title: Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia.
abstract: 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, IKKα, PKCβ, 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 IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα 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 IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα 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.
authors.affiliation: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cooper
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Cox
authors.forename: Eric I
authors.initials: EI
authors.lastname: Zimmerman
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Dewar
authors.forename: James S
authors.initials: JS
authors.lastname: Duncan
authors.forename: Martin C
authors.initials: MC
authors.lastname: Whittle
authors.forename: Thien A
authors.initials: TA
authors.lastname: Nguyen
authors.forename: Lauren S
authors.initials: LS
authors.lastname: Jones
authors.forename: Sreerupa
authors.initials: S
authors.lastname: Ghose Roy
authors.forename: David M
authors.initials: DM
authors.lastname: Smalley
authors.forename: Pei Fen
authors.initials: PF
authors.lastname: Kuan
authors.forename: Kristy L
authors.initials: KL
authors.lastname: Richards
authors.forename: Richard I
authors.initials: RI
authors.lastname: Christopherson
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Frye
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
authors.forename: Albert S
authors.initials: AS
authors.lastname: Baldwin
authors.forename: Lee M
authors.initials: LM
authors.lastname: Graves
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D000069439:Dasatinib; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D016044:Fusion Proteins, bcr-abl; D006801:Humans; D000068877:Imatinib Mesylate; D015151:Immunoblotting; D007938:Leukemia / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013844:Thiazoles / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D001549:Benzamides; D016328:NF-kappa B; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D000069439:Dasatinib