Activation Of c-Met Signaling Research Articles

ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma

BackgroundSustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive.MethodsASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein–protein interactions and protein half-life, respectively.ResultsASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown.ConclusionsThis study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression.

SOX9 in prostate cancer is upregulated by cancer-associated fibroblasts to promote tumor progression through HGF/c-Met-FRA1 signaling.

Transcription factor SOX9 was a biomarker for prostate cancer (Pca) with poor prognosis. Nevertheless, the regulatory mechanism underlying SOX9 upregulation still remains unclear. Several cytokines have been reported to be involved in the regulation of SOX9, suggesting that cancer-associated fibroblasts (CAFs), one of the main sources of secreted factors in the tumor microenvironment (TME), may play a role in regulating SOX9 expression. Herein, an invitro model of paracrine interaction between primary CAFs and Pca cells was applied to investigate the molecular mechanism of SOX9 upregulation during Pca progression. The regulatory axis was validated by invivo experiments and The Cancer Genome Atlas data. Conditional medium of CAFs (CAF-CM) upregulated the expression of SOX9, which was mutually proved to be essential for CAF-induced tumor progression. Further analysis showed that hepatocyte growth factor (HGF) secreted by CAFs was responsible for SOX9 elevation in Pca cells, via the activation of c-Met signaling. Mechanistically, HGF/c-Met signaling specifically activated MEK1/2-ERK1/2 pathway, which induced phosphorylation and upregulation of FRA1, which then transcriptionally upregulated SOX9 by binding to the promoter of SOX9 gene. Moreover, we identified that HGF/c-Met-ERK1/2-FRA1-SOX9 axis was relatively conserved between human and mouse species by validating in mouse Pca cells. Our results reveal a novel insight into the molecular mechanism that SOX9 in Pca cells is promoted by CAFs through HGF/c-Met-ERK1/2-FRA1 axis. Furthermore, SOX9 may serve as an alternative marker for the activated HGF/c-Met signaling to enroll the optimal Pca patients for HGF/c-Met inhibition treatment, since it is much more stable and easier to detect.

Abstract OT-28-04: Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test

Abstract Background: While HER2 targeted therapeutics have significantly improved outcomes of patients with HER2 overexpressed advanced breast cancer (ABC), c-Met pathway activation serves as a resistance mechanism in about 60% of patients. Neratinib is an irreversible tyrosine kinase inhibitor of the ErbB family of receptors (EGFR, HER2, and HER4) and tepotinib is a tyrosine kinase inhibitor of c-Met. From live extracted cells from patient biopsies, the CELsignia Pathway Activity Test measures in real-time ErbB and c-Met signaling activity and the dynamic response to pathway inhibition by targeted therapy ex vivo. The CELsignia test may identify patients with ABC who would benefit from the combination of neratinib and tepotinib regardless of the molecular breast cancer subtype. Trial Design: We designed a phase Ib/II multi-center, non-randomized, open-label study of combination of neratinib with tepotinib in patients with advanced breast cancer including those with inflammatory breast cancer. The study sites include UT MD Anderson Cancer Center, City of Hope, and The Ohio State University Cancer Center. Four dose levels of neratinib, and two dose levels of tepotinib will be studied in phase Ib to determine the maximum tolerated dose (MTD). Phase II portion of the study will test the early efficacy of neratinib and tepotinib combination in patients with HER2-/HR+ ABC with abnormal HER2 and c-Met signaling activity determined by the CELsignia test. Eligibility: All patients with IBC/ABC will be eligible for the phase Ib. Eligible patients must have measurable metastatic disease per RECIST v1.1 or unresectable disease amenable for biopsy and have had at >1 line of systemic therapy for advanced disease. Patients will have an Eastern Cooperative Oncology Group performance status of 0 to 2. For the phase II component, patients must have hyperactive HER2 and c-Met signaling determined by CELsignia test. Aims: The primary objective of the phase Ib component is to determine the MTD of the combination in previously treated IBC/ABC of any molecular subtype. The primary objective of the phase II component is to determine the overall response rate (ORR) in patients with HER2-/HR+ ABC who have a positive CELsignia test result (described in the Eligibility). Secondary objectives include progression free and overall survival, clinical benefit rate, duration of response, and safety and tolerability. Exploratory objectives will be to assess HER2 and c-Met driven signaling pathways as either hyperactive or abnormally active by CELsignia test. Statistical Methods: The phase I study will be conducted using a Bayesian optimal interval (BOIN) design to establish the MTD and the recommended phase II dose and assess early efficacy of the combination with a maximum of 21 patients. Two dose levels of tepotinib, and four dose levels of neratinib will be studied. The phase II study will assess the ORR using Bayesian optimal phase 2 (BOP2) design to rule out a 10% ORR in favor of a 30% target ORR, with a power of 80% and two-sided 5% significance level. Accrual: Once MTD is determined, up to 29 evaluable CELsignia test positive subjects will be enrolled to the phase II component of the study and an interim analysis will be performed when the total number of enrolled patients reaches 15. We hope to complete the accrual of patients by July 2022 and to schedule the last patient visit by July 2023. Contact information: For more information or to refer a patient, email blim@mdanderson.org, eahobbs@mdanderson.org Citation Format: Evthokia Hobbs, Eric Lindquist, Brian Sullivan, Esther Yoon, Ying Yuan, Angela Marx, Jie Willey, Huiming Sun, Rachel Layman, Daniel Stover, Yuan Yuan, Bora Lim. Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-28-04.

