Repurposing the Antihelmintic Mebendazole As a Hedgehog Inhibitor Andrew R

Published OnlineFirst November 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0755-T

Small Molecule Therapeutics Molecular Cancer Therapeutics Repurposing the Antihelmintic Mebendazole as a Hedgehog Inhibitor Andrew R. Larsen1,Ren-YuanBai2, Jon H. Chung1, Alexandra Borodovsky2, Charles M. Rudin3, Gregory J. Riggins2, and Fred Bunz1

Abstract

The hedgehog (Hh) signaling pathway is activated in many activation. The inhibition of Hh signaling by mebendazole was types of cancer and therefore presents an attractive target for new unaffected by mutants in the gene that encodes human Smooth- anticancer agents. Here, we show that mebendazole, a benzami- ened (SMO), which are selectively propagated in cell clones that dazole with a long history of safe use against nematode infesta- survive treatment with the Hh inhibitor vismodegib. Combina- tions and hydatid disease, potently inhibited Hh signaling and tion of vismodegib and mebendazole resulted in additive Hh slowed the growth of Hh-driven human medulloblastoma cells at signaling inhibition. Because mebendazole can be safely admin- clinically attainable concentrations. As an antiparasitic, meben- istered to adults and children at high doses over extended time dazole avidly binds nematode tubulin and causes inhibition of periods, we propose that mebendazole could be rapidly repur- intestinal microtubule synthesis. In human cells, mebendazole posed and clinically tested as a prospective therapeutic agent for suppressed the formation of the primary cilium, a microtubule- many tumors that are dependent on Hh signaling. Mol Cancer Ther; based organelle that functions as a signaling hub for Hh pathway 14(1); 3–13. 2014 AACR.

Introduction and evidence of elevated GLI activity in many tumors that lack pathway-activating mutations (5, 6). Activation of the hedgehog (Hh) signaling pathway is SMO antagonism has proven to be an effective strategy for required for developmental morphogenesis and is frequently treating tumors with active Hh signaling (7). The ﬁrst SMO observed in human cancers (1, 2). Canonical Hh signals are antagonist to be approved for clinical use is vismodegib (Erivedge; initiated by the interaction of Hh ligands with the receptor also known as GDC-0449). Vismodegib has been used success- PTCH1. In the unbound state, PTCH1 prevents SMO activa- fully for the treatment of locally advanced and metastatic basal tion in the primary cilium, an organelle required for the cell carcinomas (8), and is currently being tested for use in adults transduction of various chemical and mechanical signals and children with many diverse types of tumors, including (3). In the presence of ligand, PTCH1 disappears from the medulloblastomas and gliomas, which are often refractory to cilium and SMO activates downstream effectors, including the conventional therapies (9). When used as a monotherapy, vis- GLI family of transcription factors (4). Several types of cancer, modegib is associated with adverse effects that include fatigue, including basal cell carcinoma and medulloblastoma, are vomiting, weight loss, decreased appetite, dysgeusia, dehydration, frequently caused by germline or somatic mutations in PTCH1 and muscle spasm (10). Such low-grade toxicities have contrib- or by less common alterations within the pathway that lead to uted to treatment discontinuation and appear to be potentially constitutive signaling by SMO (1, 2). Alternative modes of Hh problematic when vismodegib is combined with conventional pathwayactivationinsomeofthemostcommontypesof agents (11). When used to treat a patient with metastatic medul- cancer are suggested by the widespread presence of Hh ligands loblastoma, vismodegib caused a response that was impressive but transient (12). Recurrent tumors in this patient were found to harbor a novel SMO mutation that caused drug resistance (13). 1Department of Radiation Oncology and Molecular Radiation Selection for SMO-mutant tumor cell populations can similarly Sciences, The Kimmel Cancer Center at Johns Hopkins, Baltimore, be caused by vismodegib therapy in mouse models of medullo- 2 Maryland. Department of Neurosurgery,The Kimmel Cancer Center at blastoma (13). Alternative strategies to inhibit Hh signaling have Johns Hopkins, Baltimore, Maryland. 3Memorial Hospital Research Laboratories, Memorial Sloan Kettering Cancer Center, New York, New been explored for the prevention or treatment of such recurrent York. tumors (14). Note: Supplementary data for this article are available at Molecular Cancer Benzimidazoles approved by the U.S. Food and Drug Admin- Therapeutics Online (http://mct.aacrjournals.org/). istration for the treatment of nematode infections have been Corresponding Authors: Fred Bunz, The Kimmel Cancer Center at Johns reported to have antiproliferative effects in diverse types of cancer Hopkins, 1550 Orleans Street, Room 453, Baltimore, MD 21287. Phone: 410- cells, including those derived from melanoma, non–small cell 502-7941; Fax: 410-502-2821; E-mail: [email protected]; and Gregory J. Riggins, lung cancer, ovarian cancer, adrenocortical carcinoma, and colo- The Kimmel Cancer Center at Johns Hopkins, 1550 Orleans Street, Room 257, rectal cancers (15–20). Case reports have documented responses Baltimore, MD 21287. Phone: 410-614-0477; Fax: 410-614-0478; E-mail: of a metastatic adrenocortical carcinoma (21) and a metastatic [email protected] colorectal carcinoma (22) to mebendazole (methyl N-[6-(ben- doi: 10.1158/1535-7163.MCT-14-0755-T zoly)-1H-benzamidazol-2-yl] carbamate). Our group recently 2014 American Association for Cancer Research. found that experimental brain tumors were highly sensitive to

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Larsen et al.

