Figure 1.
Application of MIB/MS to analyze the kinomes of drug-sensitive and -resistant leukemia cells.
Kinases from MYL and MYL-R cells were affinity enriched with multiplexed inhibitor beads and quantified by LC-MALDI TOF/TOF mass spectrometry (MIB/MS). Results from three independent experiments were pooled as described in Materials and Methods. (A) Over 150 kinases were quantified from MYL-R relative to MYL cells across a broad spectrum of kinase families, as depicted in the phylogenetic tree of the human protein kinase superfamily. Yellow and blue dots signify kinases that were increased or decreased in abundance, respectively, in MYL-R relative to MYL cells while gray dots signify kinases that were unchanged. Kinome illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). (B) The trend of kinase abundance changes for all kinases quantified. Dashed line, ±1.5-fold change. (C) The kinome profile of MYL-R relative to MYL was derived from the kinases that were significantly changed after statistical analysis. The kinase abundance ratios and p-values from three independent experiments were combined and adjusted for multiple hypothesis testing and ratios with a Benjamini-Hochberg q-value <0.2 were considered significant. Dashed lines, ±1.5-fold change; error bars, SE (N = 3).
Figure 2.
Validation of the MYL-R kinome profiles by immunoblotting.
(A and B) MYL and MYL-R cell lysates were analyzed by immunoblot with the antibodies indicated. Representative data are shown. (A) ATM, BCR-Abl and c-Kit were decreased in MYL-R relative to MYL cells as predicted from the kinome profile above. (B, left panel) Both total and activated Lyn and PKCβ were increased in MYL-R cells as shown using antibodies to detect phosphorylation of the activation loop of Lyn (p-Y416) or the autophosphorylation site of PKCβ. (B, right panel) Total amounts of IKKα, MEK2 and ERK1/2 were similar in MYL and MYL-R cells, however, phospho-specific antibodies demonstrated these kinases were more active in MYL-R cells. (C) Kinases from MYL and MYL-R cell lysates were captured by MIBs pull-down and analyzed by immunoblot using antibodies directed against total protein. The relative amounts of kinases captured from each cell lysate correlated with the abundance ratios predicted by the MYL-R kinome profile. MIBs exposed to MYL-R cell lysate (C, left panel) captured less ASK1, Jak1, MLTK, Yes, GSK3α, CDK2 and dCK; and (C, right panel) captured more NEK9, IKKα, RIPK2, Lyn and ERK. See also, Figures S2 and S3.
Figure 3.
Lyn drives activation of MEK and increases activation of IKKα in MYL-R cells.
(A) MYL-R cells were treated for 1 hour with dasatinib, as indicated, and lysates were analyzed by immunoblot with the antibodies indicated. Lyn phosphorylation was detected by p-SFK (Y416). For densitometry, the band densities of p-SFK, p-MEK and p-IKKα were normalized against the β-actin loading control and were plotted relative to the DMSO treatment. Error bars, SE (N = 3). (B) MYL-R cells were transduced with non-targeting shRNA (shCtrl) or shRNA directed against Lyn (shLyn) and lysates were analyzed by immunoblot with the antibodies indicated. Lyn phosphorylation was detected by p-SFK (Y416) antibody. For densitometry, the band densities of p-IKKα and p-MEK were plotted relative to total IKKα and MEK protein expression. Error bars, SE (N = 2).
Figure 4.
MYL-R cells exhibit increased NF-κB signaling.
(A) Total RNA was isolated from MYL and MYL-R cells and the expression of NF-κB target genes was evaluated by qRT-PCR. Error bars, SE (N = 3). (B) MYL and MYL-R cells were treated for two hours with DMSO or the IKK inhibitor BAY 65-1942 (BAY, 10 µM) and then analyzed by immunoblot using the antibodies indicated. The entire blot is shown in Figure S5. Error bars, SE (N = 3). (C) MYL-R cells were treated for 12 hours with DMSO or BAY (10 µM) and total RNA was isolated and evaluated by qRT-PCR. Error bars, SE (N = 3).
Figure 5.
Kinome response to targeted MEK and IKK inhibitor treatment.
(A) MYL-R cells were treated for 24 hours with AZD6244 (AZD, 5 µM), BAY 65-1942 (BAY, 10 µM) or AZD (5 µM) plus BAY (10 µM), and kinases were isolated and quantified by MIB/MS in two independent experiments. The relative abundances (Drug/DMSO) of kinases in the MEK/ERK pathway are shown. Error bars, SE (N = 2). (B) MYL-R cells were treated for 24 hours with AZD (5 µM), BAY(10 µM) or AZD (5 µM) plus BAY (10 µM), and were analyzed by immunoblot using the antibodies indicated. Data are representative of three separate experiments. (C) MYL-R cells were treated for 12 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM). Total RNA was isolated and the expression of IκBα and IL-6 were evaluated by qRT-PCR. Error bars, SE (N = 2).
Figure 6.
Targeted inhibition of kinases detected by MIB/MS leads to induction of apoptosis.
(A) MYL-R cells were treated for 48 hours with AZD6244 (AZD, 5 µM), BAY 65-1942 (BAY, 10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and cell viability was assessed by MTS assay. Error bars, SE (N = 3). (B) MYL-R cells were treated for 24 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and caspase 3/7 activity was assessed by fluorometric assay. Error bars, SE (N = 2). (C) MYL-R cells were treated for 48 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and cell lysates were analyzed by immunoblot using the antibodies indicated. Data are representative of two separate experiments.