Development of bispecific anti-c-Met/PD-1 diabodies for the treatment of solid tumors and the effect of c-Met binding affinity on efficacy

ABSTRACT Although the blockade of the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway has become a promising treatment strategy for several types of cancers, the constitutive activation of c-Met in tumors may cause a low overall response rate to PD-1 inhibitors. Increasing evidence indicates that the dual inhibition of c-Met and PD-1 could improve the efficacy of anti-PD-1/PD-L1 monoclonal antibodies for tumor immunotherapy. In this study, we developed two bispecific single-chain diabodies targeting c-Met and PD-1 for the treatment of solid tumors based on protein homology modeling, and we identified that the binding affinity of diabody-mp to c-Met was 50-folds higher than that of diabody-pm. The results of in vitro studies revealed that both diabodies suppressed HGF-induced proliferation, migration, and invasion of tumor cells, inhibiting the activation of c-Met signaling by antagonizing HGF binding to c-Met. Moreover, they promoted T cell activation by blocking the PD-1 pathway, mediating tumor cellular cytotoxicity through T cell engagement. In vivo studies with mice models demonstrated that diabody-mp exhibited higher therapeutic efficacy than other structural antibodies, greatly enhancing the survival of c-Met-positive tumor-bearing mice compared to single or combined c-Met and PD-1 blockade therapy. Furthermore, diabody-mp, which had a higher c-Met binding affinity, showed better anti-tumoral activity than diabody-pm, which had a lower c-Met binding affinity. In conclusion, bispecific anti-PD-1/c-Met diabody-mp, with high c-Met-associated affinity, inhibited tumor growth by activating T cells, suggesting its therapeutic potential for c-Met-positive solid tumors.

Abstract 3198: Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy

Abstract Background: Sub-groups of breast, lung, and colon cancer patients with genetic variants associated with dysregulated ErbB signaling benefit from treatment with ErbB targeted therapies. No sub-groups of ovarian cancer patients with actionable ErbB genetic variants have yet been found. Measurement of biological factors other than genetic variants, such as ErbB signaling activity, may identify ovarian cancer patients likely to benefit from ErbB targeted therapies. We have previously reported studies detecting dysregulated ErbB and c-Met signaling activity in 20%-25% of HER2-negative breast cancer patient tumors using the CELx Multi-Pathway (MP) Signaling Function Test. To determine whether ErbB or c-Met signaling dysregulation is involved in ovarian cancer, the CELx MP test was adapted to analyze ovarian tumor cells. The current study set out to: 1) characterize c-Met and ErbB family signaling activity in ovarian patient tumor cells and ovarian tumor cell lines; 2) evaluate in vivo response to pan-HER and c-Met inhibitors in an ovarian tumor xenograft model. Methods: For the ex vivo studies, a set of fresh tumor specimens from 15 ovarian cancer patients (90% high-grade serous, 10% endometrioid) and a set of 12 ovarian cancer cell lines was obtained. Cell samples were cultured from each patient specimen. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) with or without an antagonist (2C4, a HER2 dimerization inhibitor or tepotinib, a c-Met kinase inhibitor) was measured and quantified using an xCELLigence impedance biosensor (Agilent Technologies). Signaling activity above a previously established cut-off value was used to identify abnormal levels of EGFR, HER2 and c-Met signaling activity. For the xenograft study, OVCAR-4, an ovarian cancer cell line, determined to have abnormal HER2, EGFR, and c-Met signaling with the CELx MP Test, was studied. 40 female NSG mice were injected with two million OVCAR-4 cells. Mice were randomly assigned to either a control group that received Captisol excipient or a treatment group that received neratinib, tepotinib, or neratinib and tepotinib for 16 days. Results: Of the patient cell and cell lines samples tested ex vivo with the CELx MP test, 4 of 27 (15%; 95% CI=6%-32%) had hyperactive HER2 and c-Met signaling pathways. In the xenograft study, there was no significant difference in tumor volume between the control and tepotinib-treated or neratinib groups after 16 days of treatment. Tumor volumes relative to tumor size before treatment were reduced by 40% in the tepotinib + neratinib treated group. Conclusions: These findings suggest that a significant sub-group of ovarian cancer patients have abnormal ErbB and c-Met signaling activity that may respond to treatment with a combination of ErbB and c-Met inhibitors. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Stefano Rossetti, Catherine Kuzmicki, Aaron Broege, Joanna Sabat, Salmaan Khan, Ian MacNeil, Brian Sullivan, Lance Laing. Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3198.

