Treating cancer increasingly relies on targeting kinases, because their oncogenic activity harnesses myriad signalling cascades that drive tumorigenesis. 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 kinase 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 key steps have been achieved. First, in order to use baits relevant to human (patho-)physiology, 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 using surface-enhanced Raman spectroscopy (SERS)) is a functional system that reliably and robustly identifies the phosphorylation state of peptide probes. Specifically, peptide-tethered arrays demonstrated high sensitivity, repeatability, and specificity to discriminate between presence or absence of phosphate group on tyrosine-containing peptides densely printed on biocompatible chips. 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. Finally, in an attempt to identify hyperactive Src kinase in tumor cells, our assay discerned normal from oncogenic Src-mutated cells (wild type vs oncogenic v-Src vs Src knock-down).

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 mammary gland 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 AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C89.