Supplementary Tables 1-2, Figures 1-2 from Antibody Microarray Profiling Reveals Individual and Combined Serum Proteins Associated with Pancreatic Cancer

PDF - 39K, The ER, PR, and HER2 (ERBB2) status was determined by standardized immunohistochemical staining and pathological analysis at Spectrum Health. ER, PR, and HER2 scores are based on IHC guidelines recommended by the ASCO/CAP.

Advances in Cancer Research, Volume 137, the latest release in this ongoing, well-regarded serial provides invaluable information on the exciting and fast-moving field of cancer research. This volume presents original reviews on research bridging oncology and gene expression, with this volume covering unconventional approaches to modulating the immunogenicity of tumor cells, tumor dormancy and immunoediting, the emerging role of anti-apoptotic Bcl-2 family proteins in chemoresistance, Beclin-1 and autophagy, MDA-7/IL-24, and nanotechnology and medicine.Provides information on cancer researchOffers outstanding and original reviews on a range of cancer research topicsServes as an indispensable reference for researchers and students alike

Supplementary Figure Legends - PDF file 35K, supplementary figure legends

Serum HGF in hHGF Tg/SCID mice

Supplementary Table 1 from Phase II Trial to Evaluate Gemcitabine and Etoposide for Locally Advanced or Metastatic Pancreatic Cancer

Supplementary Figures S1-S9 - PDF file 588K, Supplementary Figure S1: The chemical structures of SGX523 and erlotinib; Supplementary Figure S2: The effect of erlotinib on basal tyrosine phosphorylation of EGFR; Supplementary Figure S3: Inhibition of MET, EGFR, and their downstream signaling by SGX523 and erlotinib in the absence of ligands; Supplementary Figure S4: Dose-dependent inhibition of H1373 and H1993 cell proliferation by SGX523 and/or erlotinib in vitro; Supplementary Figure S5: SGX523 and erlotinib combination results in a stronger ERK inhibition in the H1373 tumors; Supplementary Figure S6: The effects of SGX523 and/or erlotinib on cell viability of H358, H1373, H1993 and EBC-1; Supplementary Figure S7: EBC1-Sg3-H62 and EBC1-Sg3-H71 cells are relatively less sensitive to SGX523, compared to the parental EBC-1 cells; Supplementary Figure S8: The effect of SGX523 and/or erlotinib on MET and EGFR in the EBC-1 and its tumor derivatives; Supplementary Figure S9. Kaplan-Meier survival analyses of the mice in the drug studies

Supplementary Figures 1-6 from The Decline in U.S. Cancer Mortality in People Born since 1925

Establish MCF-10A derived cells expression of PIK3CA/MET genes

Effect of targeting PI3K/MET on acini formation and cell invasion

Supplementary figure 1. Sym015 inhibits viability of cell lines in a synergic manner; Supplementary figure 2. The Sym015 antibodies Hu9338 and Hu9006 bind to 2nd or 3rd blades of MET and block HGF binding; Supplementary figure 3. Sym015 induces MET internalization and degradation in MKN-45 cells; Supplementary figure 4. Sym015 induces MET internalization and degradation in EBC-1 cells; Supplementary figure 5. Sym015 induces MET degradation in MKN-45 and EBC-1 cells; Supplementary figure 6. Sym015 inhibits signaling by MET in EBC-1 cells; Supplementary figure 7. Sym015 inhibits motility of EBC-1

Supplementary Methods, Figures 1-4 from MET Kinase Inhibitor SGX523 Synergizes with Epidermal Growth Factor Receptor Inhibitor Erlotinib in a Hepatocyte Growth Factor–Dependent Fashion to Suppress Carcinoma Growth

Effect of HGF on cell signaling

Effect of HGF on cell signaling in MET-Y1253D/PIK3CA-H1047R cells

Abstract There is compelling evidence that oncogenic MET and PIK3CA signaling pathways contribute to breast cancer. However, the activity of pharmacologic targeting of either pathway is modest. Mechanisms of resistance to these monotherapies have not been clarified. Currently, commonly used mouse models are inadequate for studying the HGF–MET axis because mouse HGF does not bind human MET. We established human HGF–MET paired mouse models. In this study, we evaluated the cooperative effects of MET and PIK3CA in an environment with involvement of human HGF in vivo. Oncogenic MET/PIK3CA synergistically induced aggressive behavior and resistance to each targeted therapy in an HGF-paracrine environment. Combined targeting of MET and PI3K abrogates resistance. Associated cell signaling changes were explored by functional proteomics. Consistently, combined targeting of MET and PI3K inhibited activation of associated oncogenic pathways. We also evaluated the response of tumor cells to HGF stimulation using breast cancer patient-derived xenografts (PDX). HGF stimulation induced significant phosphorylation of MET for all PDX lines detected to varying degrees. However, the levels of phosphorylated MET are not correlated with its expression, suggesting that MET expression level cannot be used as a sole criterion to recruit patients to clinical trials for MET-targeted therapy. Altogether, our data suggest that combined targeting of MET and PI3K could be a potential clinical strategy for breast cancer patients, where phosphorylated MET and PIK3CA mutation status would be biomarkers for selecting patients who are most likely to derive benefit from these cotargeted therapy.