benzimidazole therapy that was administered to a mouse colony mented with 10% FBS and penicillin/streptomycin. NIH3T3 cells for control of a pinworm infestation. Follow-up studies of this were grown in DMEM supplemented with 10% calf serum. Shh- serendipitous observation showed that mebendazole inhibited Light2 cells (34) were grown in DMEM with 10% calf serum and the growth of glioma-derived neurospheres in vitro, and among 0.4 mg/mL geneticin and 0.15 mg/mL zeocin, both purchased from the benzimidazoles most effectively entered the central nervous Life Technologies. All cell lines were obtained from the ATCC system and slowed the growth of orthotopically implanted glio- within 6 months of the beginning of the project and validated by mas, which are characteristically resistant to standard modes of the supplier, except for Smo / MEFs that were a gift from James therapy (23). Kim (University of Texas Southwestern Medical Center) and were The anticancer effect of mebendazole defies a simple explana- not genetically authenticated upon their receipt in July 2013. tion. Mebendazole and related compounds have been reported to cause growth arrest and induce apoptosis in cultured cancer cells Orthotopic tumors at doses that have little effect on noncancer immortalized cells Syngeneic GL261 glioma tumors were grown intracranially in (15–17, 19, 20). It is unclear why such nonspecific antiprolifera- 4- to 6-week-old female nu/nu athymic mice (NCI-Fredrick) and tive effects would preferentially target tumor cells over the cells in treated with mebendazole as previously described (23). Each normal renewing tissues. Like all benzimidazoles used for treat- brain was snap-frozen after extraction and stored in liquid nitro- ment of helminth infestations, mebendazole binds tubulin at a gen until further analysis. Orthotopic medulloblastoma xeno- binding site also recognized by colchicine, and inhibits microtu- grafts were generated in female athymic mice, 5 to 6 weeks of age bule polymerization. The rate of dissociation of mebendazole (NCI). DAOY cells were infected with a lentivirus carrying a firefly from nematode tubulin is an order of magnitude lower than from luciferase cDNA (23) before implantation. For the implantation human tubulin (24). Inhibition of microtubule formation in the procedure, mice were anesthetized and 200,000 DAOY cells were gut of the nematode prevents the absorption of glucose and injected through a burr hole drilled 1 mm lateral to the right of the thereby leads to elimination of the parasite (25), whereas human sagittal suture and 1 mm posterior to the lambda at a depth of 2.5 cells and tissues are apparently minimally affected by the less avid mm below the dura, with the guidance of a stereotactic frame, at a mebendazole–tubulin interaction. Mebendazole is therefore well rate of 1 mL/min. Treatment was initiated at 5 days after implan- tolerated, even at high doses administered over lengthy time tation, with a daily dose of mebendazole of 25 or 50 mg/kg periods for treatment of cystic echinococcosis (26). delivered with 50% (v/v) sesame oil and PBS, by gavage. Intra- A growing body of evidence suggests that activated Hh signaling cranial luciferase activity was determined with a bioluminescence contributes to the diverse cancers that are preclinically responsive imager (Xenogen) following intraperitoneal injection of 2 mg D- to mebendazole and structurally related benzimidazoles. Recent- luciferin potassium salt (Gold Biotechnology). Animals were ly, Hh signaling has been shown to be active in many gliomas (27, scanned 15 m after injection for 1 m at a distance of 20 cm. Mice 28), while Hh ligands or markers of downstream pathway activity were euthanized when they exhibited signs of increased intracra- have been detected in melanomas, lung cancers, ovarian cancers, nial pressure. All animal protocols and procedures were per- adrenocortical cancers, and colorectal cancers (5, 6, 29–32), formed under an approved protocol and in accordance with the which are all responsive to mebendazole (15–21). Notably, Johns Hopkins Animal Care and Use Committee guidelines. For unbiased screens for novel Hh inhibitors have previously iden- RNA preparation, each brain was thawed on ice before removing tified drugs that interact with microtubules, including vinblastine, the right anterior cerebral cortex and the contralateral brain vincristine, and paclitaxel (33). These drugs potently inhibit section. Each tissue sample was suspended in 1 mL of TRizol mitosis, are highly toxic, and therefore unsuitable for long-term (Life Technologies) per 0.1 g of material, and the RNA fraction was therapy. The specific effects of these compounds on components purified according to the manufacturer's recommendations. of the Hh pathway have not been reported. In this study, we evaluated the effect of mebendazole on the Hh Plasmids and cell transfections pathway. Mebendazole treatment prevented the formation of the ShhN-conditioned media was generated by transfection of primary cilium, decreased expression of downstream Hh pathway pcDNA3 ShhN (provided by Pao Tien Chuang, University of effectors, and decreased the proliferation and survival of human California San Francisco) into 293T cells. Control media was medulloblastoma cells with constitutive Hh activation. Meben- obtained from mock-transfected 293T cells. For localization of dazole inhibited the activation of SMO-mutant proteins that give SMO, hTERT RPE-1 cells grown in chamber slides (Nunc) were rise to disease recurrence. A combination of mebendazole and transfected with pcDNA3 Smo–FLAG (provided by Chen Ming vismodegib achieved additive inhibition of canonical Hh signal- Fan, Carnegie Institute of Science). GLI1 reporter assays were ing. These results support the repurposing of mebendazole for use conducted by cotransfecting the firefly luciferase reporter pBV Luc in the many types of cancers that are initiated or maintained by 8xGli (provided by Craig Peacock, Cleveland Clinic) and TK- active Hh signaling, and suggest combinations of drugs that could Renilla luciferase reporter pGL4 74 (Promega). Overexpression of facilitate the achievement of durable responses. Hh components was achieved by transient transfection of pRK- SmoM2 (35), pCMV5 hGLI1 FLAG (provided by Peter Zaphir- opoulos, Karolinska Institute) and pCS2-MT hGLI2 FL (Addgene Materials and Methods plasmid 17648; ref. 36). Mutant-Smo expression constructs were Cell lines and cell cultures provided by James Kim. All transfections were performed with Cultures of 293T and hTERT-RPE1 cells and Smo / mouse FuGENE HD (Promega). embryo fibroblasts (MEF) were maintained in DMEM (Life Techno- logies) supplemented with 10% fetal bovine serum (FBS; Hyclone) GLI-reporter assays and drug treatment and penicillin/streptomycin. DAOY and C3H10T1/2 mouse fibro- Subconfluent Shh-Light2 cells were routinely incubated in low blast cells were grown in Eagle's MEM (Life Technologies) supple- serum conditions (0.25% FBS, 5-mm HEPES), to optimize Hh