Test identifies ovarian cancer patients with hyperactive c-Met and ErbB signaling tumors who may benefit from c-Met and pan-HER combination therapy.

e18038 Background: No sub-groups of ovarian cancer patients with clinically actionable ErbB genetic variants have yet been found. Measurement of ErbB signaling activity rather than genetic variants may identify ovarian cancer patients likely to benefit from ErbB targeted therapies. We have previously reported studies detecting dysregulated ErbB and c-Met signaling activity in 20%-25% of HER2-negative breast cancer patient tumors using the CELx Signaling Function Test. To determine whether ErbB or c-Met signaling dysregulation is involved in ovarian cancer, the CELx Test was adapted to analyze ovarian tumor cells. The current study set out to: 1) characterize c-Met and ErbB family signaling activity in ovarian patient tumor cells and tumor cell lines; 2) evaluate in vivo response to pan-HER and c-Met inhibitors in an ovarian tumor xenograft model. Methods: For the ex vivo studies, a set of fresh tumor specimens from 15 ovarian cancer patients and a set of 12 ovarian cancer cell lines was obtained. Cell samples were cultured from each. Live cell response to ErbB and c-Met agonists (NRG1b, EGF, or HGF) with or without an antagonist (2C4, a HER2 dimerization inhibitor or tepotinib, a c-Met kinase inhibitor) was measured using an xCELLigence impedance biosensor (Agilent Technologies). Signaling activity above a previously established cut-off value was used to identify abnormal levels of EGFR, HER2 and c-Met signaling activity. For the xenograft study, OVCAR-4, an ovarian cancer cell line, determined to have abnormal HER2, EGFR, and c-Met signaling with the CELx Test, was studied. 40 female NSG mice were each injected with two million cells. Mice were randomly assigned to either a control group or a treatment group that received neratinib, tepotinib, or neratinib and tepotinib for 16 days. Results: Of the patient cell and cell lines samples tested ex vivo with the CELx Test, 4 of 27 (15%; 95% CI = 6%-32%) had hyperactive HER2 and c-Met signaling pathways. In the xenograft study, tumor volume change was 40% less in the tepotinib + neratinib treated group than in the control arm change. There was no significant difference in tumor volume between the control and tepotinib or neratinib groups. Conclusions: These findings suggest that a significant sub-group of ovarian cancer patients have abnormal ErbB and c-Met signaling activity that may respond to treatment with a combination of ErbB and c-Met inhibitors. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted.

A novel non-agonist c-Met antibody drug conjugate with superior potency over a c-Met tyrosine kinase inhibitor in c-Met amplified and non-amplified cancers

ABSTRACTc-Met is a well-characterized oncogene that is associated with poor prognosis in many solid tumor types. While responses to c-Met inhibitors have been observed in clinical trials, activity appears to be limited to those with MET gene amplifications or mutations. We developed a c-Met targeted antibody-drug conjugate (ADC) with preclinical activity in the absence of MET gene amplification or mutation, and activity even in the context of moderate protein expression. The ADC utilized a high-affinity c-Met antibody (P3D12), that induced c-Met degradation with minimal activation of c-Met signaling, or mitogenic effect. P3D12 was conjugated to the tubulin inhibitor toxin MMAF via a cleavable linker (vc-MMAF). P3D12-vc-MMAF demonstrated potent in vitro activity in c-Met protein-expressing cell lines regardless of MET gene amplification or mutation status, and retained activity in cell lines with medium-low c-Met protein expression. In contrast, the c-Met tyrosine kinase inhibitor (TKI) PHA-665752 slowed tumor cell growth in vitro only in the context of MET gene amplification or very high protein expression. This differential activity was even more marked in vivo. P3D12-vc-MMAF demonstrated robust inhibition of tumor growth in the MET gene amplified MKN-45 xenograft model, and similar results in H1975, which expresses moderate levels of wild type c-Met without genomic amplification. By comparison, the c-Met TKI, PHA-665752, demonstrated modest tumor growth inhibition in MKN-45, and no inhibition at all in H1975. Taken together, these data suggest that P3D12-vc-MMAF may have a superior clinical profile in treating c-Met positive malignancies in contrast to c-Met pathway inhibitors.

Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury.

