Competitive Kinase Enrichment Proteomics Reveals That Abemaciclib Inhibits Gsk3b and Activates WNT Signaling Emily M

Home , CAMK2B, DAPK3, GSK3B, MAP2K2, MAP2K4, PKN2

Published OnlineFirst November 13, 2017; DOI: 10.1158/1541-7786.MCR-17-0468

Signal Transduction Molecular Cancer Research Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3b 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

Abstract

The cellular and organismal phenotypic response to a small- (GSK3a and b) and Ca2þ/calmodulin-dependent protein kinase molecule kinase inhibitor is defined collectively by the inhibitor's II delta and gamma (CAMKIId and g) as the most potently targets and their functions. The selectivity of small-molecule inhibited. Cell-based and in vitro kinase assays show that in kinase inhibitors is commonly determined in vitro, using purified contrast to palbociclib, abemaciclib directly inhibits GSK3a/b kinases and substrates. Recently, competitive chemical proteo- and CAMKIIg/d kinase activity at low nanomolar concentrations. mics has emerged as a complementary, unbiased, cell-based GSK3b phosphorylates b-catenin to suppress WNT signaling, methodology to define the target landscape of kinase inhibitors. while abemaciclib (but not palbociclib or ribociclib) potently Here, we evaluated and optimized a competitive multiplexed activates b-catenin-dependent WNT signaling. These data illus- inhibitor bead mass spectrometry (MIB/MS) platform using cell trate the power of competitive chemical proteomics to define lysates, live cells, and treated mice. Several clinically active kinase kinase target specificities for kinase inhibitors, thus informing inhibitors were profiled, including trametinib, BMS-777607, clinical efficacy, dose-limiting toxicities, and drug-repurposing dasatinib, abemaciclib, and palbociclib. MIB/MS competition efforts. analyses of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors abemaciclib and palbociclib revealed overlapping and Implications: This study uses a rapid and quantitative proteomics unique kinase targets. Competitive MIB/MS analysis of abema- approach to define inhibitor-target data for commonly adminis- ciclib revealed 83 target kinases, and dose–response MIB/MS tered therapeutics and provides a cell-based alternative to in vitro profiling revealed glycogen synthase kinase 3 alpha and beta kinome profiling. Mol Cancer Res; 16(2); 333–44. 2017 AACR.

Introduction positive chronic myelogenous leukemia (CML; ref. 4). Currently, 38 FDA-approved kinase inhibitors are on the market, collectively Kinases are responsible for transferring the ATP gamma phos- targeting 31 kinases, or 6.0% of the 518 human protein kinases phate onto substrates (1). Kinases are key components of signal (4–6). In addition to those already approved for patient use, there transduction pathways and play roles in a large number of cellular are 1407 open clinical trials investigating the use of kinase processes including growth, differentiation, migration, and apo- inhibitors in various patient populations either as single agents ptosis (2). Because of their varied roles in disease-relevant cellular or in combination with other compounds or biologics (clinical- phenotypes and the frequency with which kinase dysregulation trials.gov 9/25/2017). contributes to disease, kinase inhibitors have promised clinical Although kinases share low amino acid homology, a common benefit (3). Imatinib (Gleevec) was the first such small-molecule three-dimensional structure characterizes the ATP binding pocket kinase inhibitor to achieve Food and Drug Administration (FDA) (7). As such, numerous broad-spectrum and highly potent kinase approval in 2001 for Philadelphia Chromosome (BCR-ABL1) inhibitors exist. We and others have used these `dirty' kinase inhibitors as affinity tools to enrich the kinome. Specifically, 1Lineberger Comprehensive Cancer Center, University of North Carolina at covalent attachment of broad-spectrum kinase inhibitors to a fi Chapel Hill, Chapel Hill, North Carolina. 2Department of Computer Science, solid-state matrix enables the af nity capture of protein kinases, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. 3Depart- an approach referred to as kinobeads or multiplexed inhibitor ment of Pharmacology, Lineberger Comprehensive Cancer Center, University of beads (MIB; refs. 8–13). Optimization and diversification of the 4 North Carolina at Chapel Hill, Chapel Hill, North Carolina. Department of Cell inhibitor-conjugated bead composition allows detection and Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, quantitation of greater than 50% of the kinome in a single mass North Carolina. spectrometry (MS) run (8, 11). Note: Supplementary data for this article are available at Molecular Cancer Kinase inhibitors are rarely selective for a single kinase or even Research Online (http://mcr.aacrjournals.org/). kinase family (14). This low specificity limits kinase inhibitor Corresponding Author: Michael B. Major, University of North Carolina at Chapel utility in part through unintended clinical consequences and Hill, 450 West Drive, Chapel Hill, NC 27599. Phone: 9192592695; E-mail: toxicity. Multiple studies have been conducted to assess the [email protected] selectivity of various kinase inhibitors against panels of kinases doi: 10.1158/1541-7786.MCR-17-0468 using in vitro or lysate-based assays (15–18). While the resulting 2017 American Association for Cancer Research. data are valuable, they are not without caveats. Ideally, kinase