4 Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics

Repurposing Mebendazole as a Hedgehog Inhibitor

responsiveness, during a 48-hour period of drug treatment. For ogies). Stained cells were visualized with an AxioImager Z1 (Carl ShhN ligand stimulation, cells were incubated with ShhN-con- Zeiss) and images were captured with Axiovision Rel 4.6 software. ditioned media or control media, diluted at 1:5, during the The following primary antibodies were used: anti-GLI1 treatment period. Final DMSO concentration in all cultures was (C68H3; Cell Signaling Technology), anti–caspase-3 (9662; Cell 1%. For experiments involving overexpression of Hh pathway Signaling Technology) anti-cleaved caspase-3 (D175; Cell Signal- components, cells were transfected 24 hours before low serum ing Technology), anti–a-tubulin (TU-02; Santa Cruz Biotechnol- and drug treatment. Cell lysates were analyzed using the Dual- ogy), HRP-conjugated anti–b-actin (Santa Cruz Biotechnology), Luciferase Assay Reporter System (Promega). GLI-dependent anti-acetylated a-tubulin (6–11 B-1; Sigma), anti-FLAG (anti- luciferase was measured on a Victor3 V 1420 Multilabel Counter DYKDDDDK; Cell Signaling Technology). (PerkinElmer) and standardized against Renilla luciferase activity. The IC50 was calculated by Prism 5 software package (GraphPad). Quantitative real time RT-PCR RNA was isolated and purified with the TRizol reagent (Life Cell proliferation, viability, and survival assays Technologies), treated with DNase I (Thermo Scientific), and Bromodeoxyuridine (BrdUrd) incorporation during DNA syn- assayed by spectrophotometry. cDNA was synthesized with the thesis was measured using the Cell Proliferation ELISA Kit Maxima First Strand cDNA Synthesis Kit (Thermo Scientific). (Roche). Cell viability was measured with the CellTiter-Blue Cell Quantitative, real-time reverse transcription PCR (qRT-PCR) was Viability Assay Kit (Promega). Colorimetric signals were mea- performed using both Maxima Probe and Maxima SYBR Green sured on a SpectraMax M5 (Molecular Devices). For each assay, qPCR Master Mixes (Thermo Scientific) with standard cycling the IC50 was calculated with the Prism 5 software package conditions on a 7900HT Fast Real-Time PCR system (Applied (GraphPad). For assessment of clonogenic survival, cells were Biosystems). Prime Time qPCR probes and primers were used to 0 drug-treated under low serum conditions in 12-well plates. After assay the mouse genes: Ptch1:probe(5-ATTCGGACCCTGCAAG- 0 0 0 48 hours, cells were harvested in Trypsin–EDTA (Life Technolo- CATCAGT-3 ), forward primer (5 -TGTTTGCTCCCGTTCTGG-3 ), 0 0 gies), diluted at 1:4,000 in standard growth media, and seeded in reverse primer (5 -AACCAGTCCATTGAGAACCC-3 ); Gli1: probe 0 0 0 10-cm plates in triplicate. After 10 days of growth, plates were (5 -CTGGGACCCTGACATAAAGTTGGCT-3 ); forward primer (5 - 0 0 stained with 0.2% crystal violet in 50% MeOH and destained in CTTTCTGGTCTGCCCTTTTG-3 ); reverse primer (5 -TCTTTGT- 0 0 water. Colonies containing more than 50 cells were scored. For the TAATTTGACTGAACTCCG-3 ); Rpl19:probe(5-CTTCTCAGGA- 0 0 quantitation of apoptosis, cells were detached and stained with a GATACCGGGAATCCAAG-3 ); forward primer (5 -AGAAGGT- 0 0 fluorescent antibody directed against Annexin V with the Dead GACCTGGATGAGAA-3 ); reverse primer (5 -TGATACATATGGC- 0 Cell Apoptosis Kit (Life Technologies). The fraction of stained GGTCAATCT-3 ). Human transcripts were analyzed with SYBR 0 cells was determined with a FACSAria II flow cytometer (BD) in green. GLI1: forward primer (5 -CCACGGGGAGCGGAAGGAG- 0 0 0 the Sidney Kimmel Comprehensive Cancer Center Flow Cytome- 3 ); reverse primer (5 -ACTGGCATTGCTGAAGGCTTTACTG-3 ). 0 0 try Core. PTCH1: forward primer (5 -CCACAGAAGCGCTCCTACA-3 ); reverse primer (50-CTGTAATTTCGCCCCTTCC-30). PTCH2:for- Immunoblots and immunofluorescence ward primer (50-GGAATGATTGAGCGGATGATTGA-30); reverse Proteins were separated on Bis–Tris gels (Life Technologies) primer (50-CCACCTGTGCCTTGTCTAGC-30). GAPDH: forward and transferred onto Immobilion-P nylon membranes (Milli- primer (50-CGGAGTCAACGGATTTGGTCGTAT-30); reverse primer pore). Following overnight incubation with primary antibodies (50-AGCCTTCTCCATGGTGGTGAAGAC-30). All results were ana- under standard conditions, blots were developed with horserad- lyzed using SDS RQ Manager (Applied Biosystems). ish peroxidase (HRP)–conjugated secondary antibodies and visualized by chemiluminescence (Amersham). Band intensities were quantified by Image Lab software and standardized to the inten- Results sity of the loading controls anti–a-tubulin or anti–b-actin. To Inhibition of Hh signaling in vitro and in vivo assess tubulin polymerization, polymerized and unpolymerized To directly assess the effect of mebendazole on canonical Hh tubulin fractions were separated on the basis of solubility as signaling, we incubated the drug with murine Shh-Light2 cells, reported previously (37). which have stably incorporated a GLI-activated fireflylucifer- For analysis of primary cilia by immunofluorescence, cells were ase (Gli-luc) reporter construct (34). Under low serum condi- grown on poly-D-lysine (Sigma)–coated chamber slides (Nunc) tions that are optimal for Hh activation, addition of the and fixed in 0.4% paraformaldehye at 37C for 5 minutes, amino-terminal signaling domain of the secreted sonic hedge- permeabilized in 0.5% Triton X-100 at 37C for 2 minutes, hog ligand (ShhN) induced robust reporter activity that could washed in PBS, then sequentially incubated in 4% paraformal- be inhibited by mebendazole in the micromolar dose range dehyde, 37C for 5 minutes, and methanol, 20C for 5 minutes, (Fig. 1A). A similar degree of inhibition was observed in mouse as previously described (38). Nonspecific proteins were blocked C3H10T1/2 fibroblast cells transiently transfected with the with 2% bovine serum albumin in PBS for 30 minutes at room Gli-luc reporter (Fig. 1B). Mebendazole was a considerably temperature. Slides were incubated with primary antibody in more potent inhibitor of Hh pathway activation than struc- blocking buffer overnight at 4C, then washed with PBS, and turally related antihelmintic drugs (Fig. 1C), which in prior incubated with either biotinylated (Santa Cruz Biotechnology) or studies were less effective than mebendazole at inhibiting Alexa Fluor 594–conjugated (Life Technologies) secondary anti- the growth of GL261 gliomas (23). The GL261 mouse tumor bodies in blocking buffer for 20 minutes at room temperature. harbors a mutation in Pten (39); in humans, mutation of PTEN Cells were washed with PBS before adding an Alexa Fluor 488– defines a category of gliomas that are strongly associated with streptavidin conjugate (Life Technologies). Nuclei were counter- elevated Hh activity (28). We assessed endogenous Gli1 tran- stained with 40,6-diamidino-2-phenylindole (DAPI; Life Technol- script levels in mebendazole-treated and control GL261

