Abstract 1613: Characterization of the Src-regulated kinome by chemical proteomics

Abstract Enhanced Src activation has been implicated in many cancers, including those of breast, lung and pancreas. However single-agent therapies targeting Src have achieved limited success in the clinical setting. It remains unclear what regulatory influence Src has at the kinomic level, where many anti-cancer drug targets are found. This study aims to define the Src-regulated kinome by using a cutting-edge chemical proteomics strategy, and to combine this with functional genomics in order to identify kinase signaling networks that are important for Src-induced oncogenesis. This may ultimately identify potential therapeutic strategies for cancers with aberrant Src activity, that involve, for example, targeting Src in combination with a downstream kinase. We first developed a novel kinase capture reagent termed CTx-0294885 for kinase enrichment. This allows the identification of more than 230 protein kinases at the level of protein expression and phosphorylation by mass spectrometry. The combination of CTx-0294885 with 3 other existing kinase capture reagents led to the greatest kinome coverage in a single cell line reported to date (Zhang 2013). We then applied this kinase enrichment technique in combination with quantitative mass spectrometry to identify the Src-regulated kinome. This was achieved by comparing global kinase activation profiles in control MCF-10A immortalized mammary epithelial cells and MCF-10A cells expressing constitutively active Src. Nearly 300 protein kinases and 1500 kinase-derived phosphosites were identified from the kinome profiling. Importantly, over 100 kinases respond to Src activation with changes at the protein expression or phosphorylation level. This Src-regulated kinome contains: several well characterized kinases that are already being targeted in the clinical setting, such as Egfr and Braf; proto-oncogenes implicated in breast cancer but with no current functional link to Src, such as Ikkϵ; kinases involved in tumor suppression such as Lats1/2 and the Tao family kinases; and many poorly characterized kinases that may represent novel targets for drug discovery. In addition, approximately 70 other proteins co-purified with protein kinases also exhibited changes upon Src activation. These include several lipid kinases and numerous proteins involved in RNA metabolism. All of these Src-regulated proteins are currently being functionally characterized by a focused RNAi screen, specifically regarding their roles in cell viability, migration and morphology. This study will provide novel insights into the global impact of Src-induced transformation on the expressed kinome and may lead to identification of novel therapeutic targets and combination treatment strategies. Reference: Zhang L, Daly R.J et al. 2013. Characterization of a novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling. J Proteome Res. 2013 Jul 5;12(7):3104-16. Citation Format: Luxi Zhang, Jianmin Wu, Roger J. Daly. Characterization of the Src-regulated kinome by chemical proteomics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2014-1613