Abstract P4-05-02: Phospho-reactome measurements reveal heterogenic kinase signatures

Background: Treating cancer increasingly relies on targeting kinases, because their oncogenic activity drive tumorigenesis. Discovering which active mechanisms of disease progression can be efficiently targeted, and knowing whether kinase networks circumvent therapeutic interventions, are challenges researchers and clinicians face. Surprisingly however, measuring the phosphorylating activity of kinases, and potentially monitoring the functionality of the entire human phospho-reactome at once, remains largely unexplored. We developed a semi-high throughput assay to monitor the phospho-catalytic activity of kinase enzymes, using their biological targets as phospho-sensors. We successfully used this assay to identify oncogenic phospho-signatures prevalent in breast cancer, and can be used to establish drug-sensitivity profiles in models of kinase-targeted therapies. Methods: We first defined how to computationally build a library of peptide sensors established from confirmed kinase substrates’ phosphorylation sites. Precisely, we used computational methods to create a unique phospho-repertoire cataloguing 3,408 peptide sequences established from validated human proteins’ phosphorylation sites, curated from 38 public databases. Second, we experimentally used these biologically relevant probes in multiplex assays to quantify the catalytic state of kinases. Specifically, a kinome-representative 242-peptide set was developed into an ATP-consumption screen to identify the activity signatures of EGFR, MAPK, AKT, ABL and SRC family kinases, and explore 642 kinase/substrate nodes. Next, we described analysis methods to derive phospho-signatures from semi-high throughput ATP-consumption measurements. We validated the assay using isogenic culture model of basal-like breast cancer (HMT-3522 S1 and T4-2), and cell lines harboring EGFR/HER2-oncogenic alterations such as MDA-MB-231, MCF7 or T47D. Results: The differential phosphorylation activity of 25 recombinant, active kinase enzymes was successfully captured. In cancer cell extracts, hyper-activated EGFR, ERK, MEK, AKT, and SRC kinases originally identified by immuno-detection were reliably and specifically detectable using the peptide-based kinase-activity assay. The phospho-sensing assay revealed the heterogeneity of active kinase signaling circuits among different breast cancer cells. Conclusion: This unique strategy and resources allow to comprehensively measure the catalytic activity of multitude kinases at once, representing a new molecular dimension to characterize biological samples. We will use such new phospho-reactome profiling system to determine the efficacy of new combinatorial therapies, and define how chemotherapeutic interventions lead to the reprogramming of phospho-circuits. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-02.