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A B ShhN ShhN No ligand No ligand ShhN+Vis ShhN+Vis Relative Gli-luciferase Relative Gli-luciferase

[MBZ], mol/L [MBZ], mol/L C Tiabendazole

Fenbendazole

Albendazole Relative Gli-luciferase

Mebendazole [Benzimidazole], mol/L

D E 16 F 25 Vehicle 14 MBZ 20 12 MBZ Vehicle 10 15 Gli1 8 Gapdh 10 6

mRNA/Rpl19 4 5 Gli1/Rpl19 mRNA 2 0 0 Gli1 Ptch1

G 5 hTERT-RPE1 H 5 DAOY

Vehicle 4 4 0 μmol/L MBZ 0.1 μmol/L MBZ 0.1 μmol/L MBZ 3 3 1 μmol/L MBZ μ 1 mol/L MBZ 10 μmol/L MBZ μ 2 10 mol/L MBZ 2 0.2 μmol/L vismodegib mRNA/GAPDH 1 mRNA/GAPDH 1

0 0 GLI1 PTCH1 GLI1 PTCH1 GLI1 PTCH1 GLI1 PTCH1 (+) ShhN (+) ShhN

Figure 1. Mebendazole (MBZ) inhibits Hh signaling. A, Shh-Light2 cells maintained in low-serum conditions were incubated in ShhN-conditioned medium or control medium, in the presence of mebendazole at the indicated concentrations. The activity of the stably integrated Gli-luc reporter was measured after 48 hours of treatment. The effect on this assay of 0.2 mmol/L vismodegib (Vis) is indicated by the dashed line. B, C3H10T1/2 mouse ﬁbroblasts were cotransfected with the Gli-luc and Renilla luciferase reporters. After 24 hours, mebendazole was added for an additional 48 hours in low-serum media before cell lysis and measurement of luciferase activity. The effect of 0.2 mmol/L vismodegib (Vis) is indicated by the dashed line. C, the effect of mebendazole on Gli-luc reporter activity in Shh-Light2 cells was compared with that of the structurally related benzamidizoles albendazole, fenbendazole, and tiabendazole. Treatment times and conditions were as in A. D, endogenous levels of Gli1 transcripts in syngeneic, GL261 gliomas and in normal brain tissue from the contralateral region were measured by qRT-PCR. E, Gli1 expression was measured by qRT-PCR in untreated and mebendazole-treated GL261 tumors. Each measurement was standardized to a parallel measurement from a contralateral brain section that did not contain tumor tissue. F, relative protein levels of Gli1 in three untreated and three mebendazole- treated GL261 tumors were assessed by immunoblot. Immortalized hTERT-RPE1 cells (G) or DAOY medulloblastoma cells (H) growing in low serum were treated with ShhN-conditioned or control medium for 48 hours. Mebendazole was included during this treatment period at the concentrations indicated. GLI1 and PTCH1 transcript levels were assessed by qRT-PCR.

tumors. Gli1 expression was signiﬁcantly elevated in untreated signaling in the growth of these tumors. Gli1 transcript and tumors compared with nonaffected tissue from the contralat- protein expression in tumor tissues was decreased by meben- eral side of the brain (Fig. 1D), suggesting a role for Hh dazole treatment (Fig. 1E and F).

