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. 2018 Feb;16(2):333-344.
doi: 10.1158/1541-7786.MCR-17-0468. Epub 2017 Nov 13.

Competitive Kinase Enrichment Proteomics Reveals That Abemaciclib Inhibits GSK3β and Activates WNT Signaling

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Free PMC article

Competitive Kinase Enrichment Proteomics Reveals That Abemaciclib Inhibits GSK3β and Activates WNT Signaling

Emily M Cousins et al. Mol Cancer Res. .
Free PMC article

Abstract

The cellular and organismal phenotypic response to a small-molecule kinase inhibitor is defined collectively by the inhibitor's targets and their functions. The selectivity of small-molecule kinase inhibitors is commonly determined in vitro, using purified kinases and substrates. Recently, competitive chemical proteomics has emerged as a complementary, unbiased, cell-based methodology to define the target landscape of kinase inhibitors. Here, we evaluated and optimized a competitive multiplexed inhibitor bead mass spectrometry (MIB/MS) platform using cell lysates, live cells, and treated mice. Several clinically active kinase inhibitors were profiled, including trametinib, BMS-777607, dasatinib, abemaciclib, and palbociclib. MIB/MS competition analyses of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors abemaciclib and palbociclib revealed overlapping and unique kinase targets. Competitive MIB/MS analysis of abemaciclib revealed 83 target kinases, and dose-response MIB/MS profiling revealed glycogen synthase kinase 3 alpha and beta (GSK3α and β) and Ca2+/calmodulin-dependent protein kinase II delta and gamma (CAMKIIδ and γ) as the most potently inhibited. Cell-based and in vitro kinase assays show that in contrast to palbociclib, abemaciclib directly inhibits GSK3α/β and CAMKIIγ/δ kinase activity at low nanomolar concentrations. GSK3β phosphorylates β-catenin to suppress WNT signaling, while abemaciclib (but not palbociclib or ribociclib) potently activates β-catenin-dependent WNT signaling. These data illustrate the power of competitive chemical proteomics to define kinase target specificities for kinase inhibitors, thus informing clinical efficacy, dose-limiting toxicities, and drug-repurposing efforts.Implications: This study uses a rapid and quantitative proteomics approach to define inhibitor-target data for commonly administered therapeutics and provides a cell-based alternative to in vitro kinome profiling. Mol Cancer Res; 16(2); 333-44. ©2017 AACR.

