Foretinib Is Effective Therapy for Metastatic Sonic Hedgehog Medulloblastoma Claudia C

Published OnlineFirst November 12, 2014; DOI: 10.1158/0008-5472.CAN-13-3629

Cancer Therapeutics, Targets, and Chemical Biology Research

Foretinib Is Effective Therapy for Metastatic Sonic Hedgehog Medulloblastoma Claudia 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, Sidney E.Croul1, Marcel Kool5, Andrey Korshunov6,Christian A. Smith1, Michael D.Taylor1,3,7, and James T. Rutka1,7,8

Abstract

Medulloblastoma is the most common malignant pediatric toma may benefit from MET-targeted therapy. In support of this brain tumor, with metastases present at diagnosis conferring a hypothesis, we found that the approved MET inhibitor fore- poor prognosis. Mechanisms of dissemination are poorly tinib could suppress MET activation, decrease tumor cell pro- understood and metastatic lesions are genetically divergent liferation, and induce apoptosis in SHH medulloblastomas from the matched primary tumor. Effective and less toxic in vitro and in vivo. Foretinib penetrated the blood–brain barrier therapies that target both compartments have yet to be iden- and was effective in both the primary and metastatic tumor tified. Here, we report that the analysis of several large non- compartments. In established mouse xenograft or transgenic overlapping cohorts of patients with medulloblastoma reveals models of metastatic SHH medulloblastoma, foretinib admin- MET kinase as a marker of sonic hedgehog (SHH)–driven istration reduced the growth of the primary tumor, decreased medulloblastoma. Immunohistochemical analysis of phos- the incidence of metastases, and increased host survival. Taken phorylated, active MET kinase in an independent patient cohort together, our results provide a strong rationale to clinically confirmed its correlation with increased tumor relapse and evaluate foretinib as an effective therapy for patients with SHH- poor survival, suggesting that patients with SHH medulloblas- driven medulloblastoma. Cancer Res; 75(1); 134–46. 2014 AACR.

Introduction majority of survivors suffer from severe neurocognitive deficits induced by the deleterious effects of treatment on the developing Medulloblastoma, the most common malignant brain tumor nervous system (1). The discovery of novel and less toxic therapies in childhood, has a high tendency to disseminate through the has been hampered by the poor understanding of the mechanisms cerebrospinal fluid to the brain and the spinal cord leptome- of dissemination, and by the failure to account for the genetic ninges. Dissemination is a known factor of poor survival and divergence between metastatic lesions and their matched primary occurs in one third of the children at the time of diagnosis and in tumor. Recent studies have shed light into the candidate genes two thirds by the time of relapse. Affected children are treated with that drive leptomeningeal dissemination in medulloblastoma craniospinal radiation and high-dose chemotherapy but the (2, 3), but therapies that target both the primary and the metastatic compartment have not been identified. 1Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for The hepatocyte growth factor (HGF)/cMET pathway is essential Sick Children, Toronto, Canada. 2Department of Neurosurgery, Hospi- for cell proliferation and migration during embryogenesis (4) tal de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon, and, in the central nervous system, it plays a critical role in Portugal. 3Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada. 4Program in Physiology & Exper- cerebellar development (5). Aberrant cMET signaling is known imental Medicine, Hospital for Sick Children, Toronto, Canada. 5Divi- to be involved in tumor growth and metastatic behavior of several sion of Pediatric Neurooncology and Division of Molecular Genetics, human cancers (6, 7). The transmembrane receptor cMET is German Cancer Research Centre (DKFZ), University of Heidelberg, activated through phosphorylation of tyrosine residues upon Heidelberg, Germany. 6Clinical Cooperation Unit Neuropathology, German Cancer Research Centre (DKFZ), Department of Neuropa- binding of its ligand HGF, which triggers multiple downstream thology, University of Heidelberg, Heidelberg, Germany. 7Division of effector cascades including MAPK and PI3K/AKT that function in 8 Neurosurgery, Hospital for Sick Children, Toronto, Canada. Depart- various cellular processes including cell proliferation, cell surviv- ment of Surgery, University of Toronto, Toronto, Canada. al, migration, and invasion (8). In medulloblastoma, cMET Note: Supplementary data for this article are available at Cancer Research activation is associated with tumor growth and anaplastic histol- Online (http://cancerres.aacrjournals.org/). ogy (9). cMET signaling is deregulated in medulloblastoma Corresponding Author: James T. Rutka, The Hospital for Sick Children, 555 through multiple, independent molecular mechanisms including University Avenue, Suite 1503, Toronto, ON M5G 1 8, Canada. Phone: 416-813- epigenetic silencing of an upstream inhibitor, the serine protease 6425; Fax: 416-813-4975; E-mail: [email protected] inhibitor Kunitz-type 2 (SPINT2; refs. 10, 11). Previous studies have doi: 10.1158/0008-5472.CAN-13-3629 shown that cMET inhibition can effectively decrease medullo- 2014 American Association for Cancer Research. blastoma cell migration and invasion (12).