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Repurposing Mebendazole as a Hedgehog Inhibitor

A B C GLI1/GAPDH mRNA BrdUrd incorporation (%) Clonogenic survival (%)

[MBZ], mol/L [MBZ], mol/L [MBZ], mol/L D E DAOY hTERT RPE-1 MBZ (μmol/L) 0 0.1 0.5 0.75 1 0 0.1 0.5 0.75 1

GLI1 DAOY RPE-1 Caspase-3

Cleaved Cell viability (%) caspase-3

α-Tubulin

[MBZ], mol/L F G H Vehicle MBZ (1 μmol/L) Events Annexin V–positive (%)

Vehicle MBZ (1 μmol/L) [MBZ], mol/L Annexin V–FITC

Figure 2. Effect of mebendazole (MBZ) on Hh-signaling, growth, and survival of Hh-dependent medulloblastoma cells. Subconfluent DAOY cultures maintained under low- serum conditions were treated for 48 hours with mebendazole at varying concentrations. A, GLI1 expression was assayed by qRT-PCR. B, cell proliferation was assessed by measuring the incorporation of BrdUrd over 2 hours. C, cell survival was quantified by a clonogenic assay. D, the effect of mebendazole on cell viability was comparatively assessed by CellTiter-Blue in hTERT-RPE1 (blue) and DAOY (red). E, the expression of GLI1 protein and cleavage of caspase-3 was assessed by immunoblot in DAOY and hTERT-RPE1 cells treated with mebendazole for 12 hours, under low-serum conditions. a-Tubulin was probed as a loading control. F, representative nuclei from mebendazole-treated DAOY cells and untreated controls, stained with Hoechst 33258. Scale bar, 20 mm. G, Annexin V–stained cells were quantified by flow cytometry, after 24 hours of mebendazole treatment (1 mmol/L) under low-serum conditions. H, the proportion of Annexin V–positive cells after treatment with various concentrations of mebendazole, as in G.

To extend our analysis to human cells, we tested the effects tion was observed at higher mebendazole concentrations. of mebendazole on Hh-mediated gene expression in the Using the same conditions, we assessed the effect of meben- immortalized human retinal pigment epithelial cell line dazole on the human medulloblastoma cell line DAOY. In hTERT-RPE1. Unlike the majority of human cancer cell lines, boththepresenceandabsenceofShhNligand,therewas this noncancer cell line is responsive to ShhN (Fig. 1G). A partial reduction in GLI1 and PTCH1 transcripts at 0.1 mmol/L concentration of 0.1 mmol/L mebendazole was sufﬁcient to mebendazole and almost complete suppression at 1 mmol/L reduce ShhN-induced expression of endogenous GLI1 and mebendazole, similar to the effect of 0.2 mmol/L vismodegib PTCH1 to basal levels (Fig. 1G). Further Hh pathway inhibi- (Fig. 1H).

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m A Vehicle, = 75 d B 1.4 * * MBZ, m = 113 d *** 1.2 Vehicle 1 MBZ (%) 0.8 0.6

Survival 0.4

GLI1/GAPDH mRNA GLI1/GAPDH 0.2 0 GLI1 PTCH1 PTCH2 Treatment time (days)

C 16 Day 60 * 14

/sr) Vehicle 2 5.0 /sr)

12 MBZ 2 4.0

p/s/cm 10 8 p/s/cm 3.0 8 8 6 2.0 4 1.0

Radiance (x10 2 ns Radiance (x10 0 Day 0 Day 60 Vehicle MBZ-treated Days of treatment

Figure 3. Mebendazole (MBZ) suppresses the growth of medulloblastoma cells and Hh signaling in vivo. DAOY cells expressing firefly luciferase were grown as orthotopic xenograft tumors following injection into the cerebella of nude mice. After 5 days, mice were treated with 50 mg/kg mebendazole (n ¼ 6) or mock-treated (Vehicle; n ¼ 8). A, survival of mice with DAOY cell–derived orthotopic tumors. Median survival was 75 days in the control (Vehicle) group and 113 days in the mebendazole-treated group (P ¼ 0.001). One mebendazole-treated mouse was euthanized after surviving 6 months without presenting any symptoms of lethal tumor growth. B, total RNA was harvested from representative tumors at the time of death. GLI1, PTCH1,andPTCH2 transcripts were quantified by qRT-PCR. C, tumor growth in live animals was quantified by bioluminescence imaging 5 days after implantation and again following 60 days of treatment. Representative images from the 60-day time point are shown at right.

