In virtually all epithelial tumors, growth factor receptor activity is deregulated by activated mutations, genomic amplification, and autocrine loops. The dependence of tumor cell survival upon the driving oncogene has been called “oncogene addiction” and demonstrates the acute sensitivity of cancer cells to inhibition of the pathways driving their proliferation, growth and survival. Major questions regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not and why ones which respond still are able to develop resistance through the activation of other pathways. Robust, but not durable, response to receptor tyrosine kinase inhibitors (RTKIs) highlights the need to inhibit pathway activity at multiple levels.

In this study, we identify extensive signaling networks downstream of receptor tyroine kinases (RTKs) across multiple spaces, including phosphorylation, acetylation, and methylation. We used the established method of TMT labeling coupled with serial peptide immunoprecipitation applied to profile drug-treated and untreated RTK pathway-addicted cancer cell lines. Quantitative analysis was performed to look at the effects of drugs on RTK-dependent cell lines: MKN45 (Met) with Crizotinib, H3122 (Alk) with Crizotinib, H1703 (PDGFRα) with Gleevec, and H3255 (EGFR) with Iressa. Phosphotyrosine, acetyl-lysine, methyl arginine, ATM/ATR substrate (s/tQ), and AGC/CAMK/STE family kinase motif were all selected for use in peptide immunoprecipitation which allowed us to characterize and quantify a broad PTM space. In this study we were able to identify and quantify hundreds of modification sites for phosphorylation, arginine methylation, and lysine acetylation. Those sites found to be drug-sensitive, including all PTM types, were used for pathway analysis of signaling downstream of each RTK disease driver.

Citation Format: Klarisa Rikova, Benjamin Hall, Anthony Possemato, Keri Mroszczyk, Kimberly A. Lee, Jian Min Ren, Ailan Guo, Daniel Mulhern, Yi Wang, Sean A. Beausoleil, Michael J. Comb. Mapping deregulated pathways downstream of RTKs in cell line models. [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 4278. doi:10.1158/1538-7445.AM2013-4278