Identifying kinome signatures of breast cancer cell lines
Research Project by James Duncan and Marty Whittle
Traditional studies of protein kinases focus on monitoring changes in activity of single kinases or a specific subset of kinases in response to various stimuli, but are limited in scope. Our project aims to investigate kinase signaling at the kinome level to generate a more comprehensive understanding of kinase network signaling in both normal and disease states, as well as in response to kinase inhibitors. We have developed a proteo-genomic strategy combining deep sequencing and quantitative proteomics to compare global kinome activity in an array of basal-like triple negative breast cancer cell lines. We isolate endogenous protein kinases from the breast cells using Multiplexed Inhibitor Beads (MIBs), which consists of a number of immobilized pan-kinase inhibitors arranged based on inhibitor specificity (specific to non-specific). Affinity purified kinases are subsequently identified and characterized by quantitative mass spectrometry to compare the kinomes of the breast cancer cells relative to the non-tumorigenic HuMEC cells. Using proteo-genomics, we have isolated and quantified endogenous kinases (>400, genomics and >200, proteomics) from breast cancer cell lysates, identifying a kinome signature (kinotype) of elevated and down regulated kinases for each breast cancer cell line. We are currently working to characterize kinome behavior of the breast cell lines in response to specific kinase inhibitors, including lapatinib, AZD-6244, and U0126. Utilizing this proteo-genomic strategy allows us to identify novel kinase drug targets as well as to explore drug effects on the untargeted kinome.