Inhibition of Hh-dependent cell proliferation and survival, and bioluminescence imaging demonstrated a marked effect on tumor growth tumor cell proliferation in asymptomatic mice (Fig. 3C). Treat- We next assessed the effects of mebendazole on Hh-dependent ment of mice harboring DAOY-derived orthotopic tumors with cell proliferation and survival. DAOY cells have a gene expression 25 mg/kg mebendazole caused an increased median survival of 19 profile consistent with a type II (Hh-subtype) medulloblastoma days (Supplementary Fig. S1A) and similarly reduced tumor- and accordingly exhibit elevated Hh signaling (40). In these cells, specific expression of GLI1 and PTCH1 (Supplementary Fig. mebendazole caused a reduction in GLI1 expression with an S1B). Notably, DAOY xenografts exhibit large cell morphology IC50 ¼ 516 81 nmol/L (SEM; Fig. 2A). At similar concentrations, (40), which in naturally evolving tumors is associated with poor mebendazole markedly inhibited DAOY cell proliferation outcomes (41). (Fig. 2B). Clonogenic survival was affected by mebendazole at a concentration as low as 100 nmol/L (Fig. 2C). The viability of Inhibition of SMO via suppression of ciliogenesis DAOY cells, as assessed by a metabolic assay, was significantly We next tested the ability of mebendazole to counteract Hh impaired by mebendazole at concentrations approaching 1 mmol/ signaling induced by individual components of the pathway. L. In contrast, hTERT RPE-1 cells, which are Hh-responsive but not NIH3T3 cells strongly activated the Gli-luc reporter in response dependent on Hh signals for growth, were only modestly affected to ShhN-conditioned medium; this upregulation of Hh activity by this treatment (Fig. 2D). The expression of GLI1 protein was was suppressed by mebendazole and by vismodegib (Fig. 4A). similarly reduced in both cell types by increasing concentrations Hh signaling could also be strongly stimulated in these cells by of mebendazole, but only DAOY cells exhibited biochemical transient overexpression of the Ptch1-resistant SmoM2-mutant (Fig. 2E) and morphologic (Fig. 2F) evidence of apoptosis. protein (35), and by GLI1 or GLI2 (Fig. 4B). The stimulatory Accordingly, a significant proportion of DAOY cells stained with effect of SmoM2 on Gli-luc activation could be suppressed Annexin V after mebendazole treatment (Fig. 2G), and this by mebendazole, whereas reporter activation by the down- response was dose dependent (Fig. 2H). stream effectors GLI1 and GLI2 was resistant to mebendazole When injected into the cerebella of nude mice to form ortho- (Fig. 4C). topic xenograft tumors, DAOY cells were responsive to 50 mg/kg To assess the effect of mebendazole on upstream signaling by mebendazole administered by daily gavage. Mebendazole treat- SMO, we examined the cellular localization of a Smo–FLAG ment extended the median survival of tumor-bearing mice by 38 fusion protein (42) by immunofluorescence. Under low-serum days (Fig. 3A). Levels of GLI1 and PTCH2 transcripts were reduced conditions that favor primary cilium formation and robust SMO in the DAOY-derived tumors at the time of death (Fig. 3B), while activation, Smo–FLAG was predominantly localized to the

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Repurposing Mebendazole as a Hedgehog Inhibitor

A 120 B 250 Vehicle 100 0.1 μmol/L MBZ 200 1 μmol/L MBZ 80 0.2 μmol/L vismodegib 150 60 100 40 50 Relative luciferase Relative luciferase 20 0 0 Control ShhN

C 150 Vehicle 125 0.1 μmol/L MBZ 1 μmol/L MBZ 100 0.2 μmol/L vismodegib 75 50 25

Normalized luciferase 0 SmoM2 GLI1 GLI2

Figure 4. Mebendazole (MBZ) inhibits activation of SMO. A, NIH3T3 fibroblasts cotransfected with Gli-luc and Renilla reporter plasmids were maintained in low-serum conditions for 48 hours in the presence of ShhN-conditioned or control media. During this period, mebendazole or vismodegib were added at the indicated concentrations. B, a direct comparison of Gli-luc activation in NIH3T3 cells 48 hours after treatment with ShhN ligand, or 72 hours after cotransfection with plasmids that drive exogenous expression of the Ptch1-resistant Smo-mutant SmoM2, GLI1 or GLI2 in low-serum media. C, mebendazole or vismodegib was added to SmoM2-, GLI1-, or GLI2-transfected NIH3T3 cells during 48 hours of incubation in low-serum conditions. The activation of a cotransfected Gli-luc reporter byeach overexpressed gene, in the absence of drug treatment, was normalized to 100. primary cilia (Fig. 5A). Mebendazole-treated cells exhibited a Smo could be functionally suppressed by mebendazole (Fig. 6B). notable paucity of primary cilia, and in nonciliated cells Smo– Analysis of an expanded panel of all described vismodegib- FLAG was distributed throughout the cytoplasm (Fig. 5A). The resistant Smo mutants (14) confirmed that the inhibitory effects suppressive effect of mebendazole on ciliogenesis was quantified of mebendazole were unaffected by the vismodegib-selected in larger cell populations (Fig. 5B and C). The proportion of alterations in Smo (Fig. 6C). When used in combination, vismo- ciliated cells decreased in response to mebendazole within a degib and mebendazole additively suppressed the Gli-luc signal concentration range that also inhibited the polymerization of (Fig. 6D and E), a result that is consistent with two independent human a-tubulin in vitro (Fig. 5D). We next tested whether modes of pathway inhibition. allowing primary cilia to pre-form before mebendazole addition could alter the effect of mebendazole on pathway activity. Tem- Discussion porally separating serum starvation from mebendazole adminis- tration partially restored GLI1 expression (Fig. 5E) and viability The development of new drugs for the treatment of cancers is a (Fig. 5F) in mebendazole-sensitive DAOY cells. time consuming and expensive process. Unanticipated toxicities at all stages of development are a major cause of failure (43). Distinct modes of Hh-pathway inhibition by vismodegib and Repurposing well-characterized compounds for new uses can mebendazole minimize such uncertainty, save time and markedly reduce costs. As mebendazole exerts an indirect effect on SMO, we reasoned There are over 4,000 unique molecular entities approved world- that mebendazole might inhibit the activity of mutant SMO wide for human or veterinary use (44). Exploring new indications proteins that have been found to confer vismodegib resistance. for this rich compound library is a particularly attractive strategy To test this idea, we assayed the ability of mebendazole to inhibit to find new treatments for rare and neglected diseases, for which Gli-luc activation by wild-type and vismodegib-resistant mutant there are few financial incentives to justify the traditional process Smo proteins exogenously expressed in Smo / MEFs. Gli-luc of commercial drug discovery. Brain tumors are relatively rare, but activity was induced by ShhH ligand in the presence of either wild- highly lethal. The incidence of medulloblastoma, for example, is type Smo or the Smo D477G mutant. This mutation was originally approximately 1.5 per million in the United States and the found in a vismodegib-resistant allograft derived from a mouse majority of these cases occur in children (45). Economic con- medulloblastoma and corresponds to an equivalent mutation straints and the unique vulnerability of pediatric patients are found in a patient with recurrent disease (13). Activation of Gli- obstacles to the traditional drug discovery process. New strategies luc by Smo D477G was completely resistant to inhibition by to treat such diseases will likely involve agents that are first vismodegib (Fig. 6A). In contrast, both wild-type and mutant approved for other purposes.

