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

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 <em>in vitro</em>, 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 (GSK3&alpha; and &beta;) and Ca2+/calmodulin-dependent protein kinase II delta and gamma (CAMKII&delta; and &gamma;) as the most potently inhibited. Cell-based and <em>in vitro</em> kinase assays show that in contrast to palbociclib, abemaciclib directly inhibits GSK3&alpha;/&beta; and CAMKII&gamma;/&delta; kinase activity at low nanomolar concentrations. GSK3&beta; phosphorylates &beta;-catenin to suppress WNT signaling, while abemaciclib (but not palbociclib or ribociclib) potently activates &beta;-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. <strong>Implications:</strong> 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 <em>in vitro</em> kinome profiling.