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Foretinib is an orally available multikinase inhibitor that 1% v/v DMSO for 2 hours, and stimulated with human recom- targets cMET with high affinity (IC50 ¼ 0.4 nmol/L; ref. 13). binant HGF (20 ng/mL; Sigma-Aldrich) for 20 minutes or human Foretinib has demonstrated antitumor activity in preclinical PDGF-BB (20 ng/mL; Cell Signaling) for 10 minutes. models of different tumor types (14–16) and clinical trials are currently ongoing to determine its efficacy in various solid, non– Migration and invasion assays central nervous system tumors (17, 18). To date, the ability of The protocols for migration and invasion assays were as foretinib to penetrate the brain is unknown. described previously (34). Foretinib also targets other tyrosine kinases with lower affinity, For the radial migration assays, medulloblastoma cells were including the platelet-derived growth factor receptor beta seeded and allowed to migrate for 24 hours in starved media (PDGFRb) with an IC50 of 9.6 nmol/L. Interestingly, PDGFRb is (0.1% FBS) containing HGF (50 ng/mL) or PDGF-BB (50 ng/mL), known to be overexpressed in metastatic medulloblastoma (19, in the presence or absence of foretinib. The radius of the migrating 20), and targeting the receptor reduces proliferation and migra- cells was measured and compared with the initial radius using a tion of medulloblastoma cell lines (21). Activation of PDGFRb Leica Fluorescent Stereoscope (2.5 magnification). occurs through a similar mechanism to cMET receptor activation, The invasion assays were performed using Matrigel Invasion in which ligand binding (PDGF-BB) induces receptor autopho- Chambers (8 mm pore size; BD Biosciences). Daoy and ONS76 sphorylation and activates downstream signaling via MAPK and cells were incubated for 16 hours with starved media containing AKT (22). dilutions of foretinib. The number of cells per 6 random fields was Therefore, we sought to establish the subgroup-specific role of determined (10 magnification) using Volocity software (Perkin cMET and PDGFRb in medulloblastoma, and to test the efficacy Elmer). of foretinib to cross the blood–brain barrier and to target those pathways, both in the primary and in the metastatic Immunoblotting compartments. The 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/ Materials and Methods 42 MAPK (1:1,000), phospho-p44/42 MAPK (1:2,000), PARP Tumor material and patient characteristics (1:1,000), b-actin (1:10,000), anti-rabbit IgG conjugated to All tissues and clinicopathologic information were serially horseradish peroxidase (1:5,000), and anti-mouse IgG conjugat- collected in accordance with Institutional Review Boards from ed to horseradish peroxidase (1:5,000). Western blot analysis and contributing institutions. Nucleic acid extractions were carried out quantification were performed using the Fluorchem Q Imaging as previously described (23). System (ProteinSimple).