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Larsen et al.

A DAPI Acetyl-a-tubulin Smo-FLAG Merge Mebendazole was developed in 1968 for use against a broad spectrum of gastrointestinal helminth pathogens, and remains among the most widely used agents in infected adults, including pregnant women, and children (46). Doses as high as 200 Vehicle mg/kg/d have been safely and effectively administered for up to 48 weeks for the treatment of internal hydatid cysts (47, 48). Although systemic absorption is low, serum levels of meben-

MBZ dazole peak 1 to 3 hours after intake, at reported levels that range from 0.3 to 1.6 mmol/L (48, 49). We observed significant levels of Hh pathway inhibition within this dose range. The only significant side effect of these high, chronic doses is B bone marrow suppression, which has been observed in fewer than 5% of patients. Case reports suggest that this adverse effect is associated with increased therapeutic effect, and is therefore probably caused by higher blood levels of mebendazole in these patients. In supportive clinical settings, this untoward effect could be straightforwardly monitored and was reversible upon cessation of treatment (50). We show here that the low-avidity binding of mebendazole Vehicle MBZ to human tubulin can inhibit the assembly of the primary C D cilium, a tubulin-based structure. Hh pathway components are concentrated in the primary cilium (4, 51), and mutations in Vismodegib Paclitaxel murine genes necessary for cilium formation cause phenotypes consistent with deficiencies in Hh signaling (52). In mice, primary cilia are dispensable for the growth of fibroblasts and other cell types, but essential for the ongoing proliferation of cultured medulloblastoma cells and the growth of estab- lished tumors (53). The differences in mebendazole sensitivity Ciliated cells (% of total) Polymerized tubulin (%) that we observed between human medulloblastoma cells and [MBZ], mol/L [MBZ], mol/L immortalized cells (Fig. 2D and E) are consistent with these dependencies. E F Treat in low The efficiency with which mebendazole can inhibit the for- Treat in low serum serum mation of the primary cilium suggests that this anti-Hh effect Preincubate Preincubate in low serum might account for much of its activity against brain tumors and in low serum other tumor types thus far tested. Effects on other targets may also be relevant. For example, signals arising from the Wnt signaling pathway and platelet-derived growth factor (PDGF) Cell viability (%)

GLI1/GAPDH mRNA GLI1/GAPDH ligands travel through the primary cilium (3). However, the developmental abnormalities that arise in mutant mice deﬁcient

[MBZ], mol/L [MBZ], mol/L for ciliogenesis most clearly overlap with those caused by Hh signaling defects (3). Beyond the primary cilium, microtubule Figure 5. assembly plays a fundamental role in cell division. Our results Mebendazole (MBZ) inhibits formation of primary cilia. A, a Smo–FLAG fusion cannot rule out effects of mebendazole on the mitotic spindle as protein was expressed in hTERT-RPE1 cells by transient transfection. After m a cause of cytotoxicity. However, this type of antiproliferative 48-hour incubation in low serum and treatment with 1 mol/L mebendazole fi or vehicle, cells were fixed, permeabilized, and stained with antibodies activity would not be highly speci c for cancer cells. Indeed, directed against acetyl-a-tubulin (green) and FLAG (red). Nuclei were mitotic spindle checkpoint defects that are found in many counterstained with DAPI. Scale bar, 10 mm. B, cilia were numerically assessed cancers have been proposed to confer relative resistance to by acetyl-a-tubulin staining in hTERT-RPE1 cells maintained in low-serum microtubule inhibitors (54). Interestingly, mebendazole-treated conditions and treated with mebendazole. Primary cilia (indicated by arrows) xenografts have been found to exhibit reduced vascularity (17). could be visualized on individual cells (inset). Scale bar, 20 mm. C, the effects VEGF-A has accordingly been proposed as a target of benzimid- of mebendazole or 0.2 mmol/L vismodegib (red line) on the proportion of ciliated cells. D, hTERT-RPE1 cells were treated with mebendazole at the azole therapy (18). Inhibition of Hh activation potently stimu- indicated concentrations or with 10 nmol/L paclitaxel (red line) for 48 hours lates angiogenesis by the paracrine induction of VEGF-A and under low-serum conditions. Polymerized and unpolymerized tubulin other angiogenic factors (55), and could therefore account for fractions were quantified by immunobloting, normalized to the loading these seemingly disparate observations. control b-actin, and expressed as the proportion of polymerized tubulin The central role of the primary cilium in Hh pathway acti- compared with the combined polymerized and unpolymerized tubulin. E, GLI1 vation suggests that interfering with cilia formation should be expression was assessed in DAOY cells that were incubated with mebendazole for 48 hours under the low-serum conditions that allow an effective strategy for targeting Hh-driven tumors. The inhib- formation of the primary cilium ("Treat in low serum"), or that were first itory effect of microtubule inhibitors on Hh signaling has been maintained in low serum for 20 hours before adding mebendazole for an demonstrated by unbiased screens that have identified such additional 48 hours ("Preincubate in low serum"). F, cell viability was assessed agents indirectly, as inhibitors of GLI activity, or by direct by CellTiter-Blue after the treatments described in E. inhibition of cilia formation and function (33, 56, 57). Thus