Conflict of interest statement

Conflict of Interest: The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. MIB/MS competition identifies targets of trametinib
A.WB analysis of H2228 cell lysate incubated with trametinib for 1 hr prior to MIB enrichment. B. MIB/WB analysis of H2228 cells treated with trametinib for 1 hr. C. H2228 cell lysate was pre-bound to MIBs, washed, and then incubated for the indicated time with 300 nM trametinib. D. MIB/MS competition analysis of H2228 cells treated with DMSO or 30 nM trametinib for 1 hr. In the volcano plot, vertical dashed lines indicate 2-fold label free quantitation (LFQ) change, and horizontal dashed line depicts a 5% FDR threshold across 3 biological replicates. The p-values were calculated via two-tailed t-test, and the FDR was determined by the Benjamini-Hochberg procedure. 241 kinases were observed in two of three biological replicates in at least one treatment condition with a minimum of three unique peptides. E. Mice were treated with DMSO (n=4) or 0.3 mg/kg trametinib (n=5) for 2 hr. Kidneys were extracted, detergent solubilized, and subjected to MIB/MS. Volcano plot as in (D).
Figure 2
Figure 2. MIB/MS competition reveals novel targets of abemaciclib
A. MIB/AP WB of H2228 cells treated with abemaciclib or DMSO for 1 hr. B. MIB/AP WB of H2228 cells treated with palbociclib or DMSO for 1 hr. C. Volcano plot of DMSO versus 6 μM abemaciclib (3 biological replicates) in H2228 cells. In the volcano plot, vertical dashed lines indicate 2-fold label free quantitation (LFQ) change, and horizontal dashed line depicts a 5% FDR threshold across 3 biological replicates. The p-values were calculated via two-tailed t-test, and the FDR was determined by the Benjamini-Hochberg procedure. Filled red circles denote kinases meeting a 5% FDR and >2-fold LFQ decrease in abemaciclib-treated cells; of these, kinases meeting a 1% FDR are labeled. D. Volcano plot of DMSO versus 6 uM palbociclib (2 biological replicates for palbociclib and 3 biological replicates for DMSO) as described in (C). Filled purple circles denote CDK4 and 6. Filled blue circles indicate kinases that are targeted by abemaciclib but not palbociclib.
Figure 3
Figure 3. Abemaciclib dose-dependently inhibits GSK3β from binding MIBs
A. Abemaciclib dose-response in H2228 cells using isobaric TMT labeling. Data are plotted as the mean log2 fold-change compared to vehicle ± SE for 2 biological replicate experiments. TMT ratios for 128 kinases were quantified across both biological replicates. B. Abemaciclib dose-response in H2228 lysate using isobaric TMT labeling. Data are plotted as the mean log2 fold-change compared to vehicle ± SE for 2 biological replicate experiments as in (A). TMT ratios for 133 kinases were quantified across both biological replicates.
Figure 4
Figure 4. GSK3β is an abemaciclib-specific target
A. MIB/AP WB validation of GSK3β and CAMKIIβ/δ/γ following abemaciclib treatment of H2228 cells. B. MIB/AP WB confirming that palbociclib does not affect GSK3β or CAMKIIβ/δ/γ MIB binding. C. MIB/AP WB indicating that ribociclib does not preclude GSK3β from binding MIBs. D. MIB/AP WB in H1703 cells indicating abemaciclib-specific effects on GSK3β and CAMKIIβ/δ/γ MIB binding in a second cell line.
Figure 5
Figure 5
In vitro kinase activity assays indicate that abemaciclib directly inhibits GSK3β. In vitro kinase activity assays for abemaciclib and palbociclib against CDK4/cyclin D1 (A), CDK6/cyclin D1 (B), CDK4/cyclin D3 (C), CDK6/cyclin D3 (D), GSK3β (E), CAMKIIβ (F), CAMKIIδ (G), and CAMKIIγ (H).
Figure 6
Figure 6. Abemaciclib activates WNT signaling
A. Dual-Glo luciferase assays in cell lines stably expressing the WNT reporter (BAR). Bars represent mean Firefly/Renilla ratios ± SD from 4 independent wells. The following abemaciclib and palbociclib ranges were used: 0.3125 μM – 5 μM (RKO B/R and HEK293T/17 B/R) or 0.625 μM – 10 μM (H2228 B/R). CHIR-99021 was treated at 1 μM. Data are representative of one biological replicate from three independent experiments. B. IncuCyte live cell imaging of HEK293T/17 BAR-GreenFire cells treated with DMSO, palbociclib, abemaciclib, 1 μM CHIR-99021, or WNT or L-cell CM for 24 hr. Data are plotted as total green integrated intensity ± SD. Data are representative of one biological replicate from three independent experiments. C. Representative GFP fluorescent images corresponding to the experiment described in (B) at 22 hr post-treatment. Scale bar = 200 μm. D-E. Evaluation of β-catenin levels by WB analysis after 6 hr treatment of DMSO, abemaciclib (0.1–10 μM), palbociclib (0.1–10 μM), 1 μM CHIR-99021, or recombinant WNT3A (rWNT3A, 200 ng/ml) in RKO B/R cells (D) or murine L-cells (E). F-G. Evaluation of β-catenin levels by WB analysis of RKO B/R cells (F) or L-cells (G) following treatment with DMSO, 5 μM abemaciclib, 5 μM CHIR-99021, or 10 μM palbociclib over a 30 min – 6 hr time course.

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