Abstract P5-05-01: Catalogued phospho-sensing peptides identifying active, oncogenic kinase signatures
Authors: Mori M , Chen Z , Boudreau A , van't Veer L , Coppé J-P 
Published: August 2014

Background: Phospho-signaling networks represent one of the most clinically and therapeutically important frontiers of biology and medicine. Yet, measuring the phospho-catalytic activity of kinases, and monitoring the functionality of the entire kinome network at once remains largely unexplored. In particular, a highly convenient tool would be the advent of a microarray-like chip to comprehensively map phosphorylation networks to identify kinase signatures underlying cancer types and guide therapeutic interventions. In order to create such a kinase-sensing platform, we developed a library of peptide probes capable of reporting on the catalytic activity of kinases. The well-studied EGFR and SRC kinase network measurements serve as proof of concept.

Methods: We developed a human kinase protein and peptide repertoire from thousands of publications and &gt;30 public databases computationally curated, filtered and merged, suitable to act as peptide beacons to evaluate enzymes' activity. Our database catalogues 916 kinase enzymes, 1913 substrate proteins, 6173 distinctive kinase-substrate active nodes, and compiles 2702 unique biological kinase peptide targets that all represent biologically relevant, latent nano-sensors usable to comprehensively track kinase signaling networks. Here, a representative set of 33 out of 417 peptides predicted to be specifically targeted by EGFR and SRC tyrosine kinase families and 17 control peptides, is tested for their potential kinase-activity reporting ability using a common luminescence ATP-consumption assay. We further validated the peptide screen in biological extracts using an isogenic culture model of basal like breast cancer (HMT-3522 S1 and T4-2).

Results: The differential phosphorylation activity of recombinant, active kinase enzymes (EGFR, ERBB2, SRC, HCK, FYN, FRK, LYN, LCK) was successfully detected by ATP-consumption measurement using the 50 peptides in time course experiments (0.25–4 hours) with evidence of selectively. Subsets of these data were confirmed by SRC kinase inhibitor treatments and dilution assays (0.1–5ng/ul kinase enzymes). Increased presence of activated SRC kinase in biological extracts from T4-2 breast cancer cells compared to nonmalignant S1 cells was shown by significantly elevated phosphorylation of 10 tested peptides (maximum elevation was shown with a SRC -specific reporting peptide and increased from 4 to 73% ATP consumption in S1 versus T4-2 cells, respectively).

Conclusion: These data demonstrate that the tested peptides can monitor kinases' activity. The peptide database can serve as a resource to provide sensitive and specific phospho-sensing probes, used to directly and specifically evaluate the activity of kinases, and can potentially support the identification of the entire, active, oncogenic kinome. Using this resource, we were able to identify peptides with evidence of specificity in in vitro and ex culture assays. Our efforts now concentrate on expanding this approach into a functional kinomic-screening platform in multiplex array format. Ultimately, we hope to translate such device into the clinic, and map phosphorylation signatures that cause breast malignancies and to guide tailor-made therapies.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-01.