10 Mol Cancer Ther; 14(1) January 2015 Molecular Cancer Therapeutics

Repurposing Mebendazole as a Hedgehog Inhibitor

A B

Smo WT Smo D477G Smo WT Smo D477G Relative Gli-luciferase Relative Gli-luciferase

[Vismodegib], mol/L [MBZ], mol/L C 125

100

75 No drug MBZ 50 Vismodegib 25

Relative Gli-luciferase 0 WT M2 D388N D477G E522K G457S N223D L225R S391N DE

Control Control 0.1 μmol/L MBZ 0.01 μmol/L VIS 0.5 μmol/L MBZ 0.025 μmol/L VIS 1 μmol/L MB 0.05 μmol/L VIS Normalized luciferase Relative Gli-luciferase Relative Gli-luciferase

[Vismodegib], mol/L [Vismodegib], mol/L [MBZ], mol/L

Figure 6. Additive effects of mebendazole (MBZ) and vismodegib against SMO signaling. Wild-type Smo or the Smo D477G mutant were expressed with the Gli-luc and Renilla luciferase reporters by cotransfection into Smo/ MEFs. After 24 hours, cells were treated with vismodegib (A) or mebendazole (B) at the indicated concentrations, in the presence of ShhN-conditioned medium. C, the effects of 1 mmol/L mebendazole and 0.2 mmol/L vismodegib on Smo-dependent activation of the Gli-luc reporter were assessed against an expanded panel of Smo mutants. WT, wild-type Smo. D, the combined effects of vismodegib and mebendazole on relative Gli-luc activity were tested in Shh-Light2 cells under low-serum conditions with supplemental ShhN-media. The relative luciferase readout was normalized to 100 for each mebendazole concentration, so that the curves could be superimposed. The IC50 of vismodegib was unchanged by addition of mebendazole. E, a modification of the experiment shown in D, in which mebendazole was titrated into the Shh-Light2 Gli-luc assay along with fixed concentrations of vismodegib. far, the antimicrotubule agents identified in these screens are Authors' Contributions either highly toxic, or at early stages of characterization. Our Conception and design: A.R. Larsen, J.H. Chung, G.J. Riggins, F. Bunz data suggest that mebendazole might represent a simple path Development of methodology: A.R. Larsen, J.H. Chung, C.M. Rudin, F. Bunz forward for this promising therapeutic strategy. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.R. Larsen, R.-Y. Bai, A. Borodovsky A burgeoning body of evidence suggests that Hh signaling is Analysis and interpretation of data (e.g., statistical analysis, biostatistics, involved in the initiation and/or maintenance of a large pro- computational analysis): A.R. Larsen, J.H. Chung, F. Bunz portion of human cancers (2, 5, 6, 29). The repurposing of Writing, review, and/or revision of the manuscript: A.R. Larsen, J.H. Chung, mebendazole as an anticancer therapeutic will be guided by our C.M. Rudin, G.J. Riggins, F. Bunz rapidly growing understanding of the Hh pathway and how it Administrative, technical, or material support (i.e., reporting or organizing contributes to tumor growth. It will be important to understand data, constructing databases): A.R. Larsen Study supervision: F. Bunz how mebendazole interacts with other agents, including other Hh inhibitors. In particular, the additive effect of mebendazole Acknowledgments on vismodegib that we observed in vitro suggests a combina- The authors thank Dr. Verena Staedke for advice regarding the animal torial strategy that could potentially alleviate the untoward experiments. effects associated with vismodegib and also suppress recurrence. Clinical trials will be the next step in exploring these Grant Support new possibilities. This study was funded by grant R01CA157535 from the National Cancer Institute (Bethesda, MD) to F. Bunz. R-Y. Bai, A. Borodovsky, and G.J. Riggins Disclosure of Potential Conflicts of Interest were supported by the Virginia and D.K. Ludwig Fund for Cancer Research. J.H. No potential conflicts of interest were disclosed. Chung was supported by the AACR-Fight Colorectal Cancer Fellowship. G.J.

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Larsen et al.

Riggins is supported by the Irving Sherman Professorship. The Johns Hopkins advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate research core facilities were funded in part by Cancer Center grant this fact. P30CA006973 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the Received September 4, 2014; revised October 7, 2014; accepted October 27, payment of page charges. This article must therefore be hereby marked 2014; published OnlineFirst November 5, 2014.

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Repurposing the Antihelmintic Mebendazole as a Hedgehog Inhibitor

Andrew R. Larsen, Ren-Yuan Bai, Jon H. Chung, et al.

Mol Cancer Ther 2015;14:3-13. Published OnlineFirst November 5, 2014.

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