Kidney fibroblasts play a crucial role in dictating tubular cell fate and the outcome of acute kidney injury (AKI). The underlying mechanisms remain to be determined. Here, we found that mTOR signaling was activated in fibroblasts from mouse kidneys with ischemia/reperfusion injury (IRI). Ablation of fibroblast Rheb or Rictor promoted, while ablation of fibroblast Tsc1 protected against tubular cell death and IRI in mice. In tubular cells cultured with conditioned media (CM) from Rheb-/- or Rictor-/- fibroblasts, less hepatocyte growth factor (HGF) receptor c-met signaling activation or staurosporine-induced cell apoptosis was observed. While CM from Tsc1-/- fibroblasts promoted tubular cell c-met signaling activation and inhibited staurosporine-induced cell apoptosis. In kidney fibroblasts, blocking mTOR signaling downregulated the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and HGF. Downregulating fibroblast HGF expression or blocking tubular cell c-met signaling facilitated tubular cell apoptosis. Notably, renal PPARγ and HGF expression was less in mice with fibroblast Rheb or Rictor ablation, but more in mice with fibroblast Tsc1 ablation than their littermate controls, respectively. Together, these data suggest that mTOR signaling activation in kidney fibroblasts protects against tubular cell death and dictates the outcome of AKI through stimulating PPARγ and HGF expression.

A selective c-Met and Trks inhibitor Indo5 suppresses hepatocellular carcinoma growth

BackgroundHuman hepatocellular carcinoma (HCC) lacks effective curative therapy and there is an urgent need to develop a novel molecular-targeted therapy for HCC. Selective tyrosine kinase inhibitors have shown promise in treating cancers including HCC. Tyrosine kinases c-Met and Trks are potential therapeutic targets of HCC and strategies to interrupt c-Met and Trks cross-signaling may result in increased effects on HCC inhibition.MethodsThe effects of Indo5 on c-Met and Trks activity were determined with in vitro kinase activity assay, cell-based signaling pathway activation, and kinases-driven cell transformation. The in vivo anti-tumor activity was determined with xenograft mice and liver orthotopic mice models. The co-expression of c-Met and TrkB in 180 pairs of HCC and adjacent normal tissues were detected using immunohistochemical staining.ResultsIndo5, a novel lead compound displayed biochemical potency against both c-Met and Trks with selectivity over 13 human kinases. Indo5 abrogated HGF-induced c-Met signaling activation and BDNF/NGF-induced Trks signal activation, c-Met or TrkB-mediated cell transformation and migration. Furthermore, Indo5 significantly decreased the growth of HCC cells in xenograft mice and improved the survival of mice with liver orthotopic tumors. In addition, co-expression of c-Met and TrkB in HCC patients was a predictor of poor prognosis, and combined inhibition of c-Met and TrkB exerted a synergistic suppressive effect on HCC.ConclusionsThese findings indicate that Indo5 is associated with marked suppression of c-Met and Trks co-expressing HCC, supporting its clinical development as an antitumor treatment for HCC patients with co-active c-Met and Trks signaling.

Abstract P3-10-20: Sub-group of HER2- breast cancer patients with hyperactive and co-involved c-Met and ErbB pathways identified: Functional signal profiling test identifies patient group that may benefit from c-Met and pan-HER combination therapy

Abstract Background: Biological factors other than c-Met status, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. A new assay using an impedance biosensor was developed to measure c-Met and ErbB signaling activity of live tumor cells. The CELx Multi-Pathway Signaling Function (CELx MP) Test measures an individual patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists and antagonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, and c-MET signaling. This study set out to: 1) determine the prevalence of hyperactive c-Met and ErbB family signaling amongst HER2- breast cancer patients; and 2) characterize potential cross-talk between c-Met and ErbB pathways. Methods: For the prevalence study, fresh breast tumor specimens were obtained from 74 HER2- breast cancer patients. The amount of HER1, HER2, HER3, and c-Met for each specimen was determined using FACS. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without ErbB and c-Met antagonists (2C4, a HER2 mAb dimerization inhibitor, tepotinib, a c-Met TKI, or neratinib, a pan-HER TKI) was measured using an xCELLigence RTCA impedance biosensor. From these responses, HER1, HER3, and c-Met signaling initiated by their respective agonists was quantified. The net amount of HER2 participation in EGF and NRG signaling was also quantified. Signaling activity above a previously determined cutpoint was used to identify abnormal levels of HER1, HER2, HER3 and c-Met signaling activity. For the cross-talk study, three primary HER2- breast cancer specimens with hyperactive c-Met and ErbB signaling were obtained. Response to HGF, EGF, and NRG1 alone, with or without tepotinib, was measured for these specimens using an impedance biosensor. Results: The FACS analysis found all 74 tumor samples had normally expressed amounts of HER1, HER2, HER3, and c-Met. Of these samples, 20 of 74 (27.0%; 95% CI=18%-38%) had hyperactive c-Met signaling coincident with hyperactive signaling from at least one ErbB pathway. In each patient sample, neratinib combined with tepotinib inhibited virtually all signaling activity initiated with a combination EGF, NRG1 and HGF. In the cross-talk analysis of the three tumor samples, signaling response to EGF or NRG1 when combined with a c-Met antagonist was 9%-98% higher than signaling response measured with EGF or NRG1 alone. Conclusions: This test found a significant sub-set of HER2- breast cancer patients with coincidental hyperactive c-Met and ErbB signaling tumors that respond ex vivo to a combination of pan-HER and c-Met TKI's. The unexpected increase in EGF and NRG1 signaling in the presence of a c-Met antagonist provides strong evidence that c-Met and ErbB signaling is co-involved and may explain why a c-Met TKI is not an effective antagonistic when c-Met is hyperactive for this patient sub-set. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Sub-group of HER2- breast cancer patients with hyperactive and co-involved c-Met and ErbB pathways identified: Functional signal profiling test identifies patient group that may benefit from c-Met and pan-HER combination therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-20.

