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

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 , 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 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. 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 kinome profiling..

Intestinal Toxicity in Rats Following Administration of CDK4/6 Inhibitors Is Independent of Primary Pharmacology

“…The promiscuity of abemaciclib against kinases has been previously reported (18)(19)(20). In particular, abemaciclib was shown to potently inhibit GSK3b leading to activation of Wnt signaling, which is known to regulate cell proliferation/differentiation, b-catenin, and c-Myc stability in the GI system (26,27).…”

“…The ultimate effect of a drug on a given tissue should reflect the collective activity it has on its primary and any secondary molecular targets. Several publications compared the secondary pharmacology profiles of all three CDK4/6 inhibitors and all reports consistently demonstrated that abemaciclib has the greatest kinase promiscuity (17)(18)(19)(20). Therefore, any effect not purely attributable to primary pharmacology is potentially multifactorial, contributing to a complex pathogenicity that is challenging to decipher.…”

“…Understanding the cause of this GI toxicity should allow for the development of safer drugs. To this end and with the recent identification of kinases potently inhibited by abemaciclib, such as GSK3b (18,19) and the direct activation of Wnt signaling (19), we utilized transcriptomics, pathway analysis and causal reasoning to understand the pathophysiology of the intestinal changes observed in the rat. In this study, we present lines of evidence that support the potential and previously reported involvement of GSK3b inhibition in the intestinal toxicity associated with abemaciclib (18,19).…”

“…Later, Chen compared kinase selectivity among the three CDK4/6 inhibitors and confirmed that abemaciclib is the least selective (18). More recent work (19,20) utilizing kinase enrichment proteomic techniques further characterized the selectivity of abemaciclib and identified activity at 83 other target kinases and that it potently inhibits glycogen synthase kinase 3 alpha and beta (GSK3a and b), Ca 2þ /calmodulin-dependent protein kinase II delta and gamma (CAMKII d and g) and CDK9 (18)(19)(20). Because diarrhea was more prevalent in patients treated with abemaciclib, we hypothesized that it may not be mediated by the primary pharmacology (CDK4/6 inhibition), but rather through secondary pharmacologic targets specific to abemaciclib.…”

A resource for exploring the understudied human kinome for research and therapeutic opportunities

“…Profiling typically involves screening compounds against panels of recombinant enzymes (e.g. KINOMEscan) 33 or chemoproteomics in which competitive binding to ATP-like ligands on beads (so-called kinobeads 34 or multiplexed inhibitor beads – MIBs 35 ) is assayed using mass spectrometry. Such screens benefit from a comprehensive list of binding domains for which selectivity can be assayed.…”