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Therapeutics, Targets, and Chemical Biology

Foretinib Is Effective Therapy for Metastatic SonicHedgehog MedulloblastomaClaudia C. Faria1,2, Brian J. Golbourn1, Adrian M. Dubuc1,3, Marc Remke1,3, Roberto J. Diaz1,Sameer Agnihotri1, Amanda Luck1, Nesrin Sabha1, Samantha Olsen1, Xiaochong Wu1,3,Livia Garzia1,3, Vijay Ramaswamy1,3, Stephen C. Mack1,3, Xin Wang1,3, Michael Leadley4,Denis Reynaud4, Leonardo Ermini4, Martin Post4, Paul A. Northcott5, Stefan M. Pfister5,SidneyE.Croul1,MarcelKool5,AndreyKorshunov6,ChristianA.Smith1,MichaelD.Taylor1,3,7,and James T. Rutka1,7,8

Abstract

Medulloblastoma is the most common malignant pediatricbrain tumor, with metastases present at diagnosis conferring apoor prognosis. Mechanisms of dissemination are poorlyunderstood and metastatic lesions are genetically divergentfrom the matched primary tumor. Effective and less toxictherapies that target both compartments have yet to be iden-tified. Here, we report that the analysis of several large non-overlapping cohorts of patients with medulloblastoma revealsMET kinase as a marker of sonic hedgehog (SHH)–drivenmedulloblastoma. Immunohistochemical analysis of phos-phorylated, active MET kinase in an independent patient cohortconfirmed its correlation with increased tumor relapse andpoor survival, suggesting that patients with SHH medulloblas-

tomamay benefit fromMET-targeted therapy. In support of thishypothesis, we found that the approved MET inhibitor fore-tinib could suppress MET activation, decrease tumor cell pro-liferation, and induce apoptosis in SHH medulloblastomasin vitro and in vivo. Foretinib penetrated the blood–brain barrierand was effective in both the primary and metastatic tumorcompartments. In established mouse xenograft or transgenicmodels of metastatic SHH medulloblastoma, foretinib admin-istration reduced the growth of the primary tumor, decreasedthe incidence of metastases, and increased host survival. Takentogether, our results provide a strong rationale to clinicallyevaluate foretinib as an effective therapy for patients with SHH-driven medulloblastoma. Cancer Res; 75(1); 134–46. �2014 AACR.

IntroductionMedulloblastoma, the most common malignant brain tumor

in childhood, has a high tendency to disseminate through thecerebrospinal fluid to the brain and the spinal cord leptome-ninges. Dissemination is a known factor of poor survival andoccurs in one third of the children at the time of diagnosis and intwo thirds by the timeof relapse. Affected children are treatedwithcraniospinal radiation and high-dose chemotherapy but the

majority of survivors suffer from severe neurocognitive deficitsinduced by the deleterious effects of treatment on the developingnervous system (1). The discovery of novel and less toxic therapieshas beenhamperedby thepoor understanding of themechanismsof dissemination, and by the failure to account for the geneticdivergence betweenmetastatic lesions and their matched primarytumor. Recent studies have shed light into the candidate genesthat drive leptomeningeal dissemination in medulloblastoma(2, 3), but therapies that target both the primary and the meta-static compartment have not been identified.

The hepatocyte growth factor (HGF)/cMET pathway is essentialfor cell proliferation and migration during embryogenesis (4)and, in the central nervous system, it plays a critical role incerebellar development (5). Aberrant cMET signaling is knownto be involved in tumor growth andmetastatic behavior of severalhuman cancers (6, 7). The transmembrane receptor cMET isactivated through phosphorylation of tyrosine residues uponbinding of its ligand HGF, which triggers multiple downstreameffector cascades including MAPK and PI3K/AKT that function invarious cellular processes including cell proliferation, cell surviv-al, migration, and invasion (8). In medulloblastoma, cMETactivation is associated with tumor growth and anaplastic histol-ogy (9). cMET signaling is deregulated in medulloblastomathroughmultiple, independentmolecular mechanisms includingepigenetic silencing of an upstream inhibitor, the serine proteaseinhibitor Kunitz-type 2 (SPINT2; refs. 10, 11). Previous studies haveshown that cMET inhibition can effectively decrease medullo-blastoma cell migration and invasion (12).

1Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital forSick Children, Toronto, Canada. 2Department of Neurosurgery, Hospi-tal de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon,Portugal. 3Program in Developmental and StemCell Biology, Hospitalfor Sick Children, Toronto, Canada. 4Program in Physiology & Exper-imental Medicine, Hospital for Sick Children, Toronto, Canada. 5Divi-sion of Pediatric Neurooncology and Division of Molecular Genetics,German Cancer Research Centre (DKFZ), University of Heidelberg,Heidelberg, Germany. 6Clinical Cooperation Unit Neuropathology,German Cancer Research Centre (DKFZ), Department of Neuropa-thology, University of Heidelberg, Heidelberg, Germany. 7Division ofNeurosurgery, Hospital for Sick Children, Toronto, Canada. 8Depart-ment of Surgery, University of Toronto, Toronto, Canada.