Abstract P2-05-05: Evaluation of pan-HER and c-MET inhibitors tested ex vivo in primary HER2- breast cancer cells with hyperactive c-MET and ErbB family signaling

Abstract Background: To elucidate the role of c-Met signaling and its involvement with ErbB signaling as a cancer driver, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP Test measures ex vivo real-time live cell response to specific ErbB and c-Met agonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. A recent study quantified c-MET and ErbB-driven signaling activity in epithelial cell samples derived from fresh breast tumor specimens obtained from 74 HER2- breast cancer patients. Of the cell samples tested, 20 of 74, (27.0%; 95% CI=18%-38%) had both hyperactive c-MET signaling and at least one hyperactive ErbB-family receptor signaling. Using primary breast cancer cells with hyperactive c-MET and ErbB signaling and the CELx MP test, the current study set out to: 1) determine the IC50 values of six pan-HER and five c-MET inhibitors; and 2) characterize the efficacy of combinations of each pan-HER inhibitor with each c-MET inhibitor. Methods: Epithelial cells from six HER2- tumor specimens with hyperactive c-MET and ErbB-driven signaling were obtained. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without one of six pan-HER antagonists (neratinib, lapatinib, ibrutinib, dacomitinib, sapitinib, poziotinib) or one of five c-MET antagonists (tepotinib, cabozantinib, crizotinib, capmatinib, or savolitinib) was quantified using an xCELLigence RTCA impedance biosensor. Each individual drug IC50 was determined using a 1000-fold, 5-point, dose response curve with a single fixed concentration of a corresponding agonist. For the drug combination efficacy studies, fixed concentrations of the agonist mixture and clinically relevant concentrations of combinations of the antagonists were used to determine the percentage inhibition of the ErbB and c-MET signaling. Results: The IC50 values for the individual c-MET and pan-HER inhibitors ranged from 3.10nM - 28nM and 2.67nM – 137.27nM, respectively. In the drug efficacy studies, an average of at least 80% of the ErbB and c-MET signaling activated by NRG1, EGF, and HGF co-stimulation was inhibited by each combination of c-MET and pan-HER inhibitors. c-Met Inhibitor Resultsc-MET inhibitorIC50 (nM)Avg Inhibition (%) w/diff ErbBi's Capmatinib3.1094Savolitinib3.3298Tepotinib14.7096Cabozantinib27.3699Crizotinib28.21100 Pan-HER Inhibitor ResultsPan-HER inhibitorIC50 (nM)Avg Inhibition (%) w/diff c-METi's Poziotinib2.67100Neratinib4.81100Ibrutinib13.1099Dacomitinib22.06100Sapitinib41.2898Lapatinib137.2780 Conclusions: The CELx MP test using live cells measures IC50 values comparable to those derived using cell-free methods. Every combination of pan-HER and c-MET inhibitors provided comparably high (at least 80%) levels of inhibitory effect ex vivo. This suggests the sub-group of HER2- breast cancer patients diagnosed with coincident hyperactive c-MET and ErbB signaling by the CELx Test may respond to virtually any pan-HER and c-Met inhibitor combination. Studying combinations designed to minimize drug toxicities without sacrificing efficacy should thus be possible. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluation of pan-HER and c-MET inhibitors tested ex vivo in primary HER2- breast cancer cells with hyperactive c-MET and ErbB family signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-05.

