weiguo Qing, Mike Zinda, Weiguo Su, Edwin Clark. Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2014-3114

Abstract 2776: Overexpression of HGF is an effective indicator for Met-targeting therapy in hepatocellular carcinoma

Abstract The success of molecular targeted therapy against cancer depends on discovering the essential pathways that contribute to carcinogenesis and cancer progression and the molecular targets that control the pathway activity. By using genetically engineered mouse models, we show that overexpression of Hepatocyte growth factor (HGF) stimulated HBV surface antigen (HBsAg) production and accelerated the HCC progression. HGF transgenic mouse model produced spontaneous HCC with genomic profiles similar to that HBV-positive HCC patients with poor prognosis. Mechanistically, MET receptor expression is required for repair of virus-induced liver damage and HCC development. Importantly, HGF overexpression induces an autocrine activation which determines the sensitivity to small-molecule Met inhibitors. MET inhibitors are entering clinical trials against cancer, and our data provide a molecular basis for targeting the Met pathway in hepatitis B-induced HCC. Our results demonstrate that HGF is a driver of HBV-induced HCC progression and may serve as an effective biomarker for Met-targeted therapy, and that hgf-transgenic mouse model serves as a good tool for studying human HCC biology and preclinical evaluating therapeutics. Citation Format: Qian Xie. Overexpression of HGF is an effective indicator for Met-targeting therapy in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2776. doi:10.1158/1538-7445.AM2014-2776

MET and PI3K/mTOR as a potential combinatorial therapeutic target in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.

Abstract B69: Dynamic reprogramming of signaling following Met inhibition reveals pathways for cell proliferation and negative feedback in gastric cancer.

Abstract The Met receptor tyrosine kinase activates multiple signalling pathways in response to stimulation by ligand. However, the signalling events that occur under chronic Met activation in cancer cells are poorly understood. Utilizing a pharmacological small-molecule inhibitor of Met, we demonstrate that the gastric cancer cell lines, Okajima, MKN45, Snu-5 and KATO II are dependent on Met signalling for proliferation and anchorage-independent growth. We show that short-term inhibition of Met leads to waves of changes in gene expression such as a rapid loss of immediate-early genes, negative regulators, and a more delayed decrease in genes involved in the cell-cycle and proliferation. At the post-translational level, the Ras-Erk, PI3K-Akt and STAT3 pathways were all found to be dependent on Met signalling in these cells, though STAT3 signalling was the key requirement for proliferation, downstream from Met. We also observed the loss of negative regulators of Erk signalling, such as DUSP4 and DUSP6, which allowed for Mek-dependent reactivation of Erk, in the presence of Met inhibitor. As Met inhibitors are currently in clinical trials, it is essential to understand the molecular events that occur upon treatment, such that we can more effectively target sensitive tumours and avoid resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B69. Citation Format: Andrea Lai, Sean Cory, Hong Zhao, Bita Sehat, Ali Tofigh, Marie-Christine Guiot, Crista Thompson, Emily S. Bell, Nicholas Bertos, Lorenzo E. Ferri, Michael Hallett, Morag Park. Dynamic reprogramming of signaling following Met inhibition reveals pathways for cell proliferation and negative feedback in gastric cancer. [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 B69.

Abstract 4348: Antitumor activity of Tivantinib (ARQ 197) is due to inhibition of tubulin polymerization in addition to c-Met inhibition.

Abstract The receptor tyrosine kinase c-Met is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-Met axis is often dysregulated in tumors. Such c-Met activation is caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-Met inhibitors are under development as anti-cancer drugs. Tivantinib (ARQ 197) was reported as a small molecule Met inhibitor and early clinical studies suggest anti-tumor activity. To assess if the anti-tumor activity of tivantinib was due to inhibition of c-Met, we investigated the activity of tivantinib in both c-Met addicted and non-addicted cancer cells for cell viability. As expected, we observed that other Met inhibitors, crizotinib and PHA-665752, suppressed the growth of c-Met addicted cancers, but not the growth of c-Met non-addicted cells. In contrast, tivantinib inhibited cell viability similarly in both c-Met addicted and non-addicted cells. These results suggest that tivantinib exhibits its antitumor activity in a c-Met status independent manner. Tivantinib treatment increased the number of G2/M phase arrested EBC1 cells similarly as vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced cell cycle arrest at the G0/G1 phase. To identify the additional molecular target of tivantinib, we performed COMPARE analysis, in silico screening using extensive drug sensitivity database of 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib treated cells demonstrated typical microtubule disruption similar to vincristine. In vitro microtubule polymerization assay clearly demonstrated that tivantinib inhibited microtubule assembly. From these results, tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. Citation Format: Ryohei Katayama, Aki Aoyama, Takao Yamori, Jie Qi, Tomoko Ohara, Youngchul Song, Jeffrey A. Engelman, Naoya Fujita. Antitumor activity of Tivantinib (ARQ 197) is due to inhibition of tubulin polymerization in addition to c-Met inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4348. doi:10.1158/1538-7445.AM2013-4348

Abstract LB-353: Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of cabozantinib.