Abstract Purpose: MET inhibitors are in clinical trials against several cancer types, but the mechanisms toward vulnerability remain elusive. Here we characterized the molecular basis of MET amplification (METamp) and HGF-autocrine driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies. Experimental Design: METamp (MKN45 and MHCC97H) and HGF-autocrine activation (JHH5 and U87) cells were treated by the MET kinase inhibitor (INC280) and the anti-MET monoclonal antibody (MetMab) to determine the sensitivity and biological responses in vitro. Tumor inhibition was evaluated in vivo using SCID and SCIDhgf mouse models, respectively. HGF-mediated angiogenesis was measured by using the human endothelial cells (HUVEC) tube formation assay. Results: MKN45 and MHCC97H cells are more sensitive than JHH5 and U87 cells to INC280 treatment but are unresponsive to MetMab. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although INC280 and MetMab showed a moderate inhibitory effect on - JHH5 and U87 cells in vitro, both treatments potently suppressed tumor growth in mouse models. We found that HGF stimulation promotes human HUVEC cell tube formation via the Src pathway. INC280 or MetMab inhibited tube formation; thus in HGF-autocrine tumors, the endothelial cells are the secondary targets of tumor-derived HGF and MET inhibition. Conclusion: METamp and HGF-autocrine activation favor different molecular mechanisms, such as a DNA damage response or angiogenesis. Because individual types of MET oncogenic activation may respond to MET inhibitors differently, combination strategies should be developed based upon the molecular subtypes of the tumors. Citation Format: Jianqun Kou, Ben Staal, Phillip R. Musich, Liang Kang, Yuan Qin, Zhi Q. Yao, Boheng Zhang, Tam Angela, Alan Huang, Huaixiang Hao, George F. Vande Woude, Qian Xie. Differential therapeutic responses of MET oncogenic activations to Met kinase inhibitor and neutralizing antibody [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 LB-015.

MET is a tyrosine kinase receptor that transduces intracellular signaling to activate the MAPK, PI3K-Akt, and cadherin pathways (among others). In cancer cells, MET is activated upon stimulation by its only ligand, hepatocyte growth factor/scatter factor (HGF/SF), or becomes active due to mutations or amplifications that produce constitutive activation of the MET kinase. The biological consequences of HGF/SF-MET signaling include cell proliferation, cell cycle progression, increased cell motility and invasive activity, and degradation of extracellular matrices, which can lead to oncogenesis. Aberrant MET signaling contributes to the carcinogenesis of hereditary cancers and also plays a major role in the spread of cancer cells; such signaling indicates a poor prognosis for cancer patients. Genetically engineered mouse models are important tools for studying the spontaneous development of tumors mediated by HGF/SF-MET signaling. Such tumors include carcinomas, sarcomas, and lymphomas, demonstrating the breadth of MET signaling as driving force of cancer. In this chapter, we will discuss the role of HGF/SF-MET signaling in carcinogenesis and the animal models used in developing therapeutic strategies that target the HGF/SF-MET signaling pathways.

Background: Aberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive. Methods: With the goal to investigate the molecular basis of MET amplification (METamp) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring METamp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo. Results: We find that cancer cells driven by METamp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit METamp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors. Conclusions: Our results demonstrate that METamp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring METamp, a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

4573 Background: Preclinical models show that c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). The relationship between c-Met and PD-L1 in human ccRCC is not well characterized. We compared c-Met expression between primary and metastatic sites in ccRCC tissues and evaluated the association with PD-L1 expression. Methods: Pairedprimary and metastatic samples from 45 ccRCC patients were included. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab, VARI) and calculated by a combined score (CS, 0-300) as: intensity of c-Met staining (0-3) x % of positive cells (0-100). PD-L1 expression was previously assessed by IHC (PMID: 26014095). c-Met expression (average c-Met CS) between paired primary and metastatic samples were compared using Wilcoxon signed-rank test. Associations of c-Met expression with PD-L1 expression (+/-) and other clinical features were assessed with Wilcoxon rank-sum tests. Results: Our cohort included 45 primary ccRCCs and 54 corresponding metastases. c-Met expression was higher in metastatic sites compared to primary (c-Met CS: 55 vs. 28, p=0.0003) and was numerically-greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression was associated with higher FNG and T-stage in both primary and metastatic sites (Table). Conclusions: Higher c-Met expression in metastases compared to paired primary tumors in our cohort of ccRCC suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. Although the observation of higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination trials. [Table: see text]