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

Corresponding Author: James T. Rutka, The Hospital for Sick Children, 555University Avenue, Suite 1503, Toronto, ON M5G 1�8, Canada. Phone: 416-813-6425; Fax: 416-813-4975; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3629

�2014 American Association for Cancer Research.

CancerResearch

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Foretinib is an orally available multikinase inhibitor thattargets cMET with high affinity (IC50 ¼ 0.4 nmol/L; ref. 13).Foretinib has demonstrated antitumor activity in preclinicalmodels of different tumor types (14–16) and clinical trials arecurrently ongoing to determine its efficacy in various solid, non–central nervous system tumors (17, 18). To date, the ability offoretinib to penetrate the brain is unknown.

Foretinib also targets other tyrosine kinases with lower affinity,including the platelet-derived growth factor receptor beta(PDGFRb) with an IC50 of 9.6 nmol/L. Interestingly, PDGFRb isknown to be overexpressed in metastatic medulloblastoma (19,20), and targeting the receptor reduces proliferation and migra-tion of medulloblastoma cell lines (21). Activation of PDGFRboccurs through a similar mechanism to cMET receptor activation,in which ligand binding (PDGF-BB) induces receptor autopho-sphorylation and activates downstream signaling via MAPK andAKT (22).

Therefore, we sought to establish the subgroup-specific role ofcMET and PDGFRb in medulloblastoma, and to test the efficacyof foretinib to cross the blood–brain barrier and to target thosepathways, both in the primary and in the metastaticcompartments.

Materials and MethodsTumor material and patient characteristics

All tissues and clinicopathologic information were seriallycollected in accordance with Institutional Review Boards fromcontributing institutions.Nucleic acid extractionswere carriedoutas previously described (23).

Expression profiling and molecular subgroupingExpression of candidate genes was assessed using the R2 soft-

ware in independent gene expression cohorts (24–32). Expressionof reported intermediates of MET signaling was visualized usingheatmaps (7). Associations between gene expression and sub-group affiliation were evaluated using one-way ANOVA. P values< 0.05 were considered to be statistically significant.

Analysis of somatic copy-number alterationsSomatic copy-number alterations were assessed on the Affyme-

trix SNP 6.0 array platform in 1,239 cases. Raw copy-numberestimates were obtained in dChip, followed by circular binarysegmentation (CBS) in R as previously described (27).

Cell lines and animal modelsHumanmedulloblastoma cell lines (Daoy, ONS76 and D425)

were kindly provided by Dr. Annie Huang, Hospital for SickChildren, Toronto, Canada. All cell lines were authenticated andtested by PCR. Daoy-GFP/Luciferase cells were generated asdescribed previously (33). Athymic nude mice were obtainedfrom the Charles River Laboratory. Ptchþ/�/SB11/T2Onc mice(2) were generously provided by Dr. Michael Taylor, Hospitalfor Sick Children, Toronto, Canada.

Cell culture assays for cMET and PDGFRb signalingForetinib was purchased from Selleck Chemicals, dissolved in

DMSO(Sigma), and stored at�20�C.Cellswere serumstarved for24 hours in media with 0.1% (Daoy and ONS76) and 2% FBS(D425), pretreated with increasing concentrations of foretinib or

1% v/v DMSO for 2 hours, and stimulated with human recom-binant HGF (20 ng/mL; Sigma-Aldrich) for 20minutes or humanPDGF-BB (20 ng/mL; Cell Signaling) for 10 minutes.

Migration and invasion assaysThe protocols for migration and invasion assays were as

described previously (34).For the radial migration assays, medulloblastoma cells were

seeded and allowed to migrate for 24 hours in starved media(0.1%FBS) containingHGF (50 ng/mL) or PDGF-BB (50 ng/mL),in the presence or absence of foretinib. The radius of themigratingcells was measured and compared with the initial radius using aLeica Fluorescent Stereoscope (2.5� magnification).

The invasion assays were performed using Matrigel InvasionChambers (8 mm pore size; BD Biosciences). Daoy and ONS76cells were incubated for 16 hours with starved media containingdilutions of foretinib. The number of cells per 6 randomfieldswasdetermined (�10 magnification) using Volocity software (PerkinElmer).

ImmunoblottingThe following antibodies from Cell Signaling were used: cMET

(1:1,000), phospho-cMET (1:1,000), PDGFRb (1:500), phospho-PDGFRb (1:500), AKT (1:1,000), phospho-AKT (1:2,000), p44/42 MAPK (1:1,000), phospho-p44/42 MAPK (1:2,000), PARP(1:1,000), b-actin (1:10,000), anti-rabbit IgG conjugated tohorseradish peroxidase (1:5,000), and anti-mouse IgG conjugat-ed to horseradish peroxidase (1:5,000).Western blot analysis andquantification were performed using the Fluorchem Q ImagingSystem (ProteinSimple).

Cell proliferation assaysMedulloblastoma cells were treated with different concentra-

tions of foretinib or DMSO, and cell viability was determined forthe indicated time points by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)absorbance at 490 nm (CellTiter 96 Aqueous One SolutionReagent; Promega).

Active caspase assaysThe activity of caspases 3 and 7 was measured using the Apo-

ONEHomogeneous Caspase-3/7 Assay (Promega) after 12 hoursof incubation.