Cell proliferation assays Expression profiling and molecular subgrouping Medulloblastoma cells were treated with different concentra- Expression of candidate genes was assessed using the R2 soft- tions of foretinib or DMSO, and cell viability was determined for ware in independent gene expression cohorts (24–32). Expression the indicated time points by MTS (3-(4,5-dimethylthiazol-2-yl)- of reported intermediates of MET signaling was visualized using 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) heatmaps (7). Associations between gene expression and sub- absorbance at 490 nm (CellTiter 96 Aqueous One Solution group affiliation were evaluated using one-way ANOVA. P values Reagent; Promega). < 0.05 were considered to be statistically significant. Active caspase assays Analysis of somatic copy-number alterations The activity of caspases 3 and 7 was measured using the Apo- Somatic copy-number alterations were assessed on the Affyme- ONE Homogeneous Caspase-3/7 Assay (Promega) after 12 hours trix SNP 6.0 array platform in 1,239 cases. Raw copy-number of incubation. estimates were obtained in dChip, followed by circular binary segmentation (CBS) in R as previously described (27). Foretinib pharmacokinetic studies Detection and quantification of foretinib were performed using Cell lines and animal models a high-performance LC/MS/MS method (Agilent 1290 HPLC Human medulloblastoma cell lines (Daoy, ONS76 and D425) Agilent Technologies/QTRAP 5500 AB SCIEX). were kindly provided by Dr. Annie Huang, Hospital for Sick Images of foretinib distribution in various mouse organs were Children, Toronto, Canada. All cell lines were authenticated and acquired using a matrix-assisted laser desorption/ionization tested by PCR. Daoy-GFP/Luciferase cells were generated as (MALDI) time-of-flight tandem mass spectrometer (AB SCIEX described previously (33). Athymic nude mice were obtained þ TOF/TOF 5800 System; AB SCIEX). from the Charles River Laboratory. Ptch / /SB11/T2Onc mice (2) were generously provided by Dr. Michael Taylor, Hospital Medulloblastoma xenografts and transgenic mouse models for Sick Children, Toronto, Canada. All mouse studies were approved and performed in accordance to the policies and regulations of the Institutional Animal Care Cell culture assays for cMET and PDGFRb signaling and Use Committee of the University of Toronto and the Hospital Foretinib was purchased from Selleck Chemicals, dissolved in for Sick Children, in Toronto. DMSO (Sigma), and stored at 20C. Cells were serum starved for Mouse subcutaneous xenografts (Daoy and ONS76) were 24 hours in media with 0.1% (Daoy and ONS76) and 2% FBS established in athymic nude mice (Charles River Laboratories). (D425), pretreated with increasing concentrations of foretinib or Intracranial xenografts of disseminated medulloblastoma were

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A Discovery cohort (n = 199) C Validation cohort 1 (n = 439) 800 1,200 P < 0.0001 P < 0.0001 700 1,000 600 800 500

400 600

300 400 200 cMET expression cMET expression 200 100

0 0 Adult CB WNT SHH Group 3 Group 4 WNT SHH Group 3 Group 4 Fetal Adult CB CB

B Discovery cohort (n = 199) D Validation cohort 1 (n = 439) 180 400 P < 0.01 P < 0.0001 160 350

140 300 120 250 100 200 80 150 60 100 40 PDGFR b expression PDGFR b expression 50 20

0 0 Adult CB WNT SHH Group 3 Group 4 WNT SHH Group 3 Group 4 Fetal Adult CB CB

H cMET activation (Discovery) * E *** Discovery cohort (n = 199) 1.0 *** ***

Adult CB WNT SHH Group 3 Group 4 0.5 HGF

cMET Z-Score 0.0 GAB1 EGFR

–0.5 cMET activation 1)

n = 51) n = 1 –3 0 3 WNT (n = 14)SHH (n = 52) NCB ( Group 3 ( Group 4 (n = 71) F Validation cohort 1 (n = 439) I cMET activation (Validation 1) *** n.s. Fetal CB WNT SHH Group 3 Group 4 Adult CB 1.0 *** *** HGF cMET 0.5 GAB1 EGFR 0.0 Z-Score cMET activation –0.5 –3 0 3 –1.0 1)

n = 93) n = 13) n = 11 n = 164)

Validation cohort 2 (n = 285) WNT (n SHH= 53) ( G Group 3 ( Fetal CB (n = 5) Group 4 ( Adult CB ( SHH Group 3 Group 4 J cMET activation (Validation 2) 1.5 *** HGF cMET GAB1 1.0 EGFR 0.5 cMET activation Z-Score 0.0 –3 30 –0.5

n = 46) n = 188)

