Pyrvinium Attenuates Hedgehog Signaling Downstream of

Therapeutics, Targets, and Chemical Biology

Pyrvinium Attenuates Hedgehog Signaling Downstream ofSmoothened

Bin Li1, Dennis Liang Fei1, Colin A. Flaveny1, Nadia Dahmane2, Val�erie Baubet2, Zhiqiang Wang1, Feng Bai1,Xin-Hai Pei1,3, Jezabel Rodriguez-Blanco1, Brian Hang4, Darren Orton5, Lu Han1, Baolin Wang6,Anthony J. Capobianco1,3,7, Ethan Lee4, and David J. Robbins1,3,7

AbstractThe Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling

pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industryis currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of theHH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of thesecompounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquiredmutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independentactivation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trialsagainst other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms ofaction, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinwormcompound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1a (CK1a) agonist. We show here thatpyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family oftranscription factors. Consistent with CK1a agonists acting on thesemost distal components of the HH signalingpathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smomutant, aswellas the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate theutility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growthand reducing the expression of HH biomarkers. Cancer Res; 74(17); 4811–21. �2014 AACR.

IntroductionThe Hedgehog (HH) signaling pathway plays key instruc-

tional roles during embryonic development and adult tissuehomeostasis. Consistent with this pivotal instructional role,the HH signaling pathway is commonly deregulated in manyhuman cancers (1). The role HH plays in cancer was firstidentified in the inherited disorder Gorlin syndrome, whichpredisposes to basal cell carcinoma, medulloblastoma andrhabdomyosarcoma, and results from loss-of-function muta-tions in the gene encoding the HH core receptor component

Patched1 (Ptch1; ref. 2). Spontaneous cases of these tumorswere subsequently shown to result from mutations or ampli-fications of a number of HH signaling components, includingPtch1. Strong support for the role that HH signaling plays inthese cancers was provided from a number of genetic mousemodels of HH-driven medulloblastoma, in which mutations inHH signaling components lead to the genesis of the sametumors (3, 4). The growth of the tumors in thesemice could alsobe abrogated by treatment with HH signaling inhibitors (5).The pharmaceutical industry is currently focused on develop-ing small molecules targeting Smoothened (Smo), a key sig-naling effector of the HH pathway that regulates the levels andactivity of the Gli family of transcription factors (2). Althoughone of these compounds, vismodegib, is nowFDA-approved forpatients with advanced basal cell carcinoma (2), acquiredmutations in Smo can result in rapid relapse (6). Furthermore,many cancers also exhibit a Smo-independent activation of Gliproteins (7), an observation that may underlie the limitedefficacy observed for Smo inhibitors in clinical trials againstother types of cancer (2). Thus, there remains a critical need forHH inhibitorswith differentmechanismsof action, particularlythose that act downstream of Smo.

HH signaling is activated by binding of theHH ligands [Sonic(SHH), Indian, or Desert] to a receptor consisting of a Ptchprotein (Ptch1 or Ptch2) and one of three coreceptors (8). Thisresults in derepression of the G-protein–coupled seven-trans-membrane protein Smo. Ultimately, canonical HH signaling

1Molecular Oncology Program, Department of Surgery, University ofMiami, Miami, Florida. 2Department of Neurosurgery, University of Penn-sylvania, Philadelphia, Pennsylvania. 3Sylvester Cancer Center, Universityof Miami, Miami, Florida. 4Department of Cell and Developmental Biologyand Vanderbilt Ingram Cancer Center, Vanderbilt University School ofMedicine, Nashville, Tennessee. 5StemSynergy Therapeutics Inc., Miami,Florida. 6Weill Medical College, Cornell University, New York, New York.7Department of Biochemistry and Molecular Biology, University of Miami,Miami, Florida.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthor:David J. Robbins, University ofMiamiMiller Schoolof Medicine, The DeWitt Daughtry Family Department of Surgery, MolecularOncology Program, 1600 NW 10th Avenue. Miami, FL 33136. Phone: 305-243-5717; Fax: 305-243-9694; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0317

�2014 American Association for Cancer Research.

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http://cancerres.aacrjournals.org/

regulates the activity, proteolytic processing, and stability ofmembers of the Gli family of transcription factors, Gli1-3 (7).This regulation requires a number of protein kinases, includingprotein kinase A (PKA), glycogen synthase kinase 3 (GSK3), andcasein kinase1a (9–12), and the negative regulator suppressorof fused (Sufu; refs. 13, 14). Mammalian HH signaling requirestrafficking through primary cilia, a membrane-encased micro-tubule-enriched organelle located on the apical side of polar-ized cells (8, 15). Many components of the HH signalingpathway transit through the primary cilia in their basal stateand leave or enrich there in response to HH (8). During thistrafficking through the specialized environment of the primarycilia, Gli proteins, likely through their interactionwith Sufu, areconverted into their repressor forms or in response to HHconverted into their active forms (16–18). In the basal state,Gli2 andGli3 are hyperphosphorylated at their Cul1-dependentdegrons (10, 11, 19). Subsequent to ubiquitination, Gli2 andGli3 are partially cleaved by proteasomes into their repressorforms. In response to HH, Gli2 and Gli3 become differentiallyphosphorylated by PKA, at a distinct amino-terminal domain,converting them into their activated nuclear forms (19–21).Nuclear-enriched Gli2 and Gli3 are labile and are quicklydegraded by the proteasome through a Cul3-mediated ubiqui-tin proteasome system (17, 22).

We recently reported that the FDA-approved drug pyrvi-nium is a novel and potent small molecule inhibitor of theWntpathway (IC50, 10 nmol/L; ref. 23). We identified CK1a as thecritical cellular target of pyrvinium and showed that pyrviniumacts as an allosteric activator of this protein kinase. Our in vitroand cellular binding studies demonstrated that pyrviniumbinds avidly to CK1a (Kd, 1 nmol/L; ref. 23). Of the CK1 familymembers (a, g , d, and e), only CK1a is activated by pyrvinium.Pyrvinium has no effect on the activities of a panel of otherprotein kinases representing all of the major branches of thekinase superfamily (23), demonstrating that pyrvinium selec-tively binds to and activates CK1a. As CK1a is implicated as anegative regulator of the HH signaling pathway, we hypothe-sized that pyrvinium might inhibit HH signaling (24). Here, weshow that pyrvinium does indeed attenuate HH signaling, anddoes so in vitro and in vivo—attenuating the growth of a well-accepted HH-driven mouse tumor model. Furthermore, pyr-vinium acts to regulate Gli activity and stability downstream ofSmo, in a CK1a-dependent manner, including attenuating HHactivity driven by a clinically relevant, vismodegib-resistantSmo mutation (6).