Abstract P3-10-15: Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies

Abstract Background: Hyperactive c-Met signaling, including cross-talk between c-Met and ErbB family receptors, is suspected of contributing to tumor progression in a variety of cancer types. Clinical trials evaluating c-Met inhibitors alone and in combination with other targeted therapies, have produced mostly negative results in patients with amplified c-Met. This suggests other biological factors, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. To measure the c-Met and ErbB signaling activity of a patient's live tumor cells, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP test measures a patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists to diagnose breast cancer tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. In this study, to further elucidate the role of c-Met signaling and its potential involvement with ErbB signaling as a cancer driver, we studied in vivo response to a c-Met inhibitor (tepotinib), an EGFR inhibitor (erlotinib), a pan-HER inhibitor (neratinib), and a combination of these therapies using breast tumor xenograft models. Methods: HCC1954, a HER2+ cell line with hyperactive c-Met and EGFR signaling and normal HER3 and HER2-driven signaling, according to the CELx MP test, was studied. Sixty 4-5-week-old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or one of five treatment groups that received either neratinib, tepotinib, erlotinib, erlotinib and tepotinib, or neratinib and tepotinib for 17 days. Results: The most effective treatment was the combination of neratinib plus tepotinib, where the average tumor size reduction relative to the control group was 71% (p=0.0003). The average tumor size in the neratinib plus tepotinib treated group was 37% smaller (p=0.049) than the neratinib treated group, and 67% smaller (p=0.0026) than the tepotinib treated group. In the erlotinib plus tepotinib treatment group, the average tumor size was 51% smaller than the control group tumors, but the difference was not statistically significant (p=0.11). No significant difference in tumor size was found between the control group and the erlotinib or tepotinib treated groups. Conclusions: The results demonstrate that hyperactive and coincident c-Met and EGFR signaling contributes to the progression of certain breast cancers. This breast cancer sub-type is more responsive to treatment with a c-Met inhibitor plus a pan-HER inhibitor than a c-Met inhibitor plus an EGFR inhibitor or any of the single agents studied. This suggests that when hyperactive c-Met signaling and a hyperactive ErbB family member is present in a patient's tumor, each of the ErbB pathways as well as the c-Met pathway must be inhibited to treat the tumor most effectively. These findings provide strong evidence that HER2-negative breast cancer patients with coincident hyperactive c-MET and ErbB signaling may respond to treatment with a combination of pan-HER and c-Met inhibitors. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-15.

MP39-05 TRANSPOSON MUTAGENESIS DRIVES RENAL CYST FORMATION IN VIVO WHEN COMBINED WITH C-MET HYPERACTIVATION: IMPLICATIONS FOR ACQUIRED RENAL CYSTIC DISEASE

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-05 TRANSPOSON MUTAGENESIS DRIVES RENAL CYST FORMATION IN VIVO WHEN COMBINED WITH C-MET HYPERACTIVATION: IMPLICATIONS FOR ACQUIRED RENAL CYSTIC DISEASE Jason Scovell, Juan Hernandez, Adam Hollander, and Richard Link Jason ScovellJason Scovell More articles by this author , Juan HernandezJuan Hernandez More articles by this author , Adam HollanderAdam Hollander More articles by this author , and Richard LinkRichard Link More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1179AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Acquired renal cystic disease (ARCD) imparts a high risk for renal cell carcinoma (8%) in patients with end-stage renal disease. The molecular mechanism of cyst formation in ARCD remains unknown, although increased hepatocyte growth factor / C-MET signaling have been implicated in cyst formation. To explore molecular mechanisms of renal cyst development relevant to ARCD, we developed a murine model system based on tissue-selectable C-Met activation and transposon-mediated mutagenesis. METHODS To allow conditional activation of C-Met signaling, we engineered a tandem duplicated and mutated human C-MET coding sequence. Cre recombinase catalyzes exchange of the wild type C-Met locus with a constitutively active variant (M1248T). C-met+/M1248T mice were crossed with mice carrying the mutagenic Sleeping Beauty (Onc2) transposable element activated by a cre-dependent transposase (trpase). Localization to the renal epithelium was achieved by ggt-cre. Mice with activated C-Met and transposon mutagenesis (1: c-met+/M1248T; Onc2+/-, trpase+/-; ggt-cre+/-) were compared to mice with activated C-Met (2: c-met+/M1248T; ggt-cre+/-) or activated transposon mutagenesis (3: Onc2+/-, trpase+/-; ggt-cre+/-) alone. Mice were serial imaged by ultrasound and MRI. All kidneys were then examined grossly and histologically at necropsy after aging. RESULTS All mice (N=5) with both C-Met hyperactivation and transposon activity (1) developed large renal cysts by 6 months. No mice with only C-Met hyper-activation (2; N=10) or transposon activity (3; N=10) developed cysts by 6 months. Histological analysis demonstrated fluid filled and hemorrhagic renal cysts without evidence for malignancy. CONCLUSIONS C-Met hyperactivation is insufficient to drive renal cyst formation in isolation. Combining M1248T with transposon mutagenesis drove renal cyst formation with 100% penetrance. By mapping transposon insertion sites in cyst-lining epithelial cells in this model, we can now begin to isolate these interacting genes, which may improve our understanding of the molecular mechanisms underlying ARCD. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e494-e495 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Jason Scovell More articles by this author Juan Hernandez More articles by this author Adam Hollander More articles by this author Richard Link More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

