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.