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inhibitors would be evaluated in live cells or cell lysates where 100 mmol/L Tris-HCl (pH 6.8)]. Samples were then reduced with their targets reside in a native state, replete with post-translational DTT, alkylated with chloroacetamide, and concentrated prior to modifications, physiological ATP concentrations, subcellular precipitation of proteins by methanol–chloroform extraction. location, and co-complexed binding partners. Proteins were trypsinized overnight, desalted via a C18 spin Here, we utilized the MIB/MS platform to profile the kinome column, and finally extracted three times with ethyl acetate to following very short-term kinase inhibitor treatment of cell remove detergents. lysates, live cells, and mice. Inhibitor-bound kinases are compet- For TMT experiments, cells or cell lysate were treated with itively occluded from binding the MIBs and are thus easily DMSO or abemaciclib for 1 hour, and MIB/MS samples were identified in subsequent Western blots (WB) or by MS. We show prepared as above. Tryptic peptides were buffer exchanged into that MIB/MS competition provides rapid and quantitative iden- 100 mmol/L TEAB prior to the addition of TMT label reagents tification of kinases targeted by various kinase inhibitors that are (Thermo Scientific, 90111). Peptide labeling was conducted either FDA-approved or in advanced clinical trials. As such, our according to the manufacturer's instructions. Peptides from vehi- data provide inhibitor target annotation for several commonly cle and abemaciclib-treated cells were then mixed 1:1:1:1:1 prior administered drugs, thus providing clues to the molecular basis of to desalting via a C18 spin column and detergent removal by ethyl side-effect profiles and potentially offering new clinical applica- acetate extraction as described above. For the first biological tions for already approved therapies. replicate of treated H2228 cells, the following TMT reagent tags were used: DMSO (TMT10-126), 0.006 mmol/L abemaciclib Materials and Methods (TMT10-127N), 0.06 mmol/L abemaciclib (TMT10-127C), 0.6 mmol/L abemaciclib (TMT10-128N), and 6 mmol/L abemaciclib Cell culture, treatments, and lysate preparation (TMT10-128C). For the second biological replicate of treated H2228, HCC827, H1703, H358, DB, and H2228 BAR/Renilla H2228 cells, the following TMT reagent tags were used: DMSO (B/R) cells were grown in RPMI1640 supplemented with 10% (TMT10-129N), 0.006 mmol/L abemaciclib (TMT10-129C), FBS. HEK293T/17 B/R, RKO B/R, L cells, and HEK293T/17 BAR- 0.06 mmol/L abemaciclib (TMT10-130N), 0.6 mmol/L abemaciclib GreenFire cells were grown in DMEM supplemented with 10% 10 10 (TMT -130C), and 6 mmol/L abemaciclib (TMT -131). For the FBS. All cells were grown at 37 C with 5% CO . All cells were 2 TMT 10-plex experiment conducted in H2228 abemaciclib-trea- originally obtained by ATCC, thawed and grown for less than 3 ted lysates, the following TMT labels were used: DMSO-treated months, and were not further authenticated. For MIB affinity lysate [TMT10-126 (replicate 1), TMT10-127C (replicate 2)], 0.006 purification (AP) Western blots and MIB/MS experiments, cells mmol/L abemaciclib-treated lysate [TMT10-127N (replicate 1), were treated with the indicated dose of compound or vehicle for 1 TMT10-128C (replicate 2)], 0.06 mmol/L abemaciclib-treated hour. Cells were washed twice with cold PBS, scraped in PBS, lysate [TMT10-128N (replicate 1), TMT10-129C (replicate 2)], and pelleted via centrifugation. Cells were lysed in MIB lysis 0.6 mmol/L abemaciclib-treated lysate [TMT10-129N (replicate buffer [0.5% Triton X-100, 10% glycerol, 50 mmol/L Hepes- 1), TMT10-130C (replicate 2)], and 6 mmol/L abemaciclib-treated NaOH (pH 8.0), 150 mmol/L NaCl, 2 mmol/L EDTA, and 2 lysate [TMT10-130N (replicate 1), TMT10-131 (replicate 2)]. TMT- mmol/L DTT] supplemented with protease and phosphatase labeled peptides were mixed 1:1:1:1:1:1:1:1:1:1 prior to LC/MS. inhibitors (Thermo Scientific; PI78439 and PI7846). For the TMT 5-plex experiments conducted in DB cells, the following TMT labels were used: DMSO-treated lysate [TMT10- MIB kinase enrichment 126 (replicate 1), TMT10-127N (replicate 2)], 0.006 mmol/L Cells were lysed and normalized for protein concentration. For abemaciclib-treated lysate [TMT10-127N (replicate 1), TMT10- MIB affinity purification (AP) WBs, 500 to 750 mg of protein lysate 127C (replicate 2)], 0.06 mmol/L abemaciclib-treated lysate was used per sample. Lysates were incubated with MIBs and [TMT10-127C (replicate 1), TMT10-128N (replicate 2)], 0.6 nutated at 4 C for 15 minutes (8). The MIB mix contained mmol/L abemaciclib-treated lysate [TMT10-128N (replicate 1), VI16832 (22%, v/v), CTx-0294885 (22%, v/v), Purvalanol B TMT10-128C (replicate 2)], and 6 mmol/L abemaciclib-treated (14%, v/v), PP58 (14%, v/v), UNC21474 (14%, v/v), and Shokat lysate [TMT10-128C (replicate 1), TMT10-129N (replicate 2)]. For (14%, v/v) inhibitors conjugated to sepharose beads (8, 9, each replicate, TMT-labeled peptides were mixed 1:1:1:1:1 prior to 19–22). Kinase-bound MIBs were washed once each with MIB LC/MS. lysis buffer, MIB high salt buffer [0.5% Triton X-100, 50 mmol/L Hepes-NaOH (pH 8.0), 1 M NaCl, 1 mmol/L EDTA, and 1 mmol/ Antibodies, compounds, and recombinant protein L EGTA], and MIB low salt buffer [0.5% Triton X-100, 50 mmol/L The CDK4 (A304-225A) antibody was purchased from Hepes-NaOH (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, and Bethyl Laboratories. AXL (4566), CAMKII (4436), CDK6 1 mmol/L EGTA]. Proteins were then eluted from MIBs in 4 (13331), MEK1/2 (8727), MET (8198), MERTK (4319), and protein loading buffer containing DTT in a 95 C heat block for 10 SRC (2109) antibodies were obtained from Cell Signaling minutes. Standard Western blot techniques were then utilized for Technology. b-Catenin (610153) and GSK3b (610201) anti- analysis. bodies were purchased from BD Biosciences. GAPDH (G8795) For MIB/MS experiments, 5 mg of protein lysate was brought to and b-tubulin (T7816) antibodies were obtained from Sigma- 1 M NaCl and then added to gravity flow columns containing 100 Aldrich. Secondary antibodies were purchased from LI-COR mL of packed sepharose beads (8); the unbound fraction was then Biosciences: IRDye 800CW Goat anti-Mouse IgG (925-32210), passed over gravity columns containing 175 mL of packed MIBs. IRDye 680LT Goat anti-Mouse IgG (925-68020), IRDye 800CW MIB-bound proteins were washed once each with MIB high salt Goat anti-Rabbit IgG (925-32211), and IRDye 680LT Goat anti- buffer, MIB low salt buffer, and MIB low salt buffer containing Rabbit IgG (925-68021). The following chemicals were pur- 0.1% SDS. MIB-bound proteins were eluted by boiling in chased from Cayman Chemicals: abemaciclib (17740), CHIR- MIB elution buffer [0.5% SDS, 1% b-mercaptoethanol, and 99021 (13122), dasatinib (11498), and ribociclib (17666).

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Abemaciclib Inhibits GSK3b and Activates WNT Signaling