Materials and MethodsCell culture

NIH-3T3, HEK 293T, and Light-II cells were purchased fromthe American Type Culture Collection (ATCC) andwere grownin medium as indicated by ATCC's instructions. Cerebellargranular precursor cells (GPC) were isolated as previouslydescribed (25). NIH-3T3 cells stably expressing HA-Gli2 werea gift of Dr. Philip Beachy (Stanford University, Stanford, CA).Transfections were performed using Lipofectamine 2000 (Invi-trogen) according to themanufacturer's instructions.Myc- andFlag-tagged Gli1 and Myc-tagged b-Trcp were gifts of Dr.

Anthony Oro (Stanford University, Stanford, CA).Myc-Gli2 andGFP-Smo were from Addgene. Various Gli-null MEF cells weregifts of Dr. Wade Bushman (University of Wisconsin, Madison,WI; ref. 26). shRNA constructs described here were purchasedfrom Open Biosystems, and used to prepare lentivirus asdescribed therein. shRNA-expressing cell lines were selectedby 10 mg/mL puromycin. Smoothened agonist (SAG) andpyrvinium treatments were performed in the presence of0.5% FBS.

AssaysLuciferase activity was determined using a luciferase detec-

tion kit (Promega), as previously described (27). Total RNAfrom cells or tissues was extracted using the RNeasy kit(Qiagen), converted into cDNA (Applied Biosystems), thenanalyzed using real-time RT-PCR and the cognate Taqmanprobes, as per the manufacturer's instructions (Invitrogen).Immunohistochemical analysis of Smo localization to primarycilia was performed as previously described (27). Immunohis-tochemical staining of CK1a (Pierce) andGli1 (27)were carriedout using a Dako autostainer at the pathology core laboratoryof the University of Miami. In vitro phosphorylation of recom-binant human Gli1 by CK1a (Invitrogen) using radiolabeled[32P]ATP was performed as previously described (28). Hexa-histidine-tagged human Gli1 was produced using the Sf9/baculovirus system. Statistical analysis was determined by theStudent two-tailed t test, unless other stated. P values � 0.05were considered statistically significant.

BiochemistryImmunoblot analysis was carried out with the following

primary antibodies: anti-Gli1 (Cell Signaling Technology), anti-Gli2 (R&D Systems), anti-Gli3 (9), anti-HA (Santa CruzBiotechnology), anti-Myc (Santa Cruz Biotechnology), anti-Flag (Sigma), anti-Tubulin (Sigma), anti-Gapdh (Millipore),anti-ERK (Santa Cruz Biotechnology), anti-pAKT (Cell Signal-ing Technology), anti-pGSK3 (Cell Signaling Technology), anti-p-b-catenin (Cell Signaling Technology). CA-AKT plasmid(myrAkt; Addgene), Bafilomycin A1 (Sigma), and MG132 (Cal-biochem) were purchased. For immunoprecipitation, cellswere lysed in a buffer containing 50 mmol/L Tris–HCl (pH,7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, and 1% NP-40,supplemented with cOmplete Mini Protease Inhibitor Cocktail(Roche). After centrifugation, the supernatants were incubatedwith the indicated antibodies overnight at 4�C, and the immu-noprecipitates were extensively washed with lysis buffer, elut-ed with SDS sample buffer, and boiled for 5 minutes beforeanalyses by immunoblotting.

Mice and drug administrationAll mice were handled in accordance with the policies of the

University of Miami Institutional Animal Care and Use Com-mittee. Spontaneous medulloblastomas from Ptchþ/� mice(The Jackson Laboratory; Ptch1tm1Mps/J) were grafted ontoCD-1 nude mice (Charles River Laboratories) subcutaneously.Drug treatment started when the tumors reached a size ofapproximately 100 mm3. For acute treatment, pyrvinium wasdissolved in DMSO and delivered through intraperitoneal

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Cancer Res; 74(17) September 1, 2014 Cancer Research4812




injection for the indicated times. For local peritumoral deliv-ery, pyrvinium chloride was resuspended in PBS containing10% (2-Hydroxypropyl)-cyclodextrin and injected close to thetumor nodule every other day.

ResultsWe recently identified CK1a as the critical cellular target of

pyrvinium and showed that pyrvinium acts as a novel allostericactivator of its protein kinase activity (23). AsCK1a is also a keyregulator of HH signaling (24), we hypothesized that pyrviniumwould function as an inhibitor of this important developmen-tal signaling pathway. To test this hypothesis, we evaluatedpyrvinium's ability to attenuate HH signaling. We stimulatedHH activity in a HH reporter cell line (Light-II cells), usingeither SHH or the Smo agonist SAG, and in both cases

pyrvinium attenuated HH activity in a potent (IC50 of �10nmol/L), dose-dependent manner (Fig. 1A). Pyrvinium alsoinhibited HH-dependent expression of Gli1 and Ptch1, twowell-established biomarkers of HH activity (Fig. 1B; ref. 1). Incontrast, the inactive structural analog of pyrvinium VU211(23) was unable to attenuate the expression of these HHbiomarkers. We next evaluated the ability of pyrvinium toinhibit the proliferation of HH-dependent primary GPC, anoth-erwell-established readout ofHHactivity (25, 29). PrimaryGPCwere isolated and treated with SHH plus pyrvinium or the Smoantagonist cyclopamine. Pyrvinium attenuated GPC prolifer-ation, and did so in a manner similar to cyclopamine (Fig. 1C).These data indicate that pyrvinium is a potent inhibitor of HHsignaling.