Foretinib pharmacokinetic studiesDetection andquantification of foretinibwere performedusing

a high-performance LC/MS/MS method (Agilent 1290 HPLCAgilent Technologies/QTRAP 5500 AB SCIEX).

Images of foretinib distribution in various mouse organs wereacquired using a matrix-assisted laser desorption/ionization(MALDI) time-of-flight tandem mass spectrometer (AB SCIEXTOF/TOF 5800 System; AB SCIEX).

Medulloblastoma xenografts and transgenic mouse modelsAll mouse studies were approved and performed in accordance

to the policies and regulations of the Institutional Animal CareandUseCommittee of theUniversity of Toronto and theHospitalfor Sick Children, in Toronto.

Mouse subcutaneous xenografts (Daoy and ONS76) wereestablished in athymic nude mice (Charles River Laboratories).Intracranial xenografts of disseminated medulloblastoma were

Foretinib Treatment in Metastatic SHH Medulloblastoma

www.aacrjournals.org Cancer Res; 75(1) January 1, 2015 135




cMET activation (Discovery)

SHH (n =

51)

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3 (n

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6)

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Cancer Res; 75(1) January 1, 2015 Cancer Research136




established by injecting Daoy cells into the fourth ventricle ofathymic nude mice. Tumor growth and the presence of metas-tases were evaluated by weekly bioluminescence imaging usingthe IVIS Spectrum Optical In-vivo Imaging System (Caliper LifeSciences).

Osmotic pumps (Alzet; model 2004) were implanted in Ptchþ/�

micewithSleeping Beauty (SB) transposition (Ptchþ/�/SB11/T2Onc)at postnatal days 30 to 35. Pumps loaded with foretinib (6mg/kg)infused the drug into the cerebrospinal fluid (right lateral ventricle)for 28 days at a rate of 0.25 mL/hour.

ImmunohistochemistryThe protocol for immunohistochemistry was performed as

previously described (35). The following antibodies were used:Ki-67 (1:1,000; Novus), cleaved caspase 3 (1:800; Cell Signaling),phospho-cMET (1:100; Cell Signaling), and phospho-PDGFRb(1:200; Abcam).

Statistical analysisSurvival curves were generated using the Kaplan–Meier esti-

mate and a log-rank test. The comparison between binary andcategorical patient characteristics was performed using the two-sided Fisher exact test. To analyze contiguous variables, theMann–Whitney U test was used.

Results from experiments were expressed as mean � SEM. Formultiple groupcomparisons,ANOVAwas conducted followedbyapost-Tukey test or a post-Dunnett test. Direct comparisons using anunpaired two-tailed Student t test were conducted where appro-priate. Statistical analysis was performed using GraphPad Prism 5Software. We considered a P value inferior to 0.05 as significant.

ResultscMET and PDGFRb are highly expressed in SHHmedulloblastomas

Weevaluated theexpressionof cMET andPDGFRb inadiscoverycohort of primary medulloblastomas from Boston (n ¼ 199;ref. 26). cMET expression was highly upregulated in most SHHmedulloblastomas and in a subset of group 3 tumors (Fig. 1A).PDGFRb was expressed across all medulloblastoma subgroups,with higher levels of expression in SHH tumors (Fig. 1B). Weconfirmed our results using amulticenter validation cohort of 439cases profiled on the Affymetrix 133plus 2.0 arrays obtained fromthe German Cancer Research Centre, Heidelberg (validationcohort; refs. 24, 25, 31, 32, M. Kool and S.M Pfister, unpublisheddata; Fig. 1C and D).

To identify molecular markers of cMET pathway activation inSHHmedulloblastomas, we interrogated three independent data-sets using a group of genes known to activate cMET signaling (7,8). In addition to cMET, GAB1 expression was highly and specif-ically upregulated in SHH-driven medulloblastomas, across thethree nonoverlapping cohorts, when compared with normalcerebellum and with non-SHH medulloblastomas (Fig. 1E–J).

To investigate possible mechanisms leading to cMET andPDGFRb overexpression, we examined the subgroup-specificcopy number aberrations (CNA) encompassing the MET loci(7q31.2) and the PDGFRb loci (5q31), in a large cohort of1,239 medulloblastomas (27). There were no focal gains oramplifications of cMET (Supplementary Fig. S1A) and very infre-quent copy-number gains affecting the PDGFRb loci (Supplemen-tary Fig. S1B).

Identification of biologic pathways and processes associatedwith high cMET and low cMET SHH medulloblastomas

We performed gene set enrichment analysis between tumorswith the highest (n¼13) and the lowest (n¼13) cMET expressionacross a series of 51 primary SHH medulloblastomas (27). Genesets were compiled from Gene Ontology (GO), Kyoto Encyclo-pedia of Genes and Genomes (KEGG), the National CancerInstitute (NCI), Protein Families (PFAM), and Biocarta pathwaydatabases. To visualize significant gene sets (FDR < 0.25; P < 0.01)as interaction networks, we used Cytoscape and Enrichment Map(Supplementary Fig. S2). High cMET SHH medulloblastomaswere characterized by gene sets involved in organ developmentand morphogenesis, cell migration, cell cycle and DNA repair, T-cell differentiation, transcription, and mitochondrial function.Low cMET SHH medulloblastomas were defined by numerousnetworks including neural development, neurotransmission,amino acid and nucleotide metabolism, ribonucleotide biosyn-thesis, smooth muscle contraction, and Ras-GTPase activity.