SHH (n = 51) Group 3 ( Group 4 (

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established by injecting Daoy cells into the fourth ventricle of To investigate possible mechanisms leading to cMET and athymic nude mice. Tumor growth and the presence of metas- PDGFRb overexpression, we examined the subgroup-specific tases were evaluated by weekly bioluminescence imaging using copy number aberrations (CNA) encompassing the MET loci the IVIS Spectrum Optical In-vivo Imaging System (Caliper Life (7q31.2) and the PDGFRb loci (5q31), in a large cohort of Sciences). 1,239 medulloblastomas (27). There were no focal gains or þ Osmotic pumps (Alzet; model 2004) were implanted in Ptch / amplifications of cMET (Supplementary Fig. S1A) and very infre- þ mice with Sleeping Beauty (SB) transposition (Ptch / /SB11/T2Onc) quent copy-number gains affecting the PDGFRb loci (Supplemen- at postnatal days 30 to 35. Pumps loaded with foretinib (6 mg/kg) tary Fig. S1B). infused the drug into the cerebrospinal fluid (right lateral ventricle) for 28 days at a rate of 0.25 mL/hour. Identification of biologic pathways and processes associated with high cMET and low cMET SHH medulloblastomas Immunohistochemistry We performed gene set enrichment analysis between tumors ¼ ¼ The protocol for immunohistochemistry was performed as with the highest (n 13) and the lowest (n 13) cMET expression previously described (35). The following antibodies were used: across a series of 51 primary SHH medulloblastomas (27). Gene Ki-67 (1:1,000; Novus), cleaved caspase 3 (1:800; Cell Signaling), sets were compiled from Gene Ontology (GO), Kyoto Encyclo- phospho-cMET (1:100; Cell Signaling), and phospho-PDGFRb pedia of Genes and Genomes (KEGG), the National Cancer (1:200; Abcam). Institute (NCI), Protein Families (PFAM), and Biocarta pathway databases. To visualize significant gene sets (FDR < 0.25; P < 0.01) Statistical analysis as interaction networks, we used Cytoscape and Enrichment Map Survival curves were generated using the Kaplan–Meier esti- (Supplementary Fig. S2). High cMET SHH medulloblastomas mate and a log-rank test. The comparison between binary and were characterized by gene sets involved in organ development categorical patient characteristics was performed using the two- and morphogenesis, cell migration, cell cycle and DNA repair, T- sided Fisher exact test. To analyze contiguous variables, the cell differentiation, transcription, and mitochondrial function. fi Mann–Whitney U test was used. Low cMET SHH medulloblastomas were de ned by numerous Results from experiments were expressed as mean SEM. For networks including neural development, neurotransmission, multiple group comparisons, ANOVA was conducted followed by a amino acid and nucleotide metabolism, ribonucleotide biosyn- post-Tukey test or a post-Dunnett test. Direct comparisons using an thesis, smooth muscle contraction, and Ras-GTPase activity. unpaired two-tailed Student t test were conducted where appro- High p-cMET levels correlate with recurrence and poorer priate. Statistical analysis was performed using GraphPad Prism 5 survival in SHH medulloblastomas Software. We considered a P value inferior to 0.05 as significant. We stained medulloblastoma tissue microarrays comprised of an independent cohort of 385 patients for the activated cMET Results receptor or phosphorylated cMET (p-cMET). Eighty percent (104/ cMET and PDGFRb are highly expressed in SHH 128) of SHH tumors and approximately 25% (16/62) of group 3 medulloblastomas tumors showed high p-cMET staining (Fig. 2A). High p-cMET was We evaluated the expression of cMET and PDGFRb in a discovery associated with older age at diagnosis (Supplementary Fig. S3A cohort of primary medulloblastomas from Boston (n ¼ 199; and S3B) and desmoplastic histology (Supplementary Fig. S3C ref. 26). cMET expression was highly upregulated in most SHH and S3D), most likely due to a subgroup-specific enrichment with medulloblastomas and in a subset of group 3 tumors (Fig. 1A). SHH patients. There was a significant correlation between high p- PDGFRb was expressed across all medulloblastoma subgroups, cMET and an increased 5-year rate of recurrence across all sub- with higher levels of expression in SHH tumors (Fig. 1B). We groups of pediatric medulloblastomas (P ¼ 0.041; Fig. 2B) and, confirmed our results using a multicenter validation cohort of 439 particularly, across pediatric SHH tumors (P ¼ 0.011; Fig. 2C). cases profiled on the Affymetrix 133plus 2.0 arrays obtained from There was no association of p-cMET expression and the presence the German Cancer Research Centre, Heidelberg (validation of metastases or TP53 mutational status in SHH tumors (Supple- cohort; refs. 24, 25, 31, 32, M. Kool and S.M Pfister, unpublished mentary Fig. S3E and S3F). data; Fig. 1C and D). High p-cMET expression in SHH tumors was associated with To identify molecular markers of cMET pathway activation in asignificantly shorter progression-free survival (P ¼ 0.007; SHH medulloblastomas, we interrogated three independent data- Fig. 2D), particularly in the pediatric cohort (P ¼ 0.002; sets using a group of genes known to activate cMET signaling (7, Fig. 2E). A trend toward a worse overall survival was also ob- 8). In addition to cMET, GAB1 expression was highly and specif- served in these patients (Fig. 2F and G). Interestingly, consid- ically upregulated in SHH-driven medulloblastomas, across the ering the metastatic status within SHH medulloblastomas, the three nonoverlapping cohorts, when compared with normal prognostic impact of high p-cMET expression was still observed. cerebellum and with non-SHH medulloblastomas (Fig. 1E–J). 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 from Heidelberg). 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 International Consortium–MAGIC; n ¼ 285; G). H–J, Z-scores demonstrating cMET activation in SHH tumors when compared with other medulloblastoma subgroups and normal cerebellum (CB) in all datasets (, P < 0.05; , P < 0.001).