BMS-777607 (S1561) and palbociclib (S1579) were ordered 100 Å pore size) at 300 nL/min and 35C. For non-TMT experi- from Selleck Chemicals. Recombinant Wnt3A (315-20) was ments, a 180 minute gradient utilized 2% to 25% buffer B (0.1% purchased from PeproTech. formic acid in acetonitrile), and an Orbitrap Elite mass spectrom- eter (Thermo Scientific) performed the analysis. Settings for the In vitro kinase activity assays ion source and data acquisition were described previously (23). Abemaciclib and palbociclib were sent to Reaction Biology TMT experiments were performed on an Orbitrap Fusion Corporation for in vitro kinase activity assays. Briefly, kinase Lumos (Thermo Scientific) with a 180 minute gradient from substrates were diluted in base reaction buffer [20 mmol/L Hepes 2% to 30% buffer B. MS1 scans were performed in the Orbitrap (pH 7.5), 10 mmol/L MgCl2, 1 mmol/L EGTA, 0.02% Brij-35, at 120k resolution with an automated gain control (AGC) target 0.02 mg/mL BSA, 0.1 mmol/L Na3VO4, 2 mmol/L DTT, and 1% of 4e5 and max injection time of 100 milliseconds. MS2 scans DMSO]. Individual kinases were then added to the substrate were performed in the ion trap following collision induced solution and gently mixed. Compounds diluted in 100% DMSO dissociation (CID) on the 10 most intense ions. MS2 settings were added to the kinase reaction mixture by acoustic technology were AGC ¼ 1.8e4, max injection time ¼ 120 milliseconds, CID (Echo550 Liquid Handler by Labcyte; nanoliter range) and incu- collision energy ¼ 30%, and quadrupole isolation width ¼ 0.7 m/ bated for 20 minutes at room temperature prior to the addition of z. Precursors were filtered for monoisotopic peaks and charge 33P-ATP (specific activity 10 mCu/mL). Reactions were incubated states 2 to 7. Dynamic exclusion was set to 30 seconds and a mass for 2 hours at room temperature, and radioactivity was detected tolerance of 10 ppm. MS3 scans were collected on the 10 most by filter binding methods. Kinase activity data were expressed as intense MS2 fragment ions using synchronous-precursor-selec- the percent remaining kinase activity in test samples compared to tion (SPS) and performed in the Orbitrap. MS3 settings were AGC ¼ ¼ ¼ vehicle (DMSO) reactions. IC50 values and curve fits were 1.2e5, max injection time 120 milliseconds, resolution obtained using GraphPad Prism software. 60k, and higher-energy collision dissociation collision energy ¼ 55%. For MS3 scans, the isolation windows were set specifically Dual Glo Luciferase and IncuCyte live cell imaging for each precursor charge state. For precursor of z ¼ 2, the MS1 4 4 HEK293T/17 B/R (1.25 10 cells/well), RKO B/R (1.25 10 isolation width ¼ 1.3 m/z and MS2 isolation width ¼ 2 m/z. For 3 cells/well), and H2228 B/R (7.5 10 cells/well) cells were plated z ¼ 3, MS1 width ¼ 1 m/z and MS2 width ¼ 3 m/z. For z ¼ 4, MS1 in 96-well plates 1 day prior to treatment with test compounds. isolation ¼ 0.8 m/z and MS2 width ¼ 3 m/z. For z ¼ 5 to 7, MS1 Cells were treated with DMSO, 1 mmol/L CHIR-99021, L-cell or width ¼ 0.7 m/z and MS2 width ¼ 3 m/z. WNT3A conditioned media (CM, described by ATCC), abemaci- Raw MS data files were searched in MaxQuant (version 1.5.2.6) clib, or palbociclib for 20 hours. Cells were then lysed in 1X using the following parameters: specific tryptic digestion with up Passive Lysis Buffer (Promega); 10 mL of lysate was transferred to to two missed cleavages, carbamidomethyl fixed modification, white well 96-well plates with black bottoms prior to the addition variable protein N-terminal acetylation and methionine oxida- of luciferase reagents according to the manufacturer's instructions. tion, match between runs (alignment time window: 20 minutes; Plates were read on an Enspire 2300 Plate Reader (PerkinElmer). matching time window: 0.7 minutes), label free quantitation Data are plotted as the average Firefly/Renilla ratio from four (LFQ), minimum ratio count of 2, and the UniProtKB/Swiss-Prot technical replicates, and error bars represent standard deviation human canonical sequence database (release 07/2013). The two from one biological replicate. Data are representative of three TMT 5-plex biological replicates in H2228 cells were searched independent biological replicates. For live cell imaging using separately, and the utilized labels were chosen for quantitation the IncuCyte Zoom Live Cell Imaging system (Essen Instruments), (two different sets of five labels from TMT10). The H2228 abe- 4 7.5 10 HEK293T/17 BAR-GreenFire cells were seeded with 1% maciclib-treated lysate biological duplicates were shot as a single (v/v) Nuclight Red BacMam 3.0 reagent (Essen Bioscience, Cat- TMT 10-plex sample, utilizing all 10 TMT labels in a single MS run. alog No. 4621) per well in a 48-well plate one day prior to drug Data from TMT10-129N (0.6 mM abemaciclib-treated H2228 treatment. Cells were then treated with DMSO, 1 mmol/L CHIR- lysate) were dropped from the analysis due to a >10-fold reduc- 99021, L-cell CM, WNT3A CM, abemaciclib, or palbociclib. Four tion in MS3 intensity for this label. The two 5-plex TMT replicates independent wells were treated for all conditions, and the entire in DB abemaciclib-treated lysates were searched together, and experiment was conducted in three independent biological repli- utilized labels were used for quantitation (different sets of five cates. Cells were monitored for GFP (BAR activity), RFP (nuclear labels). Further bioinformatic steps are described in the Statistics staining for cell number normalization), and phase (cell density) section. The MS proteomics data have been deposited to the every hour for 24 hours using a 20 objective. Data were plotted ProteomeXchange Consortium via the PRIDE partner repository as the average total integrated intensity for green fluorescence with the dataset identifier PXD006139 (24). across four images per well and four quadruplicate wells; error bars represent standard deviations from the mean. The count fluorescent objects parameter was used in the red channel to Statistical Analysis determine the number of cells per well. Phase data were plotted as Searched MS raw files were imported into Perseus (version the percentage of the image occupied by cells. 1.5.1.6) for further filtering and data visualization. A 1% FDR was applied to all proteins; decoys, non-kinase proteins, and kinases Mass spectrometry, bioinformatics, and data filtering with <3 unique reads were removed. LFQ intensities were log2- Trypsinized peptides were separated via reverse-phase nano- transformed, and missing values were imputed from a fitted HPLC using a nanoACQUITY UPLC system (Waters Corpora- normal distribution with a down-shift of 1.8 and distribution tion). Peptides were trapped in a 2 cm column (Pepmap 100, width of 0.4. For the volcano plots depicting MS data, P-values 3 mm particle size, 100 Å pore size) and separated in a 25 cm were calculated via standard two-tailed t test, and the FDR was EASYspray analytical column (75 mm ID, 2.0 mm C18 particle size, determined by the Benjamini–Hochberg procedure. Additional