High p-cMET levels correlate with recurrence and poorersurvival in SHH medulloblastomas

We stained medulloblastoma tissue microarrays comprised ofan independent cohort of 385 patients for the activated cMETreceptor or phosphorylated cMET (p-cMET). Eighty percent (104/128) of SHH tumors and approximately 25% (16/62) of group 3tumors showed high p-cMET staining (Fig. 2A). High p-cMETwasassociated with older age at diagnosis (Supplementary Fig. S3Aand S3B) and desmoplastic histology (Supplementary Fig. S3Cand S3D),most likely due to a subgroup-specific enrichment withSHH patients. There was a significant correlation between high p-cMET and an increased 5-year rate of recurrence across all sub-groups of pediatric medulloblastomas (P ¼ 0.041; Fig. 2B) and,particularly, across pediatric SHH tumors (P ¼ 0.011; Fig. 2C).There was no association of p-cMET expression and the presenceof metastases or TP53mutational status in SHH tumors (Supple-mentary Fig. S3E and S3F).

High p-cMET expression in SHH tumors was associated witha significantly shorter progression-free survival (P ¼ 0.007;Fig. 2D), particularly in the pediatric cohort (P ¼ 0.002;Fig. 2E). A trend toward a worse overall survival was also ob-served in these patients (Fig. 2F and G). Interestingly, consid-ering the metastatic status within SHH medulloblastomas, theprognostic impact of high p-cMET expression was still observed.High p-cMET correlated (P ¼ 0.022) with a poor outcome in

Figure 1.cMET is a signature gene in SHH medulloblastoma. A and B, expression of cMET (A) and PDGFRb (B) mRNA in a discovery cohort of primary medulloblastomas(Boston; n ¼ 199). C and D, cMET (C) and PDGFRb (D) expression analysis in a validation cohort with 439 cases (validation cohort 1, multicenter cohort fromHeidelberg). E–G, heatmaps illustrating aberrant overexpression of cMET activators in SHH medulloblastomas in three nonoverlapping cohorts of patients,discovery cohort (Boston; n ¼ 199; E), validation cohort 1 (Heidelberg; n ¼ 439; F), and validation cohort 2 (Medulloblastoma Advanced Genomics InternationalConsortium–MAGIC; n¼ 285; G). H–J, Z-scores demonstrating cMET activation in SHH tumors when compared with other medulloblastoma subgroups and normalcerebellum (CB) in all datasets (� , P < 0.05; ��, P < 0.001).






nonmetastatic SHH patients (Fig. 2H and Supplementary Fig.S4A). A trend toward a worse survival was observed in patientswith high p-cMET and leptomeningeal dissemination at diag-nosis (Fig. 2I and Supplementary Fig. S4B). In the pediatriccohort, high p-cMET was correlated (P ¼ 0.014) with a poorprogression-free survival (Supplementary Fig. S4C and S4D).Survival differences could not be determined in adult patientswith SHH medulloblastoma according to p-cMET status (Sup-plementary Fig. S4E and S4F). In group 3 medulloblastomas,expression of p-cMET had no prognostic relevance and did notcorrelate with metastatic status.

Thus, we conclude that activated cMET defines distinct prog-nostic patient cohorts with higher recurrence rate and poorerprognosis in SHH subgroup of medulloblastomas.

Foretinib inhibits cMET and PDGFRb pathway activityOn the basis of the elevated expression levels of cMET and

PDGFRb in SHH and group 3 medulloblastomas, we sought toevaluate the antitumoral effect of foretinib against medullo-blastoma cell lines representative of these two subgroups.Daoy, ONS76, and D425 cells have transcriptional and cyto-genetic features that suggest they are originally derived from

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SHH / all ages / M+ (PFS)I

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Figure 2.cMET pathway activation identifiessubgroups of medulloblastoma withdistinct clinical outcomes. A,immunopositivity for activated cMET(p-cMET) is a hallmark feature ofSHH medulloblastomas identifiedby immunohistochemistry ofmedulloblastoma tissue microarraysin a cohort of 385 patients.Representative photographs of tumorswith high p-cMET and low p-cMETstaining are shown. Scale bar, 100 mm.B andC, in pediatricmedulloblastomas,high p-cMET correlates with anincreased 5-year recurrence rateacross all subgroups (P¼ 0.041; B) andin the SHH subgroup (P ¼ 0.011; C).D–I, Kaplan–Meier survival curvesdisplaying progression-free survival(PFS) and overall survival (OS) in SHHmedulloblastomas according to age(D–G) and the presence ofleptomeningeal dissemination atdiagnosis (H and I). M0, nonmetastatictumors; Mþ, metastatic tumors.

Faria et al.