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A B 5-year recurrence - pediatric MB 100 High p-cMET High p-cMET 80 Low p-cMET p-cMET 60 40 20

% Pediatric tumors 0 P = 0.041 Yes No Low p-cMET C 5-year recurrence - pediatric SHH 100 High p-cMET 80

Percentage/subgroup Low p-cMET 60 )

= 37) = 62) 40 n = 128 = 158) 4 (n P < 0.001 20 WNT ( SHH (n P = 0.011 Group 3 (n 0 Figure 2.

Group % Pediatric SHH tumors Yes No cMET pathway activation identiﬁes subgroups of medulloblastoma with SHH / all ages (PFS) SHH / pediatric (PFS) D E distinct clinical outcomes. A, 1.00 1.00 immunopositivity for activated cMET (p-cMET) is a hallmark feature of 0.75 0.75 SHH medulloblastomas identiﬁed 0.50 0.50 by immunohistochemistry of medulloblastoma tissue microarrays 0.25 0.25 in a cohort of 385 patients. Probability of PFS (%) P = 0.007 Probability of PFS (%) P = 0.002 Representative photographs of tumors 0.00 0.00 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 with high p-cMET and low p-cMET Time since diagnosis (years) Time since diagnosis (years) Number at risk Number at risk staining are shown. Scale bar, 100 mm. Low p-cMET 24 24 17 16 14 11 9 6 4 3 3 Low p-cMET 13 10 9 5 2 High p-cMET 104 95 66 48 41 31 26 17 13 9 5 High p-cMET 50 26 14 5 1 B and C, in pediatric medulloblastomas, Low p-cMET High p-cMET Low p-cMET High p-cMET high p-cMET correlates with an increased 5-year recurrence rate ¼ F SHH / all ages (OS) G SHH / pediatric (OS) across all subgroups (P 0.041; B) and in the SHH subgroup (P ¼ 0.011; C). 1.00 1.00 D–I, Kaplan–Meier survival curves 0.75 0.75 displaying progression-free survival (PFS) and overall survival (OS) in SHH 0.50 0.50 32% medulloblastomas according to age – 0.25 0.25 (D G) and the presence of Probability of OS (%) Probability of OS (%) P = 0.076 P = 0.059 leptomeningeal dissemination at 0.00 0.00 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 diagnosis (H and I). M0, nonmetastatic Time since diagnosis (years) Time since diagnosis (years) Number at risk Number at risk tumors; Mþ, metastatic tumors. Low p-cMET 24 24 19 16 14 11 9 6 4 3 3 Low p-cMET 13 10 9 5 2 1 High p-cMET 104 98 75 56 50 37 29 23 19 13 7 High p-cMET 50 31 16 7 6 1

Low p-cMET High p-cMET Low p-cMET High p-cMET

H SHH / all ages / M0 (PFS) I SHH / all ages / M+ (PFS) 1.00 1.00 0.75 0.75 0.50 0.50 0.25 0.25 Probability of PFS (%)

