Phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance.

Protein kinase signaling is fundamental to cell homeostasis and is deregulated in all cancers but varies between patients. Understanding the mechanisms underlying this heterogeneity is critical for personalized targeted therapies. Here, we used a recently established LC-MS/MS platform to profile protein phosphorylation in acute myeloid leukemia cell lines with different sensitivities to kinase inhibitors. The compounds used in this study were originally developed to target Janus kinase, phosphatidylinositol 3-kinase, and MEK. After further validation of the technique, we identified several phosphorylation sites that were inhibited by these compounds but whose intensities did not always correlate with growth inhibition sensitivity. In contrast, several hundred phosphorylation sites that correlated with sensitivity/resistance were not in general inhibited by the compounds. These results indicate that markers of pathway activity may not always be reliable indicators of sensitivity of cancer cells to inhibitors that target such pathways, because the activity of parallel kinases can contribute to resistance. By mining our data we identified protein kinase C isoforms as one of such parallel pathways being more active in resistant cells. Consistent with the view that several parallel kinase pathways were contributing to resistance, inhibitors that target protein kinase C, MEK, and Janus kinase potentiated each other in arresting the proliferation of multidrug-resistant cells. Untargeted/unbiased approaches, such as the one described here, to quantify the activity of the intended target kinase pathway in concert with the activities of parallel kinase pathways will be invaluable to personalize therapies based on kinase inhibitors.