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statistical methods were also tested and compared (permutation 1E). MEK2 protein levels were also reduced in mouse kidneys test within Perseus and the open-source software MS Stats); these treated with trametinib as compared to DMSO, although this methods yielded overly optimistic results. Thus, we chose the reduction did not meet the stringent 5% FDR as determined by the more conservative Benjamini–Hochberg procedure in an attempt Benjamini–Hochberg procedure (Fig. 1E). to limit false-positives. The competitive MIB/MS platform was also validated on two additional kinase inhibitors in Supplementary Fig. S1. Here, BMS- Study approval 777607 was shown by MIB/AP WB and MIB/MS to target its All animal handling and experiments were conducted under known targets MERTK, MET, and AXL in H2228-treated cells NIH guidelines and were approved by the UNC Institutional (Supplementary Fig. S1A and S1D). Dasatinib, a SRC family Animal Care and Use Committee (protocol number 15-332). kinase inhibitor, was effective at inhibiting SRC binding to MIBs Female (11 to 15 weeks of age) FVB/NJ (The Jackson Laboratory, in two NSCLC cell lines (Supplementary Fig. S1B and S1C). By 001800) mice were treated with DMSO (n ¼ 4) or 0.3 mg/kg MIB/MS analyses, dasatinib prevented MIB binding of several SRC trametinib (n ¼ 5) by oral gavage. Mice were sacrificed 2-hour family members (LYN, FYN, YES1, and SRC) following short-term posttreatment; kidneys were harvested and snap frozen. Tissue inhibitor treatment (Supplementary Fig. S1E). Together, these was homogenized and lysed as described above. results illustrate the power of MIB/MS competition for rapid kinase target identification in cell lysates, live cells, and in animals. Results Kinase enrichment proteomics reveals novel targets of Method validation: multiplexed inhibitor bead competition abemaciclib proteomics identifies targets of trametinib Next, we used the MIB/MS competition platform to evaluate Trametinib (GlaxoSmithKline) targets mitogen-activated pro- targets of two cyclin-dependent kinase 4 and 6 inhibitors. Abe- tein kinase kinase 1 and 2 (MAP2K1 and MAP2K2, hereby referred maciclib (LY2835219) is in late-stage clinical trials for glioblas- to as MEK1 and 2) and was approved by the FDA in 2013 as a toma, breast cancer, and NSCLC and recently received FDA þ single-agent therapy for unresectable or metastatic melanoma approval for the treatment of HR /HER2 breast cancer both as V600E V600K harboring BRAF or BRAF mutations (25). To evaluate a monotherapy and in combination with fulvestrant. Palbociclib þ and optimize the MIB competition experimental approach, we (PD-0332991) is FDA-approved for the treatment of ER /HER2 þ comparatively evaluated MIB-enriched kinases in trametinib-trea- and HR /HER2 breast cancers. Abemaciclib and palbociclib ted cell lysate, live cells, and in mice. Trametinib was chosen for were tested for their ability to inhibit CDK4 and CDK6 binding these studies due to its high specificity for MEK1 and 2 at doses in to MIBs. One hour treatment of H2228 cells with abemaciclib and the low nanomolar range (26). First, H2228 non–small cell lung palbociclib prevented the binding of CDK4/6 to MIBs, whereas cancer (NSCLC) cell lysate was incubated with trametinib for 1 AXL binding was unaffected (Fig. 2A and B). hour prior to AP with MIBs. Trametinib inhibited MEK1 and MIB/MS competition was then performed for abemaciclib and MEK2 binding at the highest dose, whereas the MIB binding of palbociclib. Across three biological replicate experiments, 1 hour AXL kinase was not affected (Fig. 1A). Second, treatment of live abemaciclib treatment decreased the binding of 83 kinases to MIBs H2228 cells for 1 hour with increasing doses of trametinib (>2-fold reduction at a 5% FDR; Fig. 2C). In addition to CDK4/6, blocked MEK1/2 binding to MIBs (Fig. 1B). Third, H2228 cell abemaciclib suppressed MIB capture of glycogen synthase kinase 3 lysate was preincubated with MIBs for 15 minutes to allow kinase a and b (GSK3A/B, hereafter referred to as GSK3 a and b) and þ binding prior to the addition of trametinib or DMSO for the several members of the CDK, MAPK, and Ca2 /calmodulin- indicated time. Trametinib was able to compete off prebound dependent protein kinase (CAMK) families (Fig. 2C and Supple- MEK1/2, but not AXL, from the MIBs (Fig. 1C). mentary Table S1). Previous studies have identified some of these To fully evaluate the kinome for responsiveness to trametinib, kinases as abemaciclib targets, including GSK3b, although no H2228 cells were treated with 30 nmol/L trametinib for an hour follow-up studies were reported for GSK3b (27, 28). Supplemen- prior to MIB/MS analysis in biological triplicate (Fig. 1D). Cumu- tary Table S2 contains a list of all potential abemaciclib kinase latively, 241 kinases were identified across all MS runs with a targets identified by the MIB/MS approach described here and in minimum of three unique peptides in at least two of three vitro kinase assays described previously (27, 28). replicates. Following short-term trametinib treatment, only MEK1 Palbociclib treatment resulted in the loss of PIP4K2B binding and MEK2 exhibited significantly decreased MIB binding. The P- to MIBs, using a two-fold cutoff and 5% FDR calculated from values in the volcano plots were calculated via a two-sided t test, biological replicate experiments (Fig. 2D). PIP4K2B has been whereas the 5% FDR was determined using the Benjamini–Hoch- previously identified as a target of palbociclib in lung squamous berg procedure. A very conservative FDR threshold was chosen for carcinoma (29). CDK4 and CDK6 exhibited a more than eight- all MS experiments to ensure that follow-up studies were con- fold reduction in MIB binding at an 11% FDR following palbo- ducted on kinase "hits" that were most likely to be true positives. ciclib treatment of H2228 cells (see Materials and Methods These results suggest that trametinib is exquisitely selective for for description of FDR calculation). These initial MIB/MS MEK1/2 and has little effect on the other 239 kinases identified competition studies suggest that abemaciclib and palbociclib across the samples. A similar experiment was conducted in tra- have very distinct kinase target profiles with the exception of metinib-treated mice to determine if MIB/MS competition could CDK4 and CDK6. identify drug targets in tissue from treated animals. Mice were treated with DMSO or 0.3 mg/kg trametinib by oral gavage, and Abemaciclib dose-dependently inhibits GSK3b MIB binding kidneys were harvested two hours posttreatment. MIB/MS To further quantify abemaciclib-responsive kinases, dose– revealed significant reduction in MEK1 binding to MIBs in tra- response MIB/MS competition experiments were conducted in metinib-treated kidneys compared to DMSO-treated kidneys (Fig. H2228 cells using isobaric tandem mass tag (TMT) technology

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Abemaciclib Inhibits GSK3b and Activates WNT Signaling

ABC Input AP:MIB Input AP:MIB Input AP:MIB Trametinib (nmol/L) Trametinib (nmol/L) Tramet. (nmol/L) Tramet. (nmol/L) Trametinib Trametinib Veh Veh 4 0.5 12 4 4 0.5 1 2 4 Time (h) 3 3,000 30 300 3,000 3 30 300 Veh Veh Veh 3 30 300 3,000 3 30 300 Veh 3,000 MEK1/2 MEK1/2 MEK1/2