Pyrvinium has been reported to exhibit biologic effects viamechanisms distinct from CK1a activation (30–33). Most

Figure 1. Pyrvinium suppresses HHsignaling in a CK1a-dependentmanner. A, Light-II cells treatedwith SHH, or the Smo agonist SAG,were subsequently incubated withthe indicated doses of pyrvinium,or vehicle (DMSO), and luciferaseactivity was determined. B, SHH-treated NIH-3T3 cells were treatedwith DMSO, pyrvinium, or theinactive pyrvinium analog VU211.Twenty-four hours later, RNA washarvested from these cells and theexpression of the HH target genesGli1 and Ptch1 determined relativeto that of the housekeeping geneGapdh. C, primary GCP cells wereincubated with BrdUrd and theindicated drugs (cyclopamine, 5mmol/L; pyrvinium, 100 nmol/L) for48 hours, followed by quantitationof BrdUrd incorporation usingmicroscopy. D, Light-II cells wereinfected with lentivirus expressingthe indicated shRNA. Seventy-twohours after infection, cells weretreated with the indicated agents(SAG, 100 nmol/L; pyrvinium, 1, 10,100 nmol/L) for 48 hours. RNA washarvested from these cells and theexpression of Gli1 determinedrelative to the expressionofGapdh.E, Light-II cells were treated withthe indicated agents in thepresence or absence of the Wntinhibitor IWR-1 (10 mmol/L) andluciferase activity was determined.F, HH or Wnt signaling activity inGli2�/�; Gli3�/�MEFs was inducedwith either SAG or Wnt3aconditioned media, in the absenceor presence of 10 nmol/Lpyrvinium. The expression of theWnt target gene Dkk1, normalizedto the expression of Gapdh, wasanalyzed as a readout of Wntactivity. Error bars, SEM (n ¼ 3);�, P < 0.05.

Casein Kinase 1a Agonists Attenuate HH Signaling

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recently, it has been suggested that pyrvinium attenuates Wntactivity by blocking activation of AKT, preventing GSK3bphosphorylation and its subsequent inactivation, which resultsin the phosphorylation and destabilization of b-catenin (34).However, we show that the expression of a constitutive activeAKT construct did not abolish pyrvinium's suppression of HHsignaling (Supplementary Fig. S1A). Furthermore, we recentlydetermined pyrvinium's ability to bind to the active site of alarge panel of protein kinases (442 purified kinases) usingAmbit's scanMAX kinase profiling service (data not shown).This screen examined the ability of pyrvinium to outcompetekinases bound to a set of immobilized active site ligands. Theseligands bind to the active site of one or more kinases with highaffinity (Kd <1 nmol/L). Different concentrations of pyrviniumwere used to outcompete the individual kinases from theimmobilized ligands. Our data suggest that pyrvinium doesnot bind to the active site of CK1a, as pyrvinium did notcompete with CK1a binding to its immobilized ligand in thisassay. Consistentwith this result, we did not observe the effectsof ATP on pyrvinium activity. We did, however, observe effectsof pyrvinium on the conformation of CK1a as detected by analtered trypsin proteolysis pattern (23). Significantly, the scan-MAX kinase screen failed to detect inhibition of any of thekinases tested (using 1 mmol/L of pyrvinium), which includedAKT1-3 isoforms and all known PI3-kinases. Furthermore,consistent with pyrvinium acting through CK1a, we show thatthe addition of pyrvinium to cells results in the time-dependentphosphorylation of a known CK1a substrate (SupplementaryFig. S1B).

Tomore directly show that pyrvinium's ability to inhibit HHsignaling is CK1a dependent, we identified two shRNAs capa-ble of reducing CK1a protein levels (Supplementary Fig. S2A).Light-II cells infected with this lentivirus were treated withSAG to stimulate HH activity, followed by pyrvinium treat-ment.CK1a knockdown reduced the capability of pyrvinium toattenuate the expression of Gli1, relative to cells infected withcontrol shRNA lentivirus (Fig. 1D). We also observed thatknockdown of CK1a reduced the overall levels of SAG-inducedHH target gene expression, as might be expected, given thepositive role that CK1a also plays in Smo activation (35).

The HH and Wnt signaling pathways negatively regulate orpositively reinforce each other depending on cell context (36).Thus, CK1a agonists could potentially attenuate HH signalingindirectly via inhibition of Wnt signaling. To test this possi-bility, we asked whether the Wnt inhibitors IWR-1 or XAV939,which mediate their activity via mechanisms distinct frompyrvinium, similarly inhibit HH signaling.We found that IWR-1and XAV939 did not suppress SAG-stimulated HH activation,nor did they potentiate the capacity of pyrvinium to inhibitHH signaling (Fig. 1E and data not shown). We then treatedGli2�/�; Gli3�/� immortalized mouse embryonic fibroblasts(MEF), which are not capable of responding to HH (26), withWnt3a in the presence and absence of pyrvinium. Althoughthese MEFs did not respond to SAG, Wnt3a-induced signalingwas still observed. Furthermore, Wnt signaling remainedpyrvinium sensitive in the absence of any HH signaling (Fig.1F). Thus, in these contexts, pyrvinium's ability to inhibit HHorWnt signaling is independent of each other.

Pyrvinium potently inhibits HH signaling stimulated by theoncogenic Smo-M2 mutant (data not shown). Given the emer-gence of vismodegib resistance in the clinic (6), due to muta-tion of its binding site on Smo, we tested whether pyrviniumcould bypass a vismodegib-resistant Smo mutant (Smo-M2-D473H; ref. 6) to inhibit HH signaling. Pyrvinium potentlyinhibited HH signaling activated by Smo-M2-D473H; vismo-degib had no observable effect (Fig. 2A). Thus, pyrvinium actsto inhibit HH signaling via a mechanism distinct from vismo-degib. We next determined the impact of pyrvinium treatmenton the localization of Smo to the primary cilia of cells, which isan indication of Smo activation (35). Our results show thatalthough pyrvinium treatment did result in a slight increase inSmo localization, in the absence of SAG treatment, thisincrease was not statistically significant (Fig. 2B). In addition,pyrvinium treatment had no impact on the increased Smolocalization observed in response to activation by SAG, whichsuggests that pyrvinium acts downstream of Smo. Consistentwith this hypothesis, pyrviniumwas able to inhibit constitutiveHH pathway activity inMEFs lacking Sufu, a negative regulatorof HH signaling (14), whereas the Smo antagonist cyclopaminewas unable to inhibit this activity (Fig. 2C).

