Abstract 5309: Phospho-reactome profiling reveals the heterogenic, targetable kinase signature of breast cancer
Authors: Jean-Philippe F. Coppé, Miki Mori, Evelyn Lee, Bo Pan, Aaron Boudreau, Zhongzhong Chen, Laura van 't Veer
Journal: Cancer Research (2014)

Abstract Background: Improper control of phosphorylation networks represents a powerful regulator and biomarker of cell dys-function leading to malignancy. 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 the active, oncogenic phospho-signature of breast cancer cells. Methods: We first built 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 measure the catalytic state of kinases. Specifically, 228 peptides were developed into an ATP-consumption screen to identify the activity signatures of AKT, EGFR, MAPK, ABL and SRC family kinases. We developed analysis methods to derive phospho-signatures from semi-high throughput measurements. Finally, we validated the bio-applicability of 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 (MDA-MB-231, AU565, MCF7, T47D). Results: The differential phosphorylation activity of 25 recombinant, active kinase enzymes was successfully captured on 228 phospho-sensing peptides. In cancer cell extracts, hyper-activated AKT, SRC, EGFR, or MAPK 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 kinase activity networks in breast cancer cells, and possible new drug-targeting opportunities. Conclusion: This unique strategy and resources allows to comprehensively measure the catalytic activity of a multitude of kinases at once, ultimately representing a new molecular dimension to characterize biological samples. Such new phospho-reactome profiling system suggests how chemotherapeutic interventions affect the activity of kinases and lead to reprogramming of phospho-circuits. Our efforts now concentrate on expanding this approach into a high-content, functional kinome-screening platform to map phosphorylation signatures that cause breast malignancies, therapeutic resistance, or recurrence. Citation Format: Jean-Philippe F. Coppé, Miki Mori, Evelyn Lee, Bo Pan, Aaron Boudreau, Zhongzhong Chen, Laura van 't Veer. Phospho-reactome profiling reveals the heterogenic, targetable kinase signature of breast cancer. [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 5309. doi:10.1158/1538-7445.AM2014-5309