AXL AXL AXL GAPDH GAPDH GAPDH H2228 Cells H2228 Cells H2228 Lysate

Trametinib vs. vehicle Trametinib vs. vehicle D H2228 cells E Mouse kidney

2.0 MAP2K1 Map2k1 5% FDR

1.5 MAP2K2 1.0 5% FDR Map2k2 q-value 1.0 q-value 10 10 −Log −Log 0.5

0.5

0.0 0.0

−4 −3 −2 −1 01234 −2 −1 0 1 2

Log2 fold change Log2 fold change

Figure 1. MIB/MS competition identiﬁes targets of trametinib. A, WB analysis of H2228 cell lysate incubated with trametinib for 1 hour prior to MIB enrichment. B, MIB/WB analysis of H2228 cells treated with trametinib for 1 hour. C, H2228 cell lysate was prebound to MIBs, washed, and then incubated for the indicated time with 300 nmol/L trametinib. D, MIB/MS competition analysis of H2228 cells treated with DMSO or 30 nmol/L trametinib for 1 hour. In the volcano plot, vertical dashed lines indicate two-fold label free quantitation (LFQ) change, and horizontal dashed line depicts a 5% FDR threshold across three biological replicates. The P-values were calculated via two-tailed t test, and the FDR was determined by the Benjamini–Hochberg procedure. A total of 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 ¼ 4) or 0.3 mg/kg trametinib (n ¼ 5) for 2 hours. Kidneys were extracted, detergent solubilized, and subjected to MIB/MS. Volcano plot as in D.

(Fig. 3; ref. 30). TMT labeling of peptides allows for direct preferentially precluded from MIB binding over CDK4/6 (Fig. quantitation at the peptide level by measuring the ratio of TMT 3B). To further confirm that these findings are not specificto labels in a MS3 scan. H2228 cells were treated for an hour with H2228 cells, a similar experiment was conducted in vehicle or increasing doses of abemaciclib prior to kinase enrichment and abemaciclib-treated DB cell lysates (diffuse large B-cell lympho- peptide labeling with TMTs (Fig. 3A). Quantitation of 128 kinases ma cell line; Supplementary Fig. S2). Again, GSK3a and GSK3b across two biological replicates revealed that abemaciclib inhibits MIB binding are inhibited at lower doses than that required to GSK3b MIB binding at concentrations lower than that required to inhibit CDK4/6 MIB binding (148 quantified kinases across both inhibit CDK4/6 MIB binding (Fig. 3A). Many previous kinase replicates; Supplementary Fig. S2). chemoproteomic studies have chosen to treat cell lysates as opposed to live cells. To address the possibility of differential GSK3b is an abemaciclib-specific target inhibitor target profiles derived from treated cells versus treated GSK3b is an integral kinase within the b-catenin destruction lysates, a similar dose–response experiment was conducted in complex. GSK3b phosphorylates b-catenin, leading to b-catenin H2228 abemaciclib-treated cell lysates (Fig. 3B). Abemaciclib ubiquitylation and proteasome-mediated degradation (31). treatment of H2228 lysates yielded similar results to H2228- Free b-catenin can then translocate to the nucleus, bind TCF/LEF, treated cells, whereby TMT ratios were quantified on 133 kinases and initiate transcription of target genes. Importantly, mutations observed in both biological replicates. As before, GSK3a and b are within the canonical WNT pathway have been linked to

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A Input AP:MIB B Input AP:MIB

Abemaciclib (μmol/L) Abemaciclib (μmol/L) Palbociclib (μmol/L) Palbociclib (μmol/L) 0.006 0.06 0.6 0.06 0.6 6 0.6 Veh Veh 6 0.006 0.06 0.6 6 Veh Veh 0.006 0.006 0.06 6 CDK6 CDK6

CDK4 CDK4

AXL AXL

GAPDH BTUBB

H2228 Cells H2228 Cells

C D Abemaciclib vs. vehicle Palbociclib vs. vehicle H2228 cells H2228 cells CAMK2G CDC42BPB CSNK2A2 1.5 TAOK3 MAP2K4 PIP4K2B 5% FDR CAMK2D CDK9 CDK17 CSNK2A1 AAK1 ROCK2 GSK3A 3 PRKCA PRKCQ CDK4 EIF2AK2 PRKD2 RPS6KA5 GSK3B ADK CDK4 CDK6 DAPK3 1.0 CDK6 CDK16 CLK2 PRKCD PKN2 2 IRAK1 PRKD3 PIKFYVE q-value q-value CAMK2G 10 10

NEK1 −Log 5% FDR −Log 0.5 CAMK2D 1 GSK3B

0 0.0

−10 −8 −6 −4 −2 0 246810 −5 −4 −3 −2 −1 012345

Log2 fold change Log2 fold change

Figure 2. MIB/MS competition reveals novel targets of abemaciclib. A, MIB/AP WB of H2228 cells treated with abemaciclib or DMSO for 1 hour. B, MIB/AP WB of H2228 cells treated with palbociclib or DMSO for 1 hour. C, Volcano plot of DMSO versus 6 mmol/L abemaciclib (three biological replicates) in H2228 cells. In the volcano plot, vertical dashed lines indicate two-fold label free quantitation (LFQ) change, and horizontal dashed line depicts a 5% FDR threshold across three 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 more than two-fold LFQ decrease in abemaciclib-treated cells; of these, kinases meeting a 1% FDR are labeled. D, Volcano plot of DMSO versus 6 mmol/L palbociclib (two biological replicates for palbociclib and three biological replicates for DMSO) as described in C. Filled purple circles denote CDK4 and CDK6. Filled blue circles indicate kinases that are targeted by abemaciclib but not palbociclib.

numerous types of cancer, autoimmune disease, and bone density was retained in the presence of both kinase inhibitors (Figs. 4A and disorders (32). For example, loss-of-function mutations in ade- B). A third CDK4/6 inhibitor was assessed for its ability to inhibit nomatous polyposis coli APC or gain-of-function mutations GSK3b binding to MIBs. Ribociclib (LEE011) was recently granted þ within CTNNB1 (gene encoding b-catenin) are causative for FDA approval for the treatment of HR /HER2 breast cancer when colorectal cancer and hepatocellular carcinoma (33–35). Because used in combination with aromatase inhibitors. Although short- of the importance of WNT signaling in cancer, we sought to test term (1 hour) treatment of H2228 cells with ribociclib inhibited the relationship between abemaciclib, GSK3b activity, and WNT CDK4 and CDK6 binding to MIBs, GSK3b was completely unaf- pathway activation. fected at doses up to 10 mmol/L (Fig. 4C). Furthermore, abemaci- MIB/AP Western blot experiments were performed to validate a clib, but not palbociclib, precluded GSK3b and CAMKIIb/d/g MIB subset of abemaciclib targets. Abemaciclib blocked the binding of binding in a second NSCLC cell line (H1703 cells; Fig. 4D). CDK6, CAMK2b/d/g (hereafter referred to as CAMKIIb/d/g), and GSK3b to MIBs (Fig. 4A). In contrast, palbociclib treatment did not Abemaciclib directly inhibits GSK3b alter GSK3b MIB binding and only moderately suppressed To test whether GSK3b kinase activity was directly inhibited by CAMKIIb/d/g at the highest dose (Fig. 4B). AXL binding to MIBs abemaciclib, in vitro kinase activity assays were performed on a