Ultimately, HH signaling is mediated by Gli transcriptionfactors, whose activity and stability are regulated by a numberof protein kinases, including CK1a (7). We, therefore, deter-minedwhether pyrvinium's ability to inhibit the expression of aHH biomarker is Gli dependent. MEFs lackingGli1,Gli2, orGli3were all capable of responding to SAG in a manner that wasattenuated by pyrvinium (Fig. 2D). Consistent with Gli2 andGli3 playing redundant roles as HH-stimulated transcriptionfactors, immortalized MEFs lacking both Gli2 and Gli3 did notrespond to SAG to promote HH pathway activation (26). A lowlevel of SAG-induced activity was observed in MEFs lackingboth Gli1 and Gli2, and this low-level activation was mediatedby the activator form of Gli3 (26). Pyrvinium did not attenuatethis low level of activity, suggesting that pyrvinium does notregulate Gli3A-mediated transcriptional activity (Fig. 2D,right). Pyrvinium attenuated constitutive HH activity in cellsoverexpressing Gli1 (Fig. 2E) or Gli2 (data not shown), incontrast with the Smo antagonist vismodegib. These resultssuggest that pyrvinium is capable of attenuating the transcrip-tional activator function of Gli1 and Gli2, which are the majordrivers of HH activity in cancer.

As CK1a has been implicated in the proteolysis of theDrosophila Gli homolog Ci (8), we tested the hypothesis thatpyrvinium attenuates Gli activity by regulating its stability. Wetransfected the NIH-3T3 cells with a plasmid expressing Gli1and treated them with increasing doses of pyrvinium. Immu-noblotting of these cell lysates showed a dose-dependentdecrease in the levels of Gli1 relative to control (Fig. 3A). Thisdecrease in Gli1 protein levels occurred at doses of pyrviniumas low as 10 nmol/L, and was not a general consequence ofinhibiting HH activity because two structurally distinct Smoantagonists did not affect Gli1 protein levels (Fig. 3A, right).Similar results were obtained with Gli2, although in this case,pyrvinium-mediated destabilization was observed only whenHH signaling was activated (Fig. 3B). We also analyzed thelevels of Gli3 protein in 3T3 cells treated with or without

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Figure 2. Pyrvinium inhibits HH signaling downstream of Smo. A, cells expressing a vismodegib-resistant, oncogenic Smo mutant (D473-M2 Smo)were treated with the indicated drugs and then assayed for luciferase activity (left). The effectiveness of vismodegib was validated on Light-II cellstreated with the Smo agonist SAG (right). B, NIH-3T3 cells stably expressing Smo–GFP were treated with vehicle, 100 nmol/L SAG, 100 nmol/Lpyrvinium, or SAG plus pyrvinium, for 24 hours. Primary cilia localization of Smo was revealed by immunocytochemistry. Smo localization wasmanually quantitated over five random images. Representative images are shown at the bottom (green, Smo-GFP; blue, DAPI; red, primary cilia).C, Sufu�/� or Sufuþ/� MEFs were treated with the indicated agents (5 mmol/L cyclopamine) for 3 days. The expression of Gli1, Ptch1, andHip was determined and normalized to that of Gapdh. D, the indicated MEFs were treated with the Smo agonist SAG in the presence or absence ofpyrvinium (10 nmol/L). Twenty-four hours later, the expression of Gli1 (left) or Ptch1 (right) was determined. E, Light-II cells expressing Myc-Gli1were treated with the indicated drugs, followed by quantitation of luciferase activity a day later. Error bars, SEM (n ¼ 3); �, P < 0.05.






pyrvinium, and did not observe decreased levels of the full-length Gli3 protein or increased levels of Gli3-R (Fig. 3C). Inresponse to HH, both full-length Gli3 and Gli3-R levels werereduced regardless of pyrvinium treatment (Fig. 3C). Thisresult is consistent with our analysis of pyrvinium's effect inGli1�/�;Gli2�/� double-knockout MEF cells, in which pyrvi-nium has no effect on HH signaling (Fig. 2D, right). We do not

know why activation of CK1a by pyrvinium is insufficient topromote additional Gli3 processing, but speculate that thebasal levels of CK1a activity are sufficient to promote maxi-mum processing or that priming by PKA is the rate-limitingevent in Gli3 processing (19).

Consistent with pyrvinium acting through CK1a to regulateGli stability,CK1a overexpression also resulted in destabilization

Figure 3. Pyrvinium enhances the degradation of Gli transcription factors. A, NIH-3T3 cells expressing Flag-Gli1 were treated with the indicated agents(vismodegib, 100 nmol/L; cyclopamine, 5mmol/L), followed by immunoblotting of these cellular lysates. B,NIH-3T3 cells expressingHA-Gli2were treatedwiththe indicated agents (SAG, 50 nmol/L; pyrvinium, 100 nmol/L), followed by immunoblotting of immunoprecipitated Gli2. C, NIH-3T3 were treated withindicated agents (SAG, 50 nmol/L; pyrvinium, 100 nmol/L), followed by immunoblotting of endogenous Gli3. Quantification of multiple replicates (left) and arepresentative immunoblot (right) are shown for A–C. Error bars, SEM; NS, not statistically different; �, P < 0.05. D, NIH-3T3 cells stably expressing theindicated shRNA were transfected with a plasmid expressing Myc-Gli1 (1 mg in control shRNA–infected cells and 0.5 mg in CK1a-knockdown cells).These cells were subsequently treated with 10 or 100 nmol/L of pyrvinium, lysed, and then analyzed by immunoblotting. E, HEK 293T cells expressing Flag-Gli1were treatedwith 200 nmol/L pyrviniumand5mmol/LofMG132overnight. Anti-Flag immunoprecipitates, from lysates of these cells,were immunoblottedusing the indicated antibodies. F, NIH-3T3 cells expressing Myc-Gli1 were treated with 100 mg/mL cycloheximide (CHX) at the indicated time points, in thepresence or absence of 100 nmol/L pyrvinium. Immunoblotting was then performed to detect Gli1 and Gapdh levels. Error bars, SEM (n ¼ 3); �, P < 0.05.