Convergent RANK- and c-Met-Mediated Signaling Components Predict Survival of Patients with Prostate Cancer: An Interracial Comparative Study

We reported (PLoS One 6 (12):e28670, 2011) that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC) tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE) tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1) expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

Radiosensitization of brain metastasis by targeting c-MET

Radiotherapy is the most widely used therapeutic modality in brain metastasis; however, it only provides palliation due to inevitable tumor recurrence. Resistance of tumor cells to ionizing radiation is a major cause of treatment failure. A critical unmet need in oncology is to develop rationale driven approaches that can enhance the efficacy of radiotherapy against metastatic tumor. Utilizing in vivo orthotopic primary tumor and brain metastasis models that recapitulate clinical situation of the patients with metastatic breast cancer, we investigated a molecular mechanism through which metastatic tumor cells acquire resistance to radiation. Recent studies have demonstrated that the hepatocyte growth factor (HGF)-c-Met pathway is essential for the pathologic development and progression of many human cancers such as proliferation, invasion and resistance to anticancer therapies. In this study, c-Met signaling activity as well as total c-Met expression was significantly upregulated in both breast cancer cell lines irradiated in vitro and ex vivo radio-resistant cells derived from breast cancer brain metastatic xenografts. To interrogate the role of c-Met signaling in radioresistance of brain metastasis, we evaluated the effects on tumor cell viability, clonogenicity, sensitivity to radiation, and in vitro/in vivo tumor growth after targeting c-Met by small-hairpin RNA (shRNA) or small-molecule kinase inhibitor (PF-2341066). Although c-Met silencing or radiation alone demonstrated a modest decrease in clonogenic growth of parental breast cancers and brain metastatic derivatives, combination of two modalities showed synergistic antitumor effects resulting in significant prolongation of overall survival in tumor-bearing mice. Taken together, optimizing c-Met targeting in combination with radiation is critical to enhance the effectiveness of radiotherapy in the treatments of brain metastasis.

Cabozantinib in Prostate Cancer: The Beginning of a Precision Paradigm?

The study reported in the article by Smith et al that accompanies this editorial presents data on a novel orally bioavailable tyrosine kinase inhibitor (TKI), cabozantinib, with activity against multiple targets including mesenchymal-epithelial transition factor (MET), also known as hepatocyte growth factor receptor, and the vascular endothelial growth factor receptor 2 (VEGFR2). The authors describe 171 men with castration-resistant prostate cancer (CRPC), a subgroup of patients from a larger phase II randomized discontinuation trial that included multiple tumor types treated with cabozantinib. The trial included a lead-in stage of open-label therapy with cabozantinib and a planned blinded random assignment to cabozantinib or placebo for patients who achieved stable disease at 12 weeks. As reported by the authors, the trial was suspended after 122 patients with CRPC were enrolled onto the open-label portion of the study because of improvements in bone scans and a decrease in pain. At the time the study was halted, a group of 31 patients had been randomly assigned. In this group, there was a marked improvement in the primary end point of progression-free survival (PFS) in the patients receiving cabozantinib compared with placebo (median, 23.9 v 5.9 weeks, respectively; hazard ratio, 0.12; P .001). Although impressive as a small phase II study, the completion of ongoing phase III studies will be critical to confirm the survival benefit. COMET-1, with a primary end point of overall survival, is a placebo-controlled trial of 960 men with CRPC and additional prior therapy randomly assigned to cabozantinib or prednisone (NCT01605227). COMET-2 is a placebo-controlled phase III trial evaluating cabozantinib versus mitoxantrone and prednisone in men with previously treated symptomatic CRPC (NCT01522443), with an emphasis on the effect of cabozantinib on pain and bone disease. Clearly, the results reported by Smith et al support the completion of these larger more definitive studies; perhaps just as important, however, is the need to understand these data as a direction toward more precise multitargeted therapy in prostate cancer. As one potential target in prostate cancer, the understanding of the role of MET, or MET in conjunction with VEGFR, is the subject of ongoing studies. Increased expression of MET protein in tumor cells compared with normal tissue has been associated with tumor grade. An evaluation of the MET ligand hepatocyte growth factor (HGF) in patients from a prior Cancer and Leukemia Group B 9480 study of suramin revealed a correlation between high plasma levels of HGF and decreased patient survival. Studies have also demonstrated that androgen receptor (AR) signaling represses the expression of MET, which supports future studies of targeting the combination of the AR and MET pathways. With the understanding that cabozantinib also inhibits the VEGF pathway, it is of interest that prior laboratory studies have supported the importance of angiogenesis in prostate cancer progression and provide a rationale for combined MET and VEGF pathway inhibition. Additional laboratory studies have demonstrated HGF-mediated angiogenesis through VEGF regulation and an association between VEGF regulation and the activation of c-MET signaling in prostate cancer. Clinical results of antiangiogenic agents alone in prostate cancer, however, have not been optimal, which supports the hypothesis that targeting multiple pathways in addition to VEGF need to be considered to fully influence the complex nature of the tumor microenvironment. In this regard, efforts to understand the effect of agents such as cabozantinib on the MET pathway with additional multitargeted effects such as VEGF inhibition are likely to be important. In fact, there are multiple agents targeting the MET pathway under investigation that have varying degrees of specificity for additional targets. In addition to MET and VEGFR2, cabozantinib has been shown in preclinical models to inhibit RET, KIT, AXL and FLT3 pathways. Cabozantinib has also been demonstrated to have distinct biologic effects on angiogenesis, invasiveness, and metastasis in animal models, as would be expected from these molecular targets. Other MET pathway–focused therapeutic approaches under investigation include antibodies against HGF such as rilotumumab (which is being evaluated in lung, gastric, and gynecologic malignancies) and antibodies against MET such as onartuzumab (which is being evaluated in a phase III trial in combination with erlotinib v erlotinib alone in patients with MET-positive non–small-cell lung carcinoma [NSCLC; NCT01456325]). Receptor TKIs targeting MET are under investigation and include nonselective TKIs such as cabozantinib (as studied in the clinical trial by Smith et al), crizotinib (which targets MET as well as ALK and is approved for the treatment of EML4-ALK positive NSCLC), and foretinib (which inhibits c-MET, VEGFR2, PDGFR, and KIT). More target-selective MET TKIs are also under investigation including agents such as tivantinib (ARQ197; Arqule, Woburn, MA), EMD 1214063 (EMD Serono, Darmstadt, Germany), EMD 1204831 (EMD Serono), AMG 337 (Amgen, Thousand Oaks, CA), and INCB028060 (Incyte, Wilmington, DE). JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 4 FEBRUARY 1 2013