SHH (Daoy and ONS76) and group 3 (D425) tumors (36).Furthermore, they express different levels of the tyrosine kinasereceptors cMET and PDGFRb (Supplementary Fig. S5A). First,we evaluated the ability of foretinib to inhibit the cMET and thePDGFRb pathway activity in the context of HGF or PDGF-BBstimulation. Foretinib potently inhibited the HGF-inducedcMET pathway activation, as evidenced by the suppression incMET phosphorylation and also by the decrease in the phos-phorylation of downstream effectors AKT and MAPK (Fig. 3Aand B). The inhibitory effect of foretinib in PDGF-BB–inducedPDGFRb pathway activation was moderate (Supplementary Fig.S5B and S5C). Furthermore, a significant antiproliferative effect(Fig. 3C; Supplementary Fig. S6A and S6B) and an induction ofapoptosis (Fig. 3D and E; Supplementary Fig. S6C and S6D)were observed in foretinib-treated medulloblastoma cells. Fore-tinib potently inhibited HGF-mediated migration (Fig. 3F andG; Supplementary Fig. S6E and S6F) and invasion (Fig. 3H andSupplementary Fig. S6G) of Daoy and ONS76 cells for all drugconcentrations. In contrast, a significant reduction in migrationand invasion mediated by PDGF-BB was only seen with 2.5mmol/L of foretinib (Supplementary Fig. S6H and S6I).

We then assessed if cMET receptor knockdown by stableshRNA expression would phenocopy the functional effects offoretinib treatment. cMET shRNA 2 was able to knockdown over70% of cMET expression in both Daoy (Fig. 3I) and ONS76(Supplementary Fig. S6J). A significant antiproliferative effectwas observed in both medulloblastoma cells (Fig. 3J and Sup-plementary Fig. S6K) stably expressing cMET shRNA comparedwith nonsilencing control cells. Furthermore, a significant inhi-bition of HGF-mediated invasion was observed (Fig. 3K andSupplementary Fig. S6L). Interestingly, the relative inhibition ofinvasion in cMET shRNA expressing Daoy cells mimics thetreatment of Daoy cells with 500 nmol/L to 1 mmol/L of fore-tinib (Fig. 3H)

Collectively, these results suggest that foretinib exhibits asignificant in vitro inhibitory activity in medulloblastoma cells,mainly through blockade of the cMET pathway.

Foretinib induces medulloblastoma regression in vivoWe sought to investigate the antitumor activity of foretinib in

vivo using subcutaneous xenograft models of SHH medullo-blastoma. Daoy and ONS76 cells were implanted in the flank ofnude mice, and animals with established tumors were treatedby oral gavage with vehicle (DMSO) or foretinib (60 and 100mg/kg), once every other day for 9 days. Therapy with foretinibsignificantly induced tumor regression at all drug concentra-tions administered. At the end of treatment, tumor volumeswere reduced by 37% and 46% with 60 and 100 mg/kg offoretinib, respectively, in Daoy xenografts (Fig. 4A and B) andby 50% in ONS76 xenografts (Supplementary Fig. S7A andS7B). Foretinib was well tolerated as mice body weights weremaintained during the treatment period for all cohorts (Fig. 4Cand Supplementary Fig. S7C).

Medulloblastoma tumor growth inhibition in mice treatedwith foretinib was associated with concomitant reduction incMET and PDGFRb pathway activity (Fig. 4D and SupplementaryFig. S7D). Foretinib treatment resulted in a potent inhibition ofcMET phosphorylation in all cohorts (Fig. 4E and SupplementaryFig. S7E) and a moderate reduction in PDGFRb activation (Fig.4F). Tumors treatedwith foretinib also displayed significant dose-dependent decrease in cell proliferation (Fig. 4G and Supplemen-

tary Fig. S7F), and increase in apoptosis (Fig. 4H and Supple-mentary Fig. S7G).

These studies demonstrate that foretinib has a significant in vivoantitumor effect in medulloblastoma allografts, through a potentsuppression of the cMET pathway activity and a moderate inhi-bition of PDGFRb signaling.

Characterization of foretinib pharmacokinetics and brainpermeability

To further evaluate the potential clinical relevance of fore-tinib in brain tumor treatment, we assessed the pharmacoki-netics of the drug and its ability to penetrate into the brain.Nude mice were treated by oral gavage with 30, 60, and 100mg/kg of foretinib, and blood and the perfused brains wereharvested at specific time points. Foretinib was detected insamples from treated animals using high-performance LC/MS/MS. Maximum concentrations of foretinib in the plasma andin the brain of mice occurred 5 hours after oral administrationwith no difference between dose levels (Fig. 5A and B). Thesame pharmacokinetic features were previously reported in aphase I clinical trial of foretinib in adult patients withadvanced solid tumors outside the central nervous system(17). Furthermore, after 5 consecutive days of treatment with60 mg/kg of oral foretinib, the penetration of the drug in thebrain was approximately 14% (Supplementary Table S1).

We then assessed the distribution of foretinib in differentmouse organs using matrix-assisted laser desorption/ionizationwith time-of-flight mass spectrometer (MALDI-TOF) imaging.Foretinib produces a strong signal (Fig. 5C and D) and five hoursafter oral administration, when it reaches the maximum plasmaconcentration, the drug is seen in the liver and also in the renalcortex (Fig. 5E). The concentration of foretinib in the brain afteroral gavage was below the threshold detection of MALDI-TOFimaging.

