title: Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays.
abstract: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect.
  
  
  
  Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models.
  
  
  
  By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation.
  
  
  
  These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683-94. ©2016 AACR.
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Apurva K
authors.initials: AK
authors.lastname: Srivastava
authors.affiliation: Biological Testing Branch, Developmental Therapeutics Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Melinda G
authors.initials: MG
authors.lastname: Hollingshead
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Weiner
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Tony
authors.initials: T
authors.lastname: Navas
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Yvonne A
authors.initials: YA
authors.lastname: Evrard
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Sonny A
authors.initials: SA
authors.lastname: Khin
authors.affiliation: National Clinical Target Validation Laboratory, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Jiuping Jay
authors.initials: JJ
authors.lastname: Ji
authors.affiliation: National Clinical Target Validation Laboratory, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: In Vivo Evaluation Group, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Borgel
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pfister
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kinders
authors.affiliation: Urologic Oncology Branch, NCI, Bethesda, Maryland.
authors.forename: Donald P
authors.initials: DP
authors.lastname: Bottaro
authors.affiliation: Urologic Oncology Branch, NCI, Bethesda, Maryland.
authors.forename: W Marston
authors.initials: WM
authors.lastname: Linehan
authors.affiliation: Division of Cancer Treatment and Diagnosis, NCI, Bethesda, Maryland.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tomaszewski
authors.affiliation: Division of Cancer Treatment and Diagnosis, NCI, Bethesda, Maryland.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland. parchmentr@mail.nih.gov.
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
mesh_terms: D000072283:A549 Cells; D000818:Animals; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D000077547:Crizotinib; D057809:HEK293 Cells; D019073:HT29 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007191:Indazoles; D007211:Indoles / Q000494:pharmacology; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: C480541:5-((2,6-dichlorobenzyl)sulfonyl)-3-((3,5-dimethyl-4-((2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-1,3-dihydro-2H-indol-2-one; D007191:Indazoles; D007211:Indoles; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D054852:Small Molecule Libraries; D013449:Sulfonamides; D013450:Sulfones; D000077547:Crizotinib; C516667:pazopanib; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met