A new anti-c-Met antibody selected by a mechanism-based dual-screening method: therapeutic potential in cancer.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

Abstract 2344: PDX from the naïve NSCLC patients containing activating EGFR mutations are resistant to erlotinib but sensitive to c-met inhibitor

Abstract NSCLC is a common cancer with the highest mortality and few effective treatment options, except for the patients with EGFR (Erlotinib, Iressa) and ALK (Crizotinib) activating mutations. Recent studies indicated that some patients with the activating EGFR mutations but without pre-exposure to EGFR tyrosine kinase inhibitors (TKIs) are resistant to TKIs. The proposed mechanisms (1) include: T790M gatekeeper mutation preventing accessing the kinase activation site, Kras mutation, pTEN loss (2), or c-met amplification (3) that activates ERBB3 signaling (4). Patient Derived Xenograft (PDX) models have been suggested to more closely represent patient tumors (5, 6). We established several PDXs, including HuPrime® LU858, LU859, LU357, LU387, LU1901 and LU2503, using tumor tissues from individual NSCLC patients. LU858/859 contain the “classic” activating EGFR mutation, L858R; LU1868 contain L858R/T790M double mutations; LU357/LUF387 contain the common exon 20 insertion (2319-2320 AACCCCCAC); and LU1901/2503 have wild type EGFR. To our surprise, the two models with single L858R mutation, just like the model with double mutations, respond to the erlotinib poorly, as seen in some of clinic cases (1). Since they do not have T790M and Kras mutation, we examined them for c-met status, finding that their c-met gene are significantly amplified with also increased c-met/HGF expression as measured by at mRNA and protein levels (IHC), consistent with those seen in clinic (3). Furthermore, LU357 is sensitive to erlotinib while LU387 is not, corresponding to no c-met amplification in LU357 but significant amplification in LU387. All these suggest that the observed erlotinib resistance in these models could potentially be attributed to c-met signaling activation via c-met/HGF autocrine. To this end, we treated LU858, LU859 and LU2503 (also with c-met amplification) with c-met inhibitor, Crizotinib. Indeed, Crizotinib caused significant tumor response as expected in all these models. In particular, the combination treatment of LU858 and LU859 with erlotinib caused complete tumor regression by overcoming the resistance, suggesting a new potential effective treatment option. The results using PDX models can potentially help to design a prospective human study to demonstrate improved efficacy by the combination treatment via patient stratification to include the naïve NSCLC patient subjects with c-met amplification/EGFR mutation. 1. A. F. Gazdar, Oncogene 28 Suppl 1, S24, 2009 2. M. L. Sos et al., Cancer Res 69, 3256, 2009 3. T. Kubo et al., Int J Cancer 124, 1778, 2009 4. A. B. Turke et al., Cancer Cell 17, 77, 2010. 5. L. Ding et al., Nature 464, 999, 2010 6. E. Marangoni et al., Clin Cancer Res 13, 3989, 2007 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 2344. doi:1538-7445.AM2012-2344