To explore the potential use of foretinib for intrathecaltherapy in neuro-oncology, we used osmotic pumps to deliverthe drug into the lateral ventricles of mice. After injection of 6mg/kg of foretinib for 28 days at a rate of 0.25 mL/hour, thedrug was distributed through the cerebrospinal fluid to thesupra- and infratentorial compartments (Fig. 5F; ref. 37).Foretinib was detected by MALDI-TOF imaging in the rightfrontal lobe of mice due to its very high concentration at thedrug delivery site (Fig. 5G). The drug was well tolerated andthe mice did not display any signs of central nervous systemtoxicity.

Collectively, these studies demonstrate for the first time, to ourknowledge, that foretinib crosses the blood–brain barrier and canbe safely administered as intrathecal therapy.

Foretinib reduces SHH medulloblastoma growth anddissemination in mouse xenografts

Having established that foretinib can effectively be delivered inthe brain, we investigated its efficacy in mouse models of dis-seminated medulloblastoma. Daoy cells expressing luciferasewere orthotopically implanted into the fourth ventricle of nudemice. Animals with a detectable signal by bioluminescence at 3days after inoculation were treated by oral gavage either withvehicle (DMSO) or 60 mg/kg of foretinib once daily, 6 days aweek, for 2 weeks. The weekly evaluation of tumor growth anddissemination by bioluminescence showed a significant






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Faria et al.





reduction in tumor size andmetastases in the animals treatedwithforetinib (Fig. 6A and B), as confirmed by the smaller increase inthe total photon flux from luciferase expressing Daoy xenografts(Fig. 6C). Histologic examination [hematoxylin and eosin (H&E)stain] of brains and spinal cords of animals in the control groupshowed a higher number of metastases in the ventricles andleptomeningeal spaces, as well as surrounding the spinal cordand the nerve roots, when compared with foretinib-treated ani-mals (Fig. 6D and E).

These results demonstrate that foretinib is effective inreducing tumor growth and treating established medulloblas-toma metastases in orthotopic mouse models of disseminatedmedulloblastoma.

Foretinib is effective against the primary and the metastaticcompartments in a transgenic model of metastatic SHHmedulloblastoma

We then tested the efficacy of foretinib in a recently publishedmetastatic mouse model of SHH medulloblastoma (2). Theauthors used the SB transposon system where random insertionevents in the cerebellar progenitor cells of Ptchþ/� mice induce avery aggressive form of medulloblastoma with high incidence ofleptomeningeal dissemination by 10 weeks of age (2). SB medul-loblastomas express high levels of cMET, HGF, and other cMETpathway activators (Fig. 7A and B). We asked whether early andcontinuous treatment with foretinib could prevent the formationofmetastases and improve survival in thismousemodel. Osmotic

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Figure 4.Foretinib induces in vivo regression ofmedulloblastoma. A and B, nude micewith Daoy hindflank xenografts weretreatedwith vehicle control (10%DMSO,n ¼ 6) or foretinib (60 and 100 mg/kg;n ¼ 6 per group) once every other dayfor 9 days. Effect of foretinib on tumorgrowthwas comparedduring treatment(A) and in the end of treatment (B).Data represent group means � SEM(�� , P < 0.001). C, average animal bodyweight before and after therapy. Datarepresent group mean � SEM. D, flanktumors treated with foretinib showinhibition of p-cMET and p-PDGFRb,decreased cell proliferation (Ki-67staining), and increased apoptosis(cleaved caspase 3 staining). Scale bar,100 mm. E–H, immunohistochemicalquantification of p-cMET (E),p-PDGFRb (F), Ki-67 (G), and cleavedcaspase 3 (H). Data representmean � SEM (�, P < 0.05; �� , P < 0.01;�� , P < 0.001).

(Continued.) Representative photographs are shown with the nuclei stained with DAPI (10� magnification). I, Western blot of Daoy cells stably expressing cMETshRNA (shRNA 1, shRNA 2) or scramble nonsilencing control (NS control shRNA). Lowest panel (I) represents quantification of cMET expression using LiCorImageStudio software. J, proliferation assay of cMET shRNA 2 expressing Daoy cells over the course of 168 hours. K, knockdown of cMET receptor significantlydecreases HGF-dependent invasion. Representative photographs are shown with the nuclei stained with DAPI (10� magnification). Data represent mean oftriplicates � SEM (� , P < 0.05; �� , P < 0.01; �� , P < 0.0001).






pumps loaded with 6 mg/kg of foretinib or vehicle (10% cremo-phor) were implanted in 4 to 5 weeks old mice and delivered thedrug for 28 days, at a rate of 0.25 mL/hour. Strikingly, foretinib-treated animals showed a significant increase in survival (Fig. 7C)and displayed medulloblastomas in the cerebellum with a lessinvasive phenotype when compared with controls (Fig. 7Dand E). Moreover, treatment with foretinib reduced the incidenceof metastases by 36% (Fig. 7F) and effectively blocked cMETactivation (Fig. 7G and H). Mice tolerated intrathecal adminis-tration of foretinib, although 4 animals presented with intratu-moral hemorrhage in the cerebellum (2 animals in the vehiclecontrol group and 2 animals in the foretinib-treated group).

