title: Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.
abstract: OBJECTIVE
  Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.
  
  
  METHODS
  We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined.
  
  
  RESULTS
  We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib-treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.
  
  
  CONCLUSIONS
  Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M.
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.|Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Guolin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Smith
authors.affiliation: Tissue Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Alex S
authors.initials: AS
authors.lastname: Lopez
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Jiannong
authors.initials: J
authors.lastname: Li
authors.affiliation: Proteomics and Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Fang
authors.affiliation: Proteomics and Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: John
authors.initials: J
authors.lastname: Koomen
authors.affiliation: Biostatistics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Bhupendra
authors.initials: B
authors.lastname: Rawal
authors.affiliation: Biostatistics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Fisher
authors.affiliation: Biostatistics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Y Ann
authors.initials: YA
authors.lastname: Chen
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kitano
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Yume
authors.initials: Y
authors.lastname: Morita
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Yamaguchi
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Shibata
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Okabe
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Okamoto
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakagawa
authors.affiliation: Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002853:Chromatography, Liquid; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000235:genetics; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D040081:Protein Array Analysis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D014443:Tyrosine; C512478:EGFR protein, human; D066246:ErbB Receptors