Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

作者：Cooper Matthew J; Cox Nathan J; Zimmerman Eric I; Dewar Brian J; Duncan James S; Whittle Martin C; Nguyen Thien A; Jones Lauren S; Roy Sreerupa Ghose; Smalley David M; Kuan Pei Fen; Richards Kristy L; Christopherson Richard I; Jin Jian; Frye Stephen V; Johnson Gary L; Baldwin Albert S; Graves Lee M^*

来源：PLos One, 2013, 8(6): e66755.

DOI：10.1371/journal.pone.0066755

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKK alpha, PKC beta, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKK alpha. Because MYL-R cells showed elevated NF-kappa B signaling relative to MYL cells, as demonstrated by increased I kappa B alpha and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKK alpha resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKK alpha as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.

• 出版日期2013-6-24