Taken together, these results demonstrate that foretinib iseffective both against the primary and the metastatic compart-ments in medulloblastoma, by reducing primary tumor invasionand preventing leptomeningeal dissemination in an aggressivemouse model of metastatic SHH medulloblastoma.

DiscussionWe show here that activation of cMET signaling is a hallmark

feature of SHH medulloblastomas and correlates with poor

outcome in pediatric patients. Targeting the cMET receptor withforetinib, a kinase inhibitor that crosses the blood–brain barrier,significantly reduces primary medulloblastoma growth and inva-sion, diminishes the incidence of metastases, and increases sur-vival in disseminated mouse models of SHH medulloblastoma.Our study identifies for the first time a subgroup-specific targeteddrug with promising efficacy against both medulloblastomaprimary tumors and metastases.

We demonstrate that cMET is highly expressed in SHHmedulloblastomas although the mechanisms leading to cMETup-regulation in this subgroup remain unclear. Of 332 medul-loblastomas recently sequenced, there were no recurrent muta-tions or amplifications of the cMET gene (32, 38–40). Theseresults suggest that the high expression of cMET is not driven byCNAs but may relate to the specific signaling events or cell oforigin of the SHH subgroup. Interestingly, our previous findingthat SPINT2, an inhibitor of cMET, is silenced by promotermethylation (10) and the recent publication that cMET is amongthe most significantly hypermethylated genes in group 4 tumors(41) suggest that cMET regulation in medulloblastomas may bedriven by epigenetic factors.

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Faria et al.





Examination of molecular pathways characterizing SHHtumors with high and low cMET expression revealed distinctpatterns of alteration. Although low cMET SHH medulloblas-tomas were associated with deregulation of biologic processesinvolved in neurogenesis and neurotransmission, high cMETSHH medulloblastomas were characterized by alterations in anumber of cancer-related networks, namely cell migration,cell cycle, DNA repair, and transcription. The activation ofdistinct biologic pathways in high cMET SHH medulloblas-tomas supports the existence of a different cell of origin forthese tumors, in keeping with a recent study that has shownthat a specific population of Nestin-expressing neuronal pro-genitors in the cerebellum can give rise to SHH-driven medul-loblastomas (42).

We identify a gene signature of cMETactivators, including cMETand GAB1, unique to SHH-driven tumors. Our results are sup-ported by a previous publication where immunoreactivity forGAB1 was reported to be a surrogate marker for SHH medullo-blastomas (43). Furthermore, we demonstrate that the cMETpathway activation status can segregate patients with SHHmedul-loblastomas into distinct prognostic outcomes. In pediatricpatients with SHH tumors, activation of cMET is correlated withan increased rate of relapse and a shorter progression-free survival.Notably, this association is not seen in adult SHH patientsconfirming the previously reported clinical and molecular dis-tinction between adult and pediatric SHH medulloblastomas(44). Furthermore, p-cMET status does not correlate with thepresence of metastases or TP53mutations, two knownmarkers of

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Figure 6.Foretinib decreases tumor growth andmetastases in medulloblastomaxenografts. A and B, representativebioluminescence imaging of nudemice intraventricular xenograftstreated with vehicle control (10%DMSO, n ¼ 11; A) or foretinib (60mg/kg, n ¼ 11; B) orally once daily for2 weeks. C, foretinib decreasesmedulloblastoma growth as denotedby a smaller change in total photonflux. Data represent group mean �SEM. D, foretinib-treatedintraventricular xenografts showsignificant reduction in the totalnumber ofmetastases. Data representgroup mean � SEM (�� , P < 0.01). E,representative H&E analysis of brainand spinal cord samples in the controlgroup and the foretinib-treatedgroup. Arrows denote metastaticdeposits in a representative animalfrom the control group, whereasarrowheads denote metastases,smaller in number and size of arepresentative animal from theforetinib-treated group. Scale bar,1,000 mm.






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Figure 7.Foretinib prevents metastases formation and increases survival in a transgenic mouse model of metastatic SHH medulloblastoma. A, heatmap illustrating aberrantoverexpression of cMET activators in medulloblastomas from Ptchþ/� mice with SB transposition (Ptchþ/�/SB11/T2Onc). B, cMET and HGF are highlyexpressed in medulloblastomas from Ptchþ/�/SB11/T2Onc mice when compared with normal cerebellum (CB). C, Kaplan–Meier survival curves demonstrate thatPtchþ/�/SB11/T2Onc mice treated with foretinib by osmotic pump infusion have an increased survival. The gray bar indicates the duration of treatment.D and E, primary medulloblastomas treated with foretinib (D) display a less invasive phenotype as denoted by the dashed line separating the tumor (T) andthe normal (N) cerebellum in H&E sections, and by the quantification (E) of the percent tumor invasion. F, foretinib decreases the incidence of metastases inan aggressive model of metastatic medulloblastoma. Data represent group mean � SEM. G and H, foretinib pump infusion inhibits cMET activation in micebearing SB medulloblastomas. Scale bar, 5 mm.