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Abemaciclib Inhibits GSK3b and Activates WNT Signaling

A Abemaciclib dose-response Abemaciclib activates WNT signaling via its inhibition of H2228 cells GSK3b Having established that abemaciclib directly inhibits GSK3b in the low nanomolar range, we tested whether abemaciclib-medi- 0 AXL ated inhibition of GSK3b resulted in stabilization of b-catenin protein and induction of b-catenin–dependent transcription. b-Catenin transcriptional activity was quantiﬁed in multiple cell lines using the b-catenin activated reporter (BAR) Fireﬂy −2 luciferase reporter (36). As expected, abemaciclib, but not palbociclib, demonstrated a dose-dependent activation of the BAR CDK4 reporter in RKO colorectal adenocarcinoma cells, HEK293T/17 (fold change from vehicle)

2 CDK6 embryonic kidney cells, and H2228 NSCLC cells across three

Log −4 biological replicates (Fig. 6A). WNT3A-conditioned media (CM) CAMK2D and the GSK3b inhibitor CHIR-99021 served as positive controls CAMK2G GSK3A (Fig. 6A). To evaluate WNT pathway activation over time, GSK3B HEK293T/17 cells carrying a b-catenin-driven GFP reporter Veh 0.006 0.06 0.6 6 (HEK293T/17 BAR-GreenFire) were treated with vehicle, CHIR- Abemaciclib concentration (μmol/L) 99021, WNT3A CM, abemaciclib, or palbociclib and monitored by live cell imaging over 24 hours. Abemaciclib, but not palbo- B Abemaciclib dose-response ciclib, induced GFP expression in a dose-dependent manner H2228 lysate (Fig. 6B and C). As expected, WNT3A CM and CHIR-99021 also activated the reporter (Fig. 6B and C). AXL Next, b-catenin protein levels were assessed by Western blot 0 analysis of RKO cells treated with vehicle, abemaciclib, palbociclib, CHIR-99021, or recombinant WNT3A (rWnt3A) for 6 hours. Abemaciclib dose-dependently stabilized b-catenin protein levels, whereas palbociclib treatment had no effect (Fig. 6D). CDK4 −2 These observations were conﬁrmed in a second cell line (Fig. 6E). CDK6 CAMK2G Finally, to determine whether abemaciclib and CHIR-99021 GSK3B induce b-catenin protein stabilization with similar kinetics, a (fold change from vehicle) 2 CAMK2D time course experiment was performed. RKO and L cells were

Log −4 treated with abemaciclib, CHIR-99021, or palbociclib for up to 6 hours. b-Catenin protein stabilization occurred within 30 min- GSK3A utes of CHIR-99021 or abemaciclib treatment and continued throughout the time course (Fig. 6F and G). Conversely, 6-hour Veh 0.006 0.06 0.6 6 vehicle or palbociclib treatment did not affect b-catenin protein Abemaciclib concentration (μmol/L) abundance (Fig. 6F and G). These data indicate that abemaciclib- Figure 3. mediated inhibition of GSK3b activates WNT signaling via b-cate- Abemaciclib dose-dependently inhibits GSK3b from binding MIBs. A, nin protein stabilization and transcriptional activity. Abemaciclib dose–response in H2228 cells using isobaric TMT labeling. Data are plotted as the mean log2 fold-change compared to vehicle SE for two biological replicate experiments. TMT ratios for 128 kinases were quantified Discussion – across both biological replicates. B, Abemaciclib dose response in H2228 lysate The MIB/MS competition platform described here provides a using isobaric TMT labeling. Data are plotted as the mean log2 fold-change compared to vehicle SE for two biological replicate experiments as in A. TMT powerful approach for identifying putative targets of kinase ratios for 133 kinases were quantified across both biological replicates. inhibitors in cell lysate, live cells, and in tissues. We discovered known and novel targets for several kinase inhibitors. Our data and conclusions support the widely-held view that understanding panel of kinases (Fig. 5). These experiments confirmed that both the kinase inhibitor target profile of clinically-active drugs may abemaciclib and palbociclib comparably suppressed their cognate help to inform toxicities associated with the inhibition of "other targets: CDK4/cyclin D1 [IC50 values of 0.46 nmol/L (abemaci- targets" and offers experimental support for drug repurposing clib, A) and 1.3 nmol/L (palbociclib, P)], CDK6/cyclin D1 [0.43 strategies (37). We suggest that target profile annotation via MIB/ nmol/L (A), 0.43 nmol/L (P)], CDK4/cyclin D3 [6.2 nmol/L (A), MS or kinobead competition might be incorporated into drug 7.0 nmol/L (P)], and CDK6/cyclin D3 [8.9 nmol/L (A), 5.1 nmol/ development pipelines prior to clinical use to minimize unin- L (P); Fig. 5A–D]. Abemaciclib was >1,000-fold more potent for tended consequences and to maximize patient safety. Future work GSK3b than palbociclib [IC50 of 8.67 nmol/L (A), 11.2 mmol/L using competitive chemical proteomics for all FDA-approved (P); Fig. 5E). Both compounds were also tested for their ability to kinase inhibitors and promising clinical candidates is warranted. inhibit CAMKIIb, CAMKIId, and CAMKIIg. Abemaciclib was >100 Previous studies have reported similar competitive kinase times more specific for the CAMKII proteins when compared to enrichment proteomic platforms (11, 13, 29). The majority of palbociclib [CAMKIIb:IC50 of 3.5 nmol/L (A), 1.6 mmol/L (P); these experiments evaluated differential kinase capture in treated CAMKIId: 2.6 nmol/L (A), 610 nmol/L (P); CAMKIIg: 52 nmol/L protein lysates as opposed to the live cell treatments used here. (A), 9.4 mmol/L (P)] as show in Fig. 5F to H. Interpretation of live cell competitive MIB data includes

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A Input AP:MIB B Input AP:MIB Abemaciclib (μmol/L) Abemaciclib (μmol/L) Palbociclib (μmol/L) Palbociclib (μmol/L) 0.06 6 Veh 0.06 0.6 6 0.006 0.6 0.006 0.06 0.6 6 0.006 0.06 0.6 6 Veh 0.006 Veh Veh CDK6 CDK6

GSK3β GSK3β

AXL AXL

CAMKII pan CAMKII pan

GAPDH GAPDH

H2228 Cells H2228 Cells

C Input AP:MIB D Input AP:MIB Ribociclib (μmol/L) Ribociclib (μmol/L) Abema. (μmol/L) Palbo. (μmol/L) Abema. (μmol/L) Palbo. (μmol/L) 1 10 0.01 0.1 10 0.01 0.1 Veh Veh 1 0.06 0.06 0.006 0.06 0.6 6 Veh 0.006 0.06 0.6 6 Veh 0.006 0.6 0.006 0.6 Veh 6 CDK6 Veh 6 CAMKII pan CDK4 GSK3β GSK3β AXL AXL GAPDH