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of the exogenous Gli1 protein (Supplementary Fig. S3A, left). Incontrast, knockdown ofCK1a attenuated pyrvinium's ability todegrade Gli1 (Fig. 3D), whereas treatment with a CK1 antag-onist (Supplementary Fig. S3A, right) increased Gli proteinlevels. To test the hypothesis that decreased stability of Glisresults from ubiquitin-mediated proteasomal and lysosomal-mediated degradation pathways, targeted via a CK1a-contain-ing protein complex, we applied pharmacologic inhibitors toblock specific protein degradation pathways and determinedtheir effect on pyrvinium-mediated Gli destabilization. Theproteasomal inhibitor MG-132, but not the lysosomal inhibitorBafilomycin A1, blocks pyrvinium-induced Gli1 destabilization(Supplementary Fig. S3B), suggesting that pyrvinium regulatesGli levels via proteasome-mediated degradation. Consistentwith this, pyrvinium greatly increased the level of Gli1 ubiqui-tination (Fig. 3E) and decreased the half-life of Gli1 protein incells treatedwith the protein synthesis inhibitor cycloheximide(Fig. 3F and Supplementary Fig. S3C).We hypothesized that CK1a might regulate Gli stability

through direct association, in a manner analogous to theassociation described between the Drosophila Gli homolog Ciand CK1a (8). To test this possibility, we lysed HEK 293T cellsexpressing Flag-tagged Gli1, Myc-tagged Gli2, or a controlplasmid, immunoprecipitated either Gli1 or Gli2, and thenimmunoblotted for associated CK1a. Endogenous CK1a asso-ciated with both Gli1 (Fig. 4A) and Gli2 (Fig. 4B), but was notobserved in the control immunoprecipitates. The associationbetween Gli1 and CK1a can be reduced following acutetreatment with pyrvinium (Fig. 4C), and this decreased asso-ciation temporally preceded any observed Gli destabilization(data not shown). The reversibility of the Gli1/CK1a associa-

tion by pyrvinium is consistent with the specificity of thisassociation.Medulloblastoma tissue harboring a constitutivelyactive HH signaling pathway coexpresses detectable levels ofendogenous Gli1 and CK1a (Fig. 4D and E). Using antibodiesspecific to either of these proteins, we also observed anassociation between endogenous CK1a and endogenous Gli1from this tissue (Fig. 4E). The decreased stability of Glis that weobserved in response to pyrvinium, combined with the asso-ciation between Gli proteins and CK1a, suggests that CK1amay directly phosphorylate Glis to regulate their stability.Consistent with this suggestion, purified CK1a is capable ofphosphorylating purified Gli1, and this phosphorylation isfurther stimulated by pyrvinium (Supplementary Fig. S1C).

To demonstrate the capacity of pyrvinium to attenuate thegrowth of HH activity–dependent cancers, we focused on awell-established mouse model of HH-dependent medulloblas-toma that is routinely used to determine the efficacy of HHinhibitors, the Ptchþ/� medulloblastoma model (2). We dis-sected a spontaneousmedulloblastoma from a Ptchþ/�mouse,and serially passaged the tumor as an allograft in CD-1 nudemice. We validated the dependency of HH signaling for thesetumors as HH (Gli1, Ptch2) but notWnt (Axin2,Dkk1, and Lgr5)biomarkers were aberrantly activated (Fig. 5A). Acute treat-ment of pyrvinium in this mousemodel attenuated the expres-sion of the HH biomarkers Gli1 and Ptch2 (Fig. 5B). Chronictreatment of suchmice, using animals subcutaneously injectedwith pyrvinium adjacent to the approximately 100-mm3

tumors, dramatically reduced the growth of medulloblastomaallografts (Fig. 5C). Hematoxylin and eosin (H&E) stainingdemonstrated that tumors from pyrvinium-treated animalsshowed decreased cancer cells and increased fibrotic tissue,

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Figure 4. CK1a associates with Glitranscription factors. A and B, HEK293T cells transfected withpcDNA3.1-Flag, Flag-Gli1 (A) orpcDNA3, Myc-Gli2 (B) weresubjected to immunoprecipitationand immunoblotting with theindicated antibodies. C,pcDNA3.1-Flag (Ctrl) or Flag-Gli1–transfected HEK 293T cells weretreated with vehicle or 200 nmol/Lpyrvinium for 1 hour, before anydetectable changes in Gli1 proteinlevels. Samples wereimmunoprecipitated with Flag M2beads and subject to immunoblotanalysis for CK1a or Flag-Gli1.D, immunohistologic staining ofCK1a and Gli1 proteins inPtchþ/�-derived medulloblastomatissue. Scale bar, 100 mm. E,endogenous CK1a (left) or Gli1(right) was immunoprecipitatedfrom homogenizedmedulloblastoma tissue using theindicated antisera (2 mg). Theseimmunoprecipitates were thenimmunoblotted for CK1a or Gli1.






comparedwith that from the vehicle-treated controls (Fig. 5D),consistent with a reduction in the size of the tumor driven bydecreased numbers of tumor cells. In the pyrvinium-treatedtumors, the expression of HH target genes was decreasedrelative to the vehicle control group (Fig. 5E). However, Wnttarget genes, which are not hyperactivated in this subtype ofmedulloblastoma (see Fig. 5A), were not different betweenpyrvinium and vehicle-treated tissue (Fig. 5E). These observa-tions are inconsistent with a generalized, nonspecific effect on

tumor cells, in which a reduction in total gene expressionmight be expected. Thus, the CK1a agonist pyrvinium attenu-ates HH pathway activity and tumor growth in vivo in a well-established model of HH-driven medulloblastoma.

DiscussionWe show here that the FDA-approved anthelmintic drug

pyrvinium can be repurposed as a potent HH inhibitor.