Faria et al.





poor prognosis across medulloblastoma subgroups (45) andwithin the SHH subgroup (46), respectively. We show that asimple immunohistochemical analysis of activated cMET pro-vides a high-quality clinical trial biomarker for identification ofpatients who could benefit from targeted therapy using cMETinhibitors.

Foretinib had dramatic therapeutic effect in SHH medullo-blastoma, both in vitro and in vivo. Furthermore, our pharma-cokinetic studies demonstrate for the first time that foretinibpenetrates the blood–brain barrier and is well tolerated anddistributed through intrathecal administration. Foretinibreduces primary medulloblastoma growth and invasion, andhas also a potent activity against medulloblastoma metastases.Administration of foretinib to an aggressive metastatic mousemodel of SHHmedulloblastoma increases survival by 45% anddiminishes the incidence of metastases by 36%. These tumorsexpress high levels of HGF, suggesting an autocrine signalingloop that maintains cMET pathway activation. Previous studieson glioblastoma have shown a correlation between HGF auto-crine expression by tumor cells and an increased sensitivity tocMET inhibition, which may explain the efficacy of foretinib(47). Interestingly, we show that foretinib also decreases acti-vation of AKT, a downstream effector of both cMET andPDGFRb signaling, and recently identified as a key contributorto leptomeningeal dissemination in medulloblastoma (2, 3).Therefore, the antimetastatic properties of foretinib may berelated to its unique ability to target three key drivers inmedulloblastoma dissemination, namely cMET, PDGFRb andindirectly, PI3K.

Given the high expression of cMET in SHH-driven medulloblas-tomas, the therapeutic regimen that may offer maximum benefitin this subset of patients is the combination of foretinib with anantagonist of SHH signaling. A significant antitumor effect wasreported in a patient with metastatic medulloblastoma after inhi-bition of smoothened (SMO), a critical component of SHH path-way, although the response was transient due to the emergence ofacquired resistance (48). Thus, we anticipate that combining fore-tinib with a SMO inhibitor may have a synergistic and an anti-resistance effect treating cMET-dependent SHHmedulloblastomas.

In summary, we show that the drug foretinib is effective treat-ment in preclinical murine models of metastatic SHH medullo-blastoma. Given the dismal outcome and lack of options for thesepatients, our results provide strong rationale for repurposingforetinib as a targeted agent in SHH-driven medulloblastoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C.C. Faria, B.J. Golbourn, A.M. Dubuc, C.A. Smith,M.D. Taylor, J.T. RutkaDevelopment ofmethodology:C.C. Faria, B.J. Golbourn, R.J. Diaz, M. Leadley,D. Reynaud, L. Ermini, M. Post, C.A. Smith, J.T. RutkaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.C. Faria, M. Remke, A. Luck, N. Sabha, X. Wu,L. Garzia, X. Wang, M. Leadley, D. Reynaud, L. Ermini, M. Post, S.M. Pfister,S.E. Croul, A. Korshunov, M.D. TaylorAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.C. Faria, A.M. Dubuc, M. Remke, S. Agnihotri,S. Olsen, V. Ramaswamy, S.C. Mack, X. Wang, M. Leadley, S.M. Pfister, M. Kool,M.D. Taylor, J.T. RutkaWriting, review, and/or revision of the manuscript: C.C. Faria, A.M. Dubuc,M. Remke, S. Agnihotri, S.C. Mack, X. Wang, M. Leadley, P.A. Northcott,S.M. Pfister, M. Kool, C.A. Smith, M.D. Taylor, J.T. RutkaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.C. Faria, A. Luck, P.A. Northcott, C.A. Smith,J.T. RutkaStudy supervision: J.T. RutkaOther (analytical method development and sample analysis—LC/MS/MSand MALDI-imaging): M. Leadley

AcknowledgmentsMass Spectrometry was performed at the Analytical Facility For Bioactive

Molecules (AFBM). The AFBM is part of the Centre for the Study of ComplexChildhood Diseases (CSCCD) at the Hospital for Sick Children, Toronto,Canada.

Grant SupportC.C. Faria was supported by a fellowship from The Hospital for Sick

Children Research Training Centre and the Garron Family Cancer Centre,and by the Programme for Advanced Medical Education, sponsored byFundac~ao Calouste Gulbenkian, Fundac~ao Champalimaud, Minist�erio daSa�ude e Fundac~ao para a Ciencia e Tecnologia, Portugal. This study wassupported by the Canadian Cancer Society Research Institute (grant #2011-70051), the Pediatric Brain Tumor Foundation of the United States, theBrain Tumour Foundation of Canada, b.r.a.i.n.child, Meagan's Walk andthe Wiley Fund at the Hospital for Sick Children. The CSCCD was supportedby the Canadian Foundation for Innovation (CFI).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 18, 2013; revised September 22, 2014; accepted October12, 2014; published OnlineFirst November 12, 2014.

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Faria et al.




2015;75:134-146. Published OnlineFirst November 12, 2014.Cancer Res Claudia C. Faria, Brian J. Golbourn, Adrian M. Dubuc, et al. MedulloblastomaForetinib Is Effective Therapy for Metastatic Sonic Hedgehog

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