Abstract 4790: Mapping the oncogenic kinome using a kinase activity screening array
Authors: Coppé Jean-Philippe F. , Chen Zhongzhong , Boudreau Aaron , Park Neil , Xu Zidar , Gray Joe , Liu Logan , van ‘t Veer Laura 
Published: August 2012

Abstract
The oncogenic activity of kinases harnesses myriad signalling cascades driving tumorigenesis. Improper control of phosphorylation networks represents one of the most relevant regulator and biomarker of cell dys-functions leading to malignancies. Surprisingly however, measuring the phosphorylation activity of kinases, and potentially monitoring the functionality of the entire kinome at once to identify molecular signatures causing cancers and guide therapeutic interventions, remains largely unexplored. Specifically, a highly convenient tool for clinical applications or research purposes would be the advent of a microarray-like chip to map phosphorylation networks. We undertook to develop such a kinase-sensing platform, and present here unique strategies, technical advances and data -including the detection of oncogenic Src and HER kinases activity in biological samples- supporting such potential breakthrough. The devised kinase activity-sensing system relies on peptide probes that act as beacons to evaluate enzymes functionality, and emit distinctive optical signals before and after (de-)phosphorylation. The following steps have been achieved. First, in order to use baits relevant to human cancer etiology, we created a protein and peptide repertoire cataloguing 6173 distinctive kinase-substrate active nodes and compiling 2702 unique biological kinase peptide targets that all represent latent nano-sensors usable to comprehensively track kinase signaling networks. Second, we established that our label-free detection approach (namely Raman scattering of probes monitored via surface-enhanced Raman spectroscopy (SERS)) is a functional system that reliably and robustly identifies the phosphorylation state of peptide probes. Third, real-time kinetics and kinase inhibitor experiments using biochemical samples and biological extracts from mammalian cells demonstrated that our approach could directly assess the functionality of kinases. Fourth, in an attempt to identify hyperactive Src kinase in tumor cells, our assay discerned normal from oncogenic Src-mutated cells. Finally, using breast cancer cell models of tumor progression and tissue samples from patients, we were able to measure EGFR kinases (hyper-)activity in vivo. Together, our strategy allows for highly sensitive, specific, rapid, one-step, label-free, multiplex capture and measurement of phosphorylation events. Our efforts will now focus on expending this approach into a fully functional kinomic-screening platform to explore oncogenic kinase networks in human cancers. We will translate such device into the clinic, and map phosphorylation signatures that cause breast malignancies to eventually guide therapeutic interventions that best match breast cancer patients’ disease characteristics and predicted responses to (chemo-)therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4790. doi:1538-7445.AM2012-4790