Abstract 5260: Identification of substrates of lung cancer-specific mutant EGFR kinases.
Authors: Maity Tapan K. , Rho Hee-Sool , Belinka Natalya , Xie Zhi , Zhang Xu , Venugopalan Abhilash , Biswas Romi , Cultraro Constance , Zhu Heng , Guha Udayan 
Published: August 2014

Abstract
Among various cancers, lung cancer is the leading cause of cancer-related mortality in both men and women in the US. The major driver mutations in lung cancer are in KRAS and EGFR. Mutations in EGFR account for 10-30% of lung adenocarcinoma. Somatic activating mutations in the kinase domain of EGFR occur in a subset of patients with lung adenocarcinoma and are associated with sensitivity to EGFR-directed tyrosine kinase inhibitors (TKIs). Unfortunately, all patients who respond dramatically to TKIs (gefitinib and erlotinib) eventually develop secondary resistance. A mutation in the gatekeeper residue in the ATP binding pocket of EGFR (T790M) occurs in around 50% of such patients.
We hypothesized that mutant EGFR kinases have altered substrate specificities in vivo; these substrates play intermediary roles in transducing mutant EGFR signaling to downstream components to promote tumorigenesis. We have employed a screening method using an in vitro kinase assay on protein arrays containing around 17,000 proteins to compare the substrate specificity of the kinase domains of wild type EGFR, L858R EGFR (the most common activating mutant), and the EGFR harboring both the L858R and T790M mutations (TKI resistant mutant). We have identified several potential substrates such as Crk, Crk-like protein, Grb2, Stat3, Nck1, PIK3R3, etc., which have altered phosphorylation status in response to mutant EGFR kinase activity. We also performed a SILAC (Stable isotope labeling by amino acids in cell culture)-based global phosphoproteomic analysis in human lung adenocarcinoma cells using mass spectrometry and observed that phosphorylation of some of the potential substrates is modulated by EGF stimulation and TKI inhibition.
We are currently validating promising substrates from the above assay by performing in vitro kinase assays in presence or absence of TKIs. Furthermore, to identify specific phosphorylation sites of the validated targets in vivo, we have adapted a SILAC-based mass spectrometry approach to immunoprecipitate specific substrates from isogenic human bronchial epithelial cells expressing various EGFR mutants followed by identification and quantification of phosphorylation. We have used siRNA-mediated gene silencing to interrogate the role of several of these substrates in survival of human lung adenocarcinoma cells. Finally, we are examining the phosphorylation status of identified substrates in tumor samples from patients of lung adenocarcinoma to correlate phosphorylation with EGFR mutation status.
Citation Format: Tapan K. Maity, Hee-Sool Rho, Natalya Belinka, Zhi Xie, Xu Zhang, Abhilash Venugopalan, Romi Biswas, Constance Cultraro, Heng Zhu, Udayan Guha. Identification of substrates of lung cancer-specific mutant EGFR kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5260. doi:10.1158/1538-7445.AM2013-5260