GAPDH H1703 Cells

H2228 Cells

Figure 4. GSK3b is an abemaciclib-specific target. A, MIB/AP WB validation of GSK3b and CAMKIIb/d/g following abemaciclib treatment of H2228 cells. B, MIB/AP WB confirming that palbociclib does not affect GSK3b or CAMKIIb/d/g MIB binding. C, MIB/AP WB indicating that ribociclib does not preclude GSK3b from binding MIBs. D, MIB/AP WB in H1703 cells indicating abemaciclib-specific effects on GSK3b and CAMKIIb/d/g MIB binding in a second cell line.

assumptions of physiological protein–protein interactions, sub- (Supplementary Table S2; refs. 27, 28). New abemaciclib targets cellular locations, and ATP/ADP concentrations. How these vari- not previously reported include several additional CDKs (12–17 ables impact a lysate-based versus cell-based competitive chem- and 19), AAK1, IKBKB, MET, PLK4, and others (Supplementary ical proteomics dataset remains to be fully examined. Data pre- Table S2). Discordance between kinase targets observed via in vitro sented here suggest that inhibitor-treated cells and lysates yield versus in vivo methods are likely driven by (i) differences in the similar results by competitive MIB/MS, although further experi- dose of abemaciclib tested, (ii) MS detection limitations, (iii) ments are necessary to comprehensively evaluate potential differ- capacity of MIBs to bind the kinase, (iv) kinase expression in a ences using multiple kinase inhibitors in lysates and in live cells. given cell line or tissue, and (v) differences in ATP and/or cofactor Second, although not tested in this study, competitive MIB concentrations. experiments in treated mice should reveal tissue-restricted kinase The value of studying target profiles of kinase inhibitors is well drug targets, owing to differential kinase expression and drug illustrated by our analysis of the clinically active and FDA- bioavailability. Third, comparative analyses of MIB- or kinobead- approved CDK4/6 inhibitors. Distinct side effect profiles are based approaches, whether in cells or lysates, must incorporate an observed in treated patients. Palbociclib leads to hematologic understanding of differences in the chemical bead mixtures toxicity and neutropenia, whereas abemeciclib treatment results employed, the length of time the beads are incubated with the in gastrointestinal (GI) toxicity and more efficient blood–brain protein extract, and the quantitative proteomic approaches used. barrier penetration (38, 39). Furthermore, abemaciclib is effective Finally, although a few kinases have been shown to bind specific as a single agent whereas palbociclib is given in combination with beads in an activity-dependent manner, the MIBs experimental letrozole (39). Neutropenia and leukopenia were the most com- platform described here reports competitive kinase capture inde- monly reported grade 3 or 4 adverse event in ribociclib-treated pendent of kinase activity. patients in a recent phase III trial (40). The differential target Comparative analysis of our abemaciclib competitive MIB/MS profiles of these compounds likely contributes to their differential data with previously reported abemaciclib targets obtained from dose-limiting toxicities. in vitro kinase activity and mobility shift assays reveal overlaps and Our data show that abemaciclib potently and directly inhibits unique discoveries (Supplementary Table S2). Common targets GSK3b and consequently activates WNT/b-catenin signal trans- across these experiments include multiple CDKs (1, 2, 5, 7, and 9), duction. Palbociclib and ribociclib do not impact GSK3b or WNT GSK3a and b, CAMKIIb and d, IRAK1, SLK, PKN1, and others signaling. Further studies are needed to determine if abemaciclib

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Abemaciclib Inhibits GSK3b and Activates WNT Signaling

A CDK4/cyclin D1 B CDK6/cyclin D1 120 Abemaciclib 120 Abemaciclib Palbociclib Palbociclib 100 100 80 80 60 60 % Activity % Activity 40 40 20 20 0 0 -11 -10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 Log [Compound] (mol/L) Log [Compound] (mol/L) Abemaciclib Palbociclib Abemaciclib Palbociclib HillSlope -1.31 -0.88 HillSlope -0.96 -1.09 IC50 [mol/L] 4.6e-010 1.3e-009 IC50 [mol/L] 4.3e-010 4.3e-010 C CDK4/cyclin D3 D CDK6/cyclin D3 120 120 Abemaciclib Abemaciclib 100 Palbociclib 100 Palbociclib 80 80 60 60

% Activity 40 % Activity 40 20 20 0 0 -11 -10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 Log [Compound] (mol/L) Log [Compound] (mol/L) Figure 5. Abemaciclib Palbociclib Abemaciclib Palbociclib In vitro kinase activity assays indicate that HillSlope -1.81 -1.41 HillSlope -0.76 -0.79 6.2e-009 7.0e-009 IC [mol/L] 8.9e-009 5.1e-009 abemaciclib directly inhibits GSK3b. In vitro kinase IC50 [mol/L] 50 activity assays for abemaciclib and palbociclib against E GSK3β F CAMKIIβ CDK4/cyclin D1 (A), CDK6/cyclin D1 (B), CDK4/cyclin 120 120 D3 (C), CDK6/cyclin D3 (D), GSK3b (E), CAMKIIb (F), Abemaciclib Abemaciclib CAMKIId (G), and CAMKIIg (H). 100 Palbociclib 100 Palbociclib 80 80 60 60

% Activity 40 % Activity 40 20 20 0 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3 Log [Compound] (mol/L) Log [Compound] (mol/L) Abemaciclib Palbociclib Abemaciclib Palbociclib HillSlope -0.95 -0.84 HillSlope -0.77 -0.80 8.7e-009 1.1e-005 3.5e-009 1.6e-006 IC50 [mol/L] IC50 [mol/L] G CAMKIIδ H CAMKIIγ 120 120 Abemaciclib Abemaciclib 100 Palbociclib 100 Palbociclib 80 80 60 60

% Activity 40 % Activity 40 20 20 0 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3 Log [Compound] (mol/L) Log [Compound] (mol/L) Abemaciclib Palbociclib Abemaciclib Palbociclib HillSlope -0.86 -0.84 HillSlope -0.81 -0.93 2.6e-009 6.1e-007 5.2e-008 9.4e-006 IC50 [mol/L] IC50 [mol/L] activates WNT signaling in patients and whether this activation generally dosed continuously in patients at up to 200 mg every contributes to observed toxicities, particularly within the GI 12 hours for 2 to 3 weeks (41). As such, long-term exposure to tract. WNT signaling contributes signiﬁcantly to bone density abemaciclib may lead to continuous WNT signaling in these disorders, a myriad of cancers, metabolic disorders, and neuro- patients, a potentially harmful side effect that should be developmental and degenerative diseases (32). Abemaciclib is considered.