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Figure 5. Pyrvinium attenuates the growth of a Ptchþ/�-derived medulloblastoma allograft. A, RNA was extracted from normal mouse brains (N1 or N2) orPtchþ/�-derived medulloblastoma tissue (T1 or T2). Real-time RT-PCR was then used to detect the expression of the indicated HH or Wnt targetgenes, normalized to 18S ribosomal RNA. B, allografts ofPtchþ/�-derivedmedulloblastomawere grown in nudemice until they were approximately 100mm3

in size. Thesemicewere subsequently treatedwith 5mg/kg pyrvinium, or vehicle, for the indicated times. The expression ofGli1 orPtch2was thenquantitatedby real-time RT-PCR. Error bars, SEM (n ¼ 5); �, P < 0.05. C, allografts of similar medulloblastoma were grown in nude mice until they were approximately100 mm3 in size. These mice were subsequently treated with vehicle or pyrvinium (0.8 mg/kg) by subcutaneous injection every 2 days. Tumor volumeswere measured at the indicated time points. Error bars, SEM (n¼ 6); �, P < 0.05. D, representative H&E staining of tumors from vehicle and pyrvinium-treatedmice in C. Scale bar, 100 mm. E, following the chronic administration of pyrvinium in mice harboring Ptchþ/� medulloblastoma allografts in C, tumors wereharvested and subjected to real time RT-PCR analysis for HH or Wnt target genes. Error bars, SEM (n ¼ 6); �, P < 0.05.

Li et al.





Consistent with pyrvinium acting as a CK1a agonist (23), weshow that it attenuates HH signaling in a CK1a-dependentmanner.We further show that CK1a andGli proteins associateand propose a model by which pyrvinium induces Gli desta-bilization downstream of Smo. Unlike vismodegib, the onlySmo inhibitor currently FDA approved (2), pyrvinium is able toattenuate increased Gli activity resulting from loss of Sufu,overexpression of a Gli protein, or a Smo protein harboring aclinically relevant vismodegib-resistant mutation. Pyrviniumwas also able to attenuate the growth of a well-established HH-driven cancer model, a Ptchþ/�-driven medulloblastoma allo-graftmousemodel (5, 37). Interestingly, a significant number ofhuman medulloblastomas result from loss of Sufu or amplifi-cation of Gli2 (1), and thus would be resistant to vismodegibbut might conceivably respond to a CK1a agonist such aspyrvinium. In its current method of dosing and formulation,pyrvinium likely lacks the pharmacokinetic properties to beused in the clinic against HH-driven tumors. However,improvements in its formulation or delivery, such as perhapsdirect ventricular delivery into the brain, might rapidly beadapted to treat such patients with late-stage cancer for whomfew therapeutic options remain.Importantly, given the emergence of noncanonical Gli acti-

vation in human cancers (1), pyrvinium's effects on HH sig-naling occurred downstream of Smo, where it induces thedestabilization of Gli proteins. A number of other Gli inhibitorshave now been described (2). For example, although Gliantagonists (GANT) are now widely used Gli inhibitors in thelaboratory setting, their IC50s are more than 100-times higherthan the CK1a agonists described here. Similar potency con-cerns also exist for another class of HH inhibitor HPI (IC50, 10mmol/L), which perturb Gli processing and stability. Arsenictrioxide is another FDA-approved drug that has been repur-posed as aGli inhibitor in vitro and in vivo.Although this drug isalready clinically available, its limited potency and well-described dose-limiting toxicities in humans, which occur atdoses similar to those required to inhibit HH signaling, maylimit its usefulness in the clinic.Although CK1 was initially thought to be constitutively

active, recent evidence suggests that individual members ofthis protein kinase family are regulated via an array of tran-scriptional and posttranslational mechanisms (38). For CK1a,such mechanisms include the frequent silencing of CK1a inmelanoma and loss-of-heterozygosity of the CK1a gene in ap-proximately 30% of all human tumors (http://www.sanger.ac.uk/cgi-bin/genetics/CGP/cghviewer/CghHome.cgi)—consis-tent with CK1a activity being growth suppressing in manyhuman cancers (39, 40). The identification of pyrvinium as anallosteric activator of CK1a led to the speculation that pyrvi-nium may be mimicking an endogenous regulator of CK1a.This was confirmed with the recent identification of a novelfamily of proteins that regulate the activity of CK1 kinasesin vivo, and the demonstration that CK1a activity can beattenuated by extracellular ligands, such as Wnts (41, 42).These newly identified levels of regulation could potentiallybe usurped for the development of novel anticancer therapeu-tics that target individual CK1 isoforms. The excitement of aclass of anticancer agents such as pyrvinium comes from the

recognition that drugs that act via allosteric mechanisms arenot constrained by active site chemistry and are much moreselective (sites may not be conserved even between proteins inthe same family) in their mechanism of action.

CK1a participates in a number of cellular processes otherthan HH and Wnt signaling, including retinoid X receptorregulation (43). Interestingly, similar to its role in HH signaling,CK1a plays a dual function inNF-kB signaling, both promotingand repressing receptor-induced NF-kB activity (44). However,this is unlikely to be an impediment for its further developmentas a target for treating cancer. In fact, inhibitors that targetother distinct CK1 isoforms (d and e) are currently in preclin-ical development in both academia and industry, to treat avariety of human pathologies (45, 46). In addition, there issignificant precedence for the development of very effectiveanticancer agents that target proteins involved in basic cellularprocesses, which takes advantage of a tumor's increasedsensitivity to such agents. Many of these have already beenapproved by the FDA or are in clinical trials. These includepaclitaxel (microtubule stabilizer), vorinostat (HDAC inhibi-tor), velcade (proteasome inhibitor), temsirolimus (mTORinhibitor), and geldanamycin derivatives (targets HSP90 andcurrently in phase II clinical trials).

