Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.
Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence ofEGFRTKI-sensitizing mutations. However, 30-40% of patients withEGFRmutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Xu
X
Zhang
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Tapan
T
Maity
§Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205.
Manoj K
MK
Kashyap
¶Department of System Biology, Columbia University, New York, New York, 10032.|‖PsychoGenics Inc., Tarrytown, New York, 10591.
Mukesh
M
Bansal
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Abhilash
A
Venugopalan
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Sahib
S
Singh
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Shivangi
S
Awasthi
§Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205.
Arivusudar
A
Marimuthu
§Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205.
Harrys Kishore
HK
Charles Jacob
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Natalya
N
Belkina
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Stephanie
S
Pitts
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Constance M
CM
Cultraro
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Shaojian
S
Gao
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Guldal
G
Kirkali
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892.
Romi
R
Biswas
§Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205.|**Medimmune LLC, Gaithersburg, Maryland, 20878.
Raghothama
R
Chaerkady
¶Department of System Biology, Columbia University, New York, New York, 10032.
Andrea
A
Califano
§Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205.
Akhilesh
A
Pandey
From the ‡Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, 20892; udayan.guha@nih.gov.
Udayan
U
Guha
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000077716:Afatinib; D014408:Biomarkers, Tumor / Q000378:metabolism*; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D007553:Isotope Labeling; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D040901:Proteomics / Q000379:methods*; D011799:Quinazolines / Q000494:pharmacology / Q000627:therapeutic use; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism
D014408:Biomarkers, Tumor; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D019000:Phosphotyrosine; D000077716:Afatinib; D014443:Tyrosine; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors