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Competitive Kinase Enrichment Proteomics Reveals That Abemaciclib Inhibits GSK3 And

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Competitive Kinase Enrichment Proteomics Reveals That Abemaciclib Inhibits GSK3 And Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3 and Activates WNT Signaling Emily M. Cousins1, Dennis Goldfarb1,2, Feng Yan1, Jose Roques1, David Darr1, Gary L. Johnson1,3, and Michael B. Major1,2,3,4* 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 2Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 3Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 4Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA Running title: Abemaciclib Inhibits GSK3β and Activates WNT Signaling Key Words: Kinase, chemoproteomics, GSK3β, WNT signaling Additional Information Financial support: This work was supported by the following: V Foundation grant number T2014-009 to MBM and GLJ, Gabrielle’s Angel Foundation (grant number 85) to MBM, NIH/NCI grant (5R01CA187799) to MBM, and the NIH T32 Postdoctoral Training Grant in Pulmonology (5T32HL007106-39) to EMC. 1 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. *To whom correspondence should be addressed: Michael B. Major, Department of Cell Biology and Physiology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 450 West Drive, Lineberger Building, CB#7295, Chapel Hill, NC, 27599, USA. Telephone: (919)-259-2695. Fax: (919)-966- 9673. Email: [email protected] Conflict of Interest: The authors declare no potential conflicts of interest. Abstract word count: 250 Total word count: 6482 including figure legends (excluding references and abtract) Total number of figures and tables in manuscript: 6 Total number of supplemental figures and tables: 4 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 2 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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. Introduction Kinases are responsible for transferring the ATP gamma phosphate onto substrates (1). Kinases are key components of signal transduction pathways and play roles in a large number of cellular processes including growth, differentiation, migration, and apoptosis (2). Due to their varied roles in disease-relevant cellular phenotypes and the frequency with which kinase dysregulation contributes to disease, kinase inhibitors have promised clinical benefit (3). Imatinib (Gleevec®) was the first such small-molecule kinase inhibitor to achieve Food and Drug Administration (FDA) approval in 2001 for Philadelphia Chromosome (BCR-ABL1) positive chronic 3 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. myelogenous leukemia (CML) (4). Currently, 38 FDA-approved kinase inhibitors are on the market, collectively targeting 31 kinases, or 6.0% of the 518 human protein kinases (4-6). In addition to those already approved for patient use, there are 1407 open clinical trials investigating the use of kinase inhibitors in various patient populations either as single agents or in combination with other compounds or biologics (clinicaltrials.gov 9/25/2017). Though kinases share low amino acid homology, a common three-dimensional structure characterizes the ATP binding pocket (7). As such, numerous broad-spectrum and highly potent kinase inhibitors exist. We and others have used these ‘dirty’ kinase inhibitors as affinity tools to enrich the kinome. Specifically, covalent attachment of broad-spectrum kinase inhibitors to a solid-state matrix enables the affinity capture of protein kinases, an approach referred to as kinobeads or multiplexed-inhibitor beads (MIBs) (8-13). Optimization and diversification of the inhibitor-conjugated bead composition allows detection and quantitation of greater than 50% of the kinome in a single mass spectrometry run (8,11). Kinase inhibitors are rarely selective for a single kinase or even kinase family (14). This low specificity limits kinase inhibitor utility in part through unintended clinical consequences and toxicity. Multiple studies have been conducted to assess the selectivity of various kinase inhibitors against panels of kinases using in vitro or lysate-based assays (15-18). While the resulting data are valuable, they are not without caveats. Ideally, kinase inhibitors would be evaluated in live cells or cell lysates where their targets reside in a native state, replete with post- translational modifications, physiological ATP concentrations, subcellular location, and co- complexed binding partners. Here, we utilized the MIB/MS platform to profile the kinome following very short-term kinase inhibitor treatment of cell lysates, live cells, and mice. Inhibitor-bound kinases are 4 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. competitively occluded from binding the MIBs and are thus easily identified in subsequent Western blots or by mass spectrometry (MS). We show that MIB/MS competition provides rapid and quantitative identification of kinases targeted by various kinase inhibitors that are either FDA-approved or in advanced clinical trials. As such, our data provide inhibitor target annotation for several commonly administered drugs, thus providing clues to the molecular basis of side-effect profiles and potentially offering new clinical applications for already approved therapies. Materials and Methods Cell culture, treatments, and lysate preparation: H2228, HCC827, H1703, H358, DB, and H2228 BAR/Renilla (B/R) cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). HEK293T/17 B/R, RKO B/R, L-cells, and HEK293T/17 BAR-GreenFire cells were grown in DMEM supplemented with 10% FBS. All cells were grown at 37˚C with 5% CO2. All cells were originally obtained by ATCC, thawed and grown for less than 3 months, and were not further authenticated. For MIB affinity purification Western blots and MIB/MS experiments, cells were treated with the indicated dose of compound or vehicle for 1 hr. Cells were washed twice with cold PBS, scraped in PBS, and pelleted via centrifugation. Cells were lysed in MIB lysis buffer (0.5% Triton X-100, 10% glycerol, 50 mM Hepes-NaOH [pH 8.0], 150 mM NaCl, 2 mM EDTA and 2 mM DTT) supplemented with protease and phosphatase inhibitors (Thermo Scientific, PI78439 and PI7846). MIB kinase enrichment: 5 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published
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