Disclosure of Potential Conflicts of InterestD. Orton received a commercial research grant from and has ownership

interest (including patents) in StemSynergy Therapeutics, Inc. E. Lee is thefounder of and has ownership interest (including patents) in StemSynergyTherapeutics, Inc. D.J. Robbins has ownership interest (including patents) inand is a consultant/advisory board member for StemSynergy Therapeutics, Inc.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B. Li, D.L. Fei, C.A. Flaveny, D. Orton, A.J. Capobianco,D.J. RobbinsDevelopment of methodology: B. Li, D.L. Fei, C.A. Flaveny, F. Bai, A.J.CapobiancoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Li, D.L. Fei, C.A. Flaveny, N. Dahmane, Z. Wang,X.-H. Pei, B. Hang, L. Han, A.J. Capobianco, E. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Li, D.L. Fei, C.A. Flaveny, N. Dahmane, J. Rodri-guez-Blanco, B. Hang, D. Orton, L. Han, A.J. Capobianco, E. Lee, D.J. RobbinsWriting, review, and/or revision of the manuscript: B. Li, C.A. Flaveny,N. Dahmane, D. Orton, A.J. Capobianco, E. Lee, D.J. RobbinsAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Li, C.A. Flaveny, V. Baubet, D. Orton,A.J. Capobianco, D.J. RobbinsStudy supervision: B. Li, C.A. Flaveny, A.J. Capobianco, D.J. RobbinsOther (provided Gli3 antibody): B. Wang

AcknowledgmentsThe authors thankDrs. Karoline Briegel and Teresa Zimmers andmembers of

the Robbins, Capobianco, and Lee laboratories for providing insight duringdiscussions about this article. The authors also thank Dr. Rune Toftga

�rd

(Karolinska Institute) for providing Sufu�/� MEF cells.

Grant SupportThis work was supported by the following grants: NIH: CA082628, GM64011,

GM103926, GM081635, and P50CA95103; Alex Lemonade Stand Foundation, andThe University of Miami Women's Cancer Association.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 5, 2014; revised May 19, 2014; accepted June 4, 2014;published OnlineFirst July 3, 2014.






References1. Teglund S, Toftgard R. Hedgehog beyondmedulloblastoma and basal

cell carcinoma. Biochim Biophys Acta 2010;1805:181–208.2. AmakyeD, Jagani Z, DorschM. Unraveling the therapeutic potential of

the Hedgehog pathway in cancer. Nat Med 2013;19:1410–22.3. Goodrich LV, Milenkovic L, Higgins KM, Scott MP. Altered neural cell

fates and medulloblastoma in mouse patched mutants. Science1997;277:1109–13.

4. Zibat A, Uhmann A, Nitzki F, Wijgerde M, Frommhold A, Heller T, et al.Time-point and dosage of gene inactivation determine the tumorspectrum in conditional Ptch knockouts. Carcinogenesis 2009;30:918–26.

5. Berman DM, Karhadkar SS, Hallahan AR, Pritchard JI, Eberhart CG,Watkins DN, et al. Medulloblastoma growth inhibition by hedgehogpathway blockade. Science 2002;297:1559–61.

6. YauchRL,DijkgraafGJ, AlickeB, Januario T, AhnCP,Holcomb T, et al.Smoothened mutation confers resistance to a Hedgehog pathwayinhibitor in medulloblastoma. Science 2009;326:572–4.

7. Hui CC, Angers S. Gli proteins in development and disease. Annu RevCell Dev Biol 2011;27:513–37.

8. Robbins DJ, Fei DL, Riobo NA. The Hedgehog signal transductionnetwork. Sci Signal 2012;5:re6.

9. Wang B, Fallon JF, Beachy PA. Hedgehog-regulated processing ofGli3 produces an anterior/posterior repressor gradient in the devel-oping vertebrate limb. Cell 2000;100:423–34.

10. Pan Y, Bai CB, Joyner AL, Wang B. Sonic hedgehog signaling reg-ulates Gli2 transcriptional activity by suppressing its processing anddegradation. Mol Cell Biol 2006;26:3365–77.

11. Tempe D, Casas M, Karaz S, Blanchet-Tournier MF, Concordet JP.Multisite protein kinase A and glycogen synthase kinase 3beta phos-phorylation leads to Gli3 ubiquitination by SCFbetaTrCP. Mol Cell Biol2006;26:4316–26.

12. Bhatia N, Thiyagarajan S, Elcheva I, SaleemM, Dlugosz A, Mukhtar H,et al. Gli2 is targeted for ubiquitination and degradation by beta-TrCPubiquitin ligase. J Biol Chem 2006;281:19320–6.

13. Chen MH, Wilson CW, Li YJ, Law KK, Lu CS, Gacayan R, et al.Cilium-independent regulation of Gli protein function by Sufu inHedgehog signaling is evolutionarily conserved. Genes Dev 2009;23:1910–28.

14. Svard J, Heby-Henricson K, Persson-Lek M, Rozell B, Lauth M,Bergstrom A, et al. Genetic elimination of Suppressor of fused revealsan essential repressor function in the mammalian Hedgehog signalingpathway. Dev Cell 2006;10:187–97.

15. Rohatgi R, Milenkovic L, Scott MP. Patched1 regulates hedgehogsignaling at the primary cilium. Science 2007;317:372–6.

16. Humke EW, Dorn KV, Milenkovic L, Scott MP, Rohatgi R. The outputof Hedgehog signaling is controlled by the dynamic associationbetween Suppressor of Fused and the Gli proteins. Genes Dev2010;24:670–82.

17. Wen X, Lai CK, Evangelista M, Hongo JA, de Sauvage FJ, Scales SJ.Kinetics of hedgehog-dependent full-length Gli3 accumulation inprimary cilia and subsequent degradation. Mol Cell Biol 2010;30:1910–22.

18. Tukachinsky H, Lopez LV, Salic A. Amechanism for vertebrate Hedge-hog signaling: recruitment to cilia and dissociation of SuFu-Gli proteincomplexes. J Cell Biol 2010;191:415–28.

19. Wang B, Li Y. Evidence for the direct involvement of {beta}TrCPin Gli3 protein processing. Proc Natl Acad Sci U S A 2006;103:33–8.

20. Pan Y, Wang C, Wang B. Phosphorylation of Gli2 by protein kinaseA is required for Gli2 processing and degradation and the SonicHedgehog-regulated mouse development. Dev Biol 2009;326:177–89.

21. Niewiadomski P, Kong JH, Ahrends R,MaY, Humke EW, Khan S, et al.Gli protein activity is controlled by multisite phosphorylation in verte-brate hedgehog signaling. Cell Rep 2014;6:168–81.