Abstract Introduction: Cabozantinib is an oral, small-molecule inhibitor of MET, VEGFR2, and RET, and it also inhibits AXL, KIT and TIE-2. These targets are known to play important roles in tumor cell proliferation and/or angiogenesis. Cabozantinib is FDA-approved for the treatment of patients with progressive metastatic medullary thyroid cancer. Methods: Cabozantinib was tested against the PPTP's in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 μM using the PPTP's standard 96 hour exposure period. It was tested against the PPTP solid tumor xenografts using a dose of 30 mg/kg administered by the oral route daily for 3 or 4 weeks with a total treatment/observation period of 6 weeks. Results: The median relative IC50 value for the PPTP cell lines was 8.1μM, with a range from 0.034 μM to greater than 10.0 μM and with only 3 cell lines showing IC50 values less than 1 μM. The most sensitive cell line, Kasumi-1, is an AML cell line that is known to have an activating KIT mutation. Cabozantinib was generally well tolerated in vivo, with only a 1.8% toxicity rate in treated animals. Thirty-four of 34 tested xenograft models were considered evaluable for efficacy. Cabozantinib induced significant differences in EFS distribution compared to control in 31 of 34 (91%) of the evaluable solid tumor xenografts. Cabozantinib induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C greater than 2) in 22 of 31 (71%) evaluable solid tumor xenografts. Intermediate activity for the EFS T/C metric was observed across all tumor panels evaluated. Partial responses were observed for two of the solid tumor xenografts, the rhabdoid tumor xenograft BT-29 and the slow-growing ependymoma xenograft BT-41. Tumor growth control was most pronounced for the alveolar rhabdomyosarcoma xenografts (ARMS), with 4 of 5 ARMS lines showing less than 20% increase in tumor volume during the 21 days of treatment. Two of 3 Wilms tumor lines (KT-11 and KT-13) also showed complete tumor growth control at Day 21, as did the medulloblastoma xenograft (BT-28) that was studied. Conclusions: The in vitro and in vivo pattern of activity observed for cabozantinib against the PPTP preclinical models is similar to that of agents previously studied by the PPTP that inhibit angiogenesis (e.g., sunitinib, cediranib, and sorafenib). The absence of in vitro activity for most solid tumor cell lines at concentrations associated with specific kinase inhibition together with widespread in vivo tumor growth inhibition and infrequent tumor regression suggest that for most pediatric cancers the in vivo effects of cabozantinib will primarily result from indirect effects on tumor growth (e.g., inhibition of VEGFR2-mediated angiogenesis). Exceptions would be those pediatric cancers with activating genomic alterations in kinases specifically inhibited by cabozantinib (e.g., medullary thyroid cancer with RET mutations). (Supported by NO1-CM-42216 from the NCI). Citation Format: Malcolm Smith, Min Kang, Patrick Reynolds, Richard Gorlick, Anders Kolb, John Maris, Stephen Keir, Catherine Billups, Raushan Kurmasheva, Peter Houghton. Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of cabozantinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-353. doi:10.1158/1538-7445.AM2013-LB-353

Abstract 2162: Inhibition of pancreatic cancer cell and tumor growth using MET inhibitors in combination with inhibitors of downstream signaling mediators, MEK and PI3K.

Abstract Oncogenic signaling through the receptor tyrosine kinase (RTK) MET has been implicated in driving tumor cell invasive growth. High expression of MET or its cognate ligand, the hepatocyte growth factor (HGF) have been associated with poor prognosis in multiple cancer types, including pancreatic cancer. MET has also been implicated in co-operative signaling with other RTKs, including those in the HER family. We examined the expression of MET and HGF in a panel of pancreatic cancer cell lines and examined the impact of MET activation on cell signaling. While most lines were uniformly positive for MET protein, 6/12 lines not co-expressing HGF and MET, and all sub-lines of the autocrine KP4 line, demonstrated constitutive phosphorylation of MET (p-MET) in normal growth conditions. All lines were positive for epidermal growth factor receptor (EGFR) protein and had basal levels of phosphorylated-EGFR (p-EGFR) with evidence of downstream activity in the MAPK and PI3K signaling pathways. Treatment of some lines expressing constitutive p-MET with the small molecule MET inhibitor GDC-0712 resulted in suppression of both p-MET and p-EGFR. However, treatment of these same lines with erlotinib resulted in no inhibition of p-EGFR or p-MET, strongly suggesting that MET activates EGFR phosphorylation. Co-suppression of MET with GDC-0712 or onartuzumab (MetMAb) and erlotinib resulted in greater inhibition than either agent alone. We assessed whether anti-tumor activity could be further improved by targeting downstream molecules of MET or EGFR signaling, including modulators of the Ras or PI3K pathways, in combination with MET inhibitors. Improved cell inhibition was seen with combinations of MET inhibitors and the MEK inhibitor GDC-0973 compared with either agent alone, with greater activity than combinations of MET and EGFR inhibitors. Analysis of the combination of MET inhibitors with GDC-0973 revealed that Ras signaling contributed to the stability of total MET levels, resulting in lower total p-MET burden when MEK was inhibited. The combination of MET and MEK inhibitors produced greater suppression of downstream signaling through ERK and the ribosomal S6 kinase, which resulted in better induction of the pro-apoptotic mediator Bim and induction of cleaved caspase 3. These effects were not seen with combinations of MET and PI3K inhibitors in vitro. Lastly, MET inhibitors were evaluated in combination with MEK, PI3K or EGFR inhibitors in pancreatic xenograft tumor models. The combination of MET and MEK inhibitors resulted in additive anti-tumor activity whereas there was little to no apparent increase in activity with combinations of MET and PI3K or EGFR inhibitors. These data provide evidence for an interplay between MET and mutant Ras signal transduction and provide a rationale for combination of MET and MEK inhibitors in the treatment of pancreatic cancer. Citation Format: Nai-Ying Yang, Jing Peng, Janet Tien, Mark Merchant. Inhibition of pancreatic cancer cell and tumor growth using MET inhibitors in combination with inhibitors of downstream signaling mediators, MEK and PI3K. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2162. doi:10.1158/1538-7445.AM2013-2162