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RKO B/R HEK293T/17 B/R H2228 B/R A 8 0.9 0.35 7 0.8 0.30 6 0.7 0.6 0.05 5 0.5 0.04 4 0.4 0.03 3 0.3 0.02 2 0.2 1 0.1 0.01 Firefly/ Renilla (arbitrary units) Firefly/ Renilla (arbitrary units)

0 0 Firefly/ Renilla (arbitrary units) 0

Palbociclib Abemaciclib Palbociclib Abemaciclib Palbociclib Abemaciclib CHIR-99021 WNT3A CM Veh L cell CM Veh L cell CM CHIR-99021 WNT3A CM Veh CHIR-99021 WNT3A CM L cell CM Treatment Treatment Treatment

6x105 WNT CM L-cell CM 1 uM CHIR-99021 DMSO B C DMSO L-cell CM Palbociclib (2.5 μmol/L) 0.625 uM Abema 0.625 uM Palbo

5 1.25 uM Abema 1.25 uM Palbo 5x10 2.5 uM Abema 2.5 uM Palbo 5 uM Abema 5 uM Palbo

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Total green integrated intensity 1x105

0 02 4 6 81012141618 20 22 Hours posttreatment

Abema. (μmol/L) Palbo. (μmol/L) Abemaciclib (5 μmol/L) CHIR-99021 (5 μmol/L) D F Veh Palbociclib 6 0.5123460.5123466 Time (h) 2.5 Untreated Veh 0.1 5 10 0.1 2.5 5 CHIR-99021 rWnt3A 10 β-catenin β-catenin

GSK3β GSK3β

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RKO B/R cells RKO B/R cells

E Abema. (μmol/L) Palbo. (μmol/L) G Abemaciclib (5 μmol/L) CHIR-99021 (5 μmol/L) Veh Palbociclib 6 0.5 1 2 3 4 6 0.5 1 2 3 4 6 6 Time (h) 5 5 CHIR-99021 Untreated 0.1 2.5 10 rWnt3A Veh 0.1 2.5 10 Ctnnb1 Ctnnb1

Gsk3β Gsk3β

Gapdh Gapdh

L-cells (murine) L-cells (murine)

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 four independent wells. The following abemaciclib and palbociclib ranges were used: 0.3125 to 5 mmol/L (RKO B/R and HEK293T/17 B/R) or 0.625 to 10 mmol/L (H2228 B/R). CHIR-99021 was treated at 1 mmol/L. 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 mmol/L CHIR-99021, or WNT or L-cell CM for 24 hours. 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-hour posttreatment. Scale bar ¼ 200 mm. D and E, Evaluation of b-catenin levels by WB analysis after 6-hour treatment of DMSO, abemaciclib (0.1–10 mmol/L), palbociclib (0.1–10 mmol/L), 1 mmol/L CHIR-99021, or recombinant WNT3A (rWNT3A, 200 ng/mL) in RKO B/R cells (D) or murine L-cells (E). F and G, Evaluation of b-catenin levels by WB analysis of RKO B/R cells (F)or L-cells (G) following treatment with DMSO, 5 mmol/L abemaciclib, 5 mmol/L CHIR-99021, or 10 mmol/L palbociclib over a 30 minutes to 6 hour time course.

In addition to GSK3b, our data and those of previous reports occurs in the low nanomolar range (27, 28). Four human CAMKII þ indicate that abemaciclib inhibits members of the Ca2 /calmod- isoforms exist (a, b, d, and g), and they are serine/threonine ulin-dependent kinase II (CAMKII) family; in vitro studies kinases that perform many functions and are responsive to described here demonstrate that this inhibition is direct and calcium signaling (42, 43). CAMKIIg has been shown to play

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roles in cell growth and survival in liver cancer and CML, whereas Authors' Contributions CAMKIIa depletion led to reduced growth of osteosarcoma cell Conception and design: E.M. Cousins, M.B. Major lines (44–46). In T-cell lymphoma, CAMKIIg phosphorylates Development of methodology: E.M. Cousins, D. Goldfarb, F. Yan, G.L. Johnson, cMYC at serine 62, leading to stabilization of cMYC protein M.B. Major Acquisition of data (provided animals, acquired and managed patients, (47). Furthermore, inhibition of CAMKIIg resulted in decreased provided facilities, etc.): E.M. Cousins, D. Goldfarb, J. Roques, M.B. Major tumor loads, suggesting that CAMKIIg could be exploited as a Analysis and interpretation of data (e.g., statistical analysis, biostatistics, therapeutic target in T-cell lymphoma (47). Phosphorylation of computational analysis): E.M. Cousins, D. Goldfarb, M.B. Major CAMKII at threonine 286 was increased in breast cancer tissue Writing, review, and/or revision of the manuscript: E.M. Cousins, G.L. Johnson, compared to matched normal tissue, and phosphorylated T286 M.B. Major CAMKII regulated metastatic potential of breast cancer cells (48). Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.M. Cousins, D. Darr, M.B. Major Because of the roles of CAMKII in cell growth and invasion in Study supervision: M.B. Major multiple tumor types, CAMKII is a rational therapeutic target. Berbamine is a natural product derived from the Berberis amurensis Acknowledgments shrub and has been shown to inhibit CAMKII in Huh7 cells with an This work was supported by the following: V Foundation grant number IC50 of 5.2 mg/mL (7.6 mmol/L given berbamine MW of 681.65; T2014-009 to M.B. Major and G.L. Johnson, Gabrielle's Angel Foundation ref. 45). Data presented here suggest that abemaciclib is a better (grant number 85) to M.B. Major, NIH/NCI grant (5R01CA187799) to M.B. inhibitor of CAMKII as in vitro kinase activity assays calculated IC50 Major, and the NIH T32 Postdoctoral Training Grant in Pulmonology values between 2 and 52 nmol/L depending on the CAMKII (5T32HL007106-39) to E.M. Cousins. isoform. Furthermore, treatment of live H2228 NSCLC cells with The authors thank members of the Major Laboratory and Johnny Castillo for 60 nmol/L abemaciclib led to a >60% reduction in CAMKIId feedback, reagents, and expertise regarding project design and experimental procedures. and CAMKIIg MIB binding (Fig. 3 and Supplementary Table S1). Based on these data and previous reports, future studies The costs of publication of this article were defrayed in part by the should be conducted to determine whether abemaciclib could be payment of page charges. This article must therefore be hereby marked repurposed as a CAMKII inhibitor for the treatment of CAMKII- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate driven disease. this fact.

Disclosure of Potential Conﬂicts of Interest Received August 25, 2017; revised October 17, 2017; accepted November 1, No potential conﬂicts of interest were disclosed. 2017; published OnlineFirst November 13, 2017.

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344 Mol Cancer Res; 16(2) February 2018 Molecular Cancer Research

Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3 β and Activates WNT Signaling

Emily M. Cousins, Dennis Goldfarb, Feng Yan, et al.

Mol Cancer Res 2018;16:333-344. Published OnlineFirst November 13, 2017.

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