22. ZhangQ,ShiQ,ChenY,YueT, Li S,WangB, et al.Multiple Ser/Thr-richdegrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3ubiquitin ligase. Proc Natl Acad Sci U S A 2009;106:21191–6.

23. Thorne CA, Hanson AJ, Schneider J, Tahinci E, Orton D, Cselenyi CS,et al. Small-molecule inhibition of Wnt signaling through activation ofcasein kinase 1alpha. Nat Chem Biol 2010;6:829–36.

24. LumL, YaoS,Mozer B, Rovescalli A, VonKessler D, NirenbergM, et al.Identification of Hedgehog pathway components by RNAi inDrosoph-ila cultured cells. Science 2003;299:2039–45.

25. Dahmane N, Sanchez P, Gitton Y, Palma V, Sun T, Beyna M, et al. TheSonic Hedgehog-Gli pathway regulates dorsal brain growth andtumorigenesis. Development 2001;128:5201–12.

26. Lipinski RJ, Bijlsma MF, Gipp JJ, Podhaizer DJ, Bushman W. Estab-lishment and characterization of immortalized Gli-null mouse embry-onic fibroblast cell lines. BMC Cell Biol 2008;9:49.

27. Fei DL, Li H, Kozul CD, Black KE, Singh S,Gosse JA, et al. Activation ofHedgehog signaling by the environmental toxicant arsenic may con-tribute to the etiology of arsenic-induced tumors. Cancer Res 2010;70:1981–8.

28. Cselenyi CS, Jernigan KK, Tahinci E, Thorne CA, Lee LA, Lee E. LRP6transduces a canonical Wnt signal independently of Axin degradationby inhibiting GSK3's phosphorylation of beta-catenin. Proc Natl AcadSci U S A 2008;105:8032–7.

29. Wechsler-Reya RJ, Scott MP. Control of neuronal precursor pro-liferation in the cerebellum by Sonic Hedgehog. Neuron 1999;22:103–14.

30. ShenM,BellaousovS, HillerM, de LaGrangeP,Creamer TP,MalinaO,et al. Pyrvinium pamoate changes alternative splicing of the serotoninreceptor 2C by influencing its RNA structure. Nucleic Acids Res 2013;41:3819–32.

31. Tomitsuka E, Kita K, Esumi H. An anticancer agent, pyrviniumpamoateinhibits the NADH-fumarate reductase system–a uniquemitochondrialenergy metabolism in tumour microenvironments. J Biochem 2012;152:171–83.

32. Harada Y, Ishii I, Hatake K, Kasahara T. Pyrvinium pamoate inhibitsproliferation of myeloma/erythroleukemia cells by suppressing mito-chondrial respiratory complex I and STAT3. Cancer Lett 2012;319:83–8.

33. Yu DH, Macdonald J, Liu G, Lee AS, Ly M, Davis T, et al. Pyrviniumtargets the unfolded protein response to hypoglycemia and its anti-tumor activity is enhancedby combination therapy. PLoSONE2008;3:e3951.

34. Venerando A, Girardi C, Ruzzene M, Pinna LA. Pyrvinium pamoatedoes not activate protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation. Biochem J 2013;452:131–7.

35. Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J, et al. Sonic Hedgehogdependent phosphorylation by CK1alpha and GRK2 is required forciliary accumulation and activation of smoothened. PLoS Biol 2011;9:e1001083.

36. Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev MolCell Biol 2005;6:306–17.

37. Wong H, Alicke B, West KA, Pacheco P, La H, Januario T, et al.Pharmacokinetic-pharmacodynamic analysis of vismodegib in pre-clinical models of mutational and ligand-dependent Hedgehog path-way activation. Clin Cancer Res 2011;17:4682–92.

38. Cheong JK, VirshupDM.Casein kinase 1: complexity in the family. Int JBiochem Cell Biol 2011;43:465–9.

39. Elyada E, Pribluda A, Goldstein RE, Morgenstern Y, Brachya G,Cojocaru G, et al. CKIalpha ablation highlights a critical role for p53in invasiveness control. Nature 2011;470:409–13.

40. Sinnberg T, Menzel M, Kaesler S, Biedermann T, Sauer B, Nahnsen S,et al. Suppression of casein kinase 1alpha inmelanoma cells induces aswitch in beta-catenin signaling to promote metastasis. Cancer Res2010;70:6999–7009.

41. Cruciat CM, Dolde C, de Groot RE, Ohkawara B, Reinhard C,Korswagen HC, et al. RNA helicase DDX3 is a regulatory subunitof casein kinase 1 in Wnt-beta-catenin signaling. Science 2013;339:1436–41.

42. Hernandez AR, Klein AM, Kirschner MW. Kinetic responses ofbeta-catenin specify the sites of Wnt control. Science 2012;338:1337–40.

Li et al.





43. Zhao Y, Qin S, Atangan LI, Molina Y, Okawa Y, Arpawong HT, et al.Casein kinase 1alpha interacts with retinoid X receptor and inter-feres with agonist-induced apoptosis. J Biol Chem 2004;279:30844–9.

44. BidereN,NgoVN, Lee J,CollinsC, ZhengL,WanF, et al. Casein kinase1alpha governs antigen-receptor-induced NF-kappaB activation andhuman lymphoma cell survival. Nature 2009;458:92–6.

45. Arey R, McClung CA. An inhibitor of casein kinase 1 epsilon/deltapartially normalizes the manic-like behaviors of the ClockDelta19mouse. Behav Pharmacol 2012;23:392–6.

46. Behrend L, Milne DM, Stoter M, Deppert W, Campbell LE, Meek DW,et al. IC261, a specific inhibitor of the protein kinases casein kinase1-delta and -epsilon, triggers the mitotic checkpoint and inducesp53-dependent postmitotic effects. Oncogene 2000;19:5303–13.






2014;74:4811-4821. Published OnlineFirst July 3, 2014.Cancer Res Bin Li, Dennis Liang Fei, Colin A. Flaveny, et al. SmoothenedPyrvinium Attenuates Hedgehog Signaling Downstream of

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