Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Abstract

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinasemediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies. [Cancer Res 2009;69(14):5987-95]

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…This new array platform permits interrogation of kinase activity in small volume, low concentration samples, using a chip preprinted with consensus phosphorylation sequences for specific kinases. Two chips are available, one for tyrosine kinases (STK), and another for serine/threonine kinases (STK) (Hilhorst et al, 2009;Sikkema et al, 2009). Each chip has B140 unique kinase targets; thus, the pattern and intensity of phosphorylation on the chip yields activity data for potentially hundreds of protein kinases.…”

Acute Stress Elicited by Bungee Jumping Suppresses Human Innate Immunity

“…Comprehensive descriptions of mammalian kinomes have become possible through the sequential spotting of kinase substrates, encompassing the entire human kinome, on a carrier. When such peptide arrays are incubated with cellular lysates and radioactive ATP, kinases active in the lysate will phosphorylate their respective substrates, and upon determining substrate phosphorylation using a phosphoimager, comprehensive descriptions of cellular signaling may be generated (27,28). From the kinome profiles obtained before and after bungee jumping, a noncanonical (also termed nongenomic) glucocorticoid signature stands out.…”

Dual inhibition of Jak2 and STAT5 enhances killing of myeloproliferative neoplasia cells

“…First, pimozide does not inhibit Jak family kinases in an isolated in vitro kinase assays (Table 1). Second, its pattern of inhibition of substrate phosphorylation catalyzed by extracts from Jak2 V617F-expressing cells using the PamChip tyrosine kinase microarray system (PamGene, Den Bosch, Netherlands) 11 is clearly distinct from that mediated by a Jak inhibitor (including no inhibition of the phopshorylation of the Jak2 peptide by pimozide; Figure 1b). Pimozide did inhibit phosphorylation of a peptide derived from Jak1, though this likely occurred through an indirect mechanism.…”