P = 0.022 Probability of PFS (%) P = 0.208 0.00 0.00 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time since diagnosis (years) Time since diagnosis (years) Number at risk Number at risk Low p-cMET 21 21 16 15 13 10 8 6 4 3 3 Low p-cMET 33111110000 High p-cMET 89 80 58 42 36 28 23 15 11 7 3 High p-cMET 1515865332222

Low p-cMET High p-cMET Low p-cMET High p-cMET

nonmetastatic SHH patients (Fig. 2H and Supplementary Fig. Thus, we conclude that activated cMET deﬁnes distinct prog- S4A). A trend toward a worse survival was observed in patients nostic patient cohorts with higher recurrence rate and poorer with high p-cMET and leptomeningeal dissemination at diag- prognosis in SHH subgroup of medulloblastomas. nosis (Fig. 2I and Supplementary Fig. S4B). In the pediatric cohort, high p-cMET was correlated (P ¼ 0.014) with a poor Foretinib inhibits cMET and PDGFRb pathway activity progression-free survival (Supplementary Fig. S4C and S4D). On the basis of the elevated expression levels of cMET and Survival differences could not be determined in adult patients PDGFRb in SHH and group 3 medulloblastomas, we sought to with SHH medulloblastoma according to p-cMET status (Sup- evaluate the antitumoral effect of foretinib against medullo- plementary Fig. S4E and S4F). In group 3 medulloblastomas, blastoma cell lines representative of these two subgroups. expression of p-cMET had no prognostic relevance and did not Daoy, ONS76, and D425 cells have transcriptional and cyto- correlate with metastatic status. genetic features that suggest they are originally derived from

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SHH (Daoy and ONS76) and group 3 (D425) tumors (36). tary Fig. S7F), and increase in apoptosis (Fig. 4H and Supple- Furthermore, they express different levels of the tyrosine kinase mentary Fig. S7G). receptors cMET and PDGFRb (Supplementary Fig. S5A). First, These studies demonstrate that foretinib has a significant in vivo we evaluated the ability of foretinib to inhibit the cMET and the antitumor effect in medulloblastoma allografts, through a potent PDGFRb pathway activity in the context of HGF or PDGF-BB suppression of the cMET pathway activity and a moderate inhi- stimulation. Foretinib potently inhibited the HGF-induced bition of PDGFRb signaling. cMET pathway activation, as evidenced by the suppression in cMET phosphorylation and also by the decrease in the phos- Characterization of foretinib pharmacokinetics and brain phorylation of downstream effectors AKT and MAPK (Fig. 3A permeability and B). The inhibitory effect of foretinib in PDGF-BB–induced To further evaluate the potential clinical relevance of fore- PDGFRb pathway activation was moderate (Supplementary Fig. tinib in brain tumor treatment, we assessed the pharmacoki- S5B and S5C). Furthermore, a significant antiproliferative effect netics of the drug and its ability to penetrate into the brain. (Fig. 3C; Supplementary Fig. S6A and S6B) and an induction of Nude mice were treated by oral gavage with 30, 60, and 100 apoptosis (Fig. 3D and E; Supplementary Fig. S6C and S6D) mg/kg of foretinib, and blood and the perfused brains were were observed in foretinib-treated medulloblastoma cells. Fore- harvested at specific time points. Foretinib was detected in tinib potently inhibited HGF-mediated migration (Fig. 3F and samples from treated animals using high-performance LC/MS/ G; Supplementary Fig. S6E and S6F) and invasion (Fig. 3H and MS. Maximum concentrations of foretinib in the plasma and Supplementary Fig. S6G) of Daoy and ONS76 cells for all drug in the brain of mice occurred 5 hours after oral administration concentrations. In contrast, a significant reduction in migration with no difference between dose levels (Fig. 5A and B). The and invasion mediated by PDGF-BB was only seen with 2.5 same pharmacokinetic features were previously reported in a mmol/L of foretinib (Supplementary Fig. S6H and S6I). phase I clinical trial of foretinib in adult patients with We then assessed if cMET receptor knockdown by stable advanced solid tumors outside the central nervous system shRNA expression would phenocopy the functional effects of (17). Furthermore, after 5 consecutive days of treatment with foretinib treatment. cMET shRNA 2 was able to knockdown over 60 mg/kg of oral foretinib, the penetration of the drug in the 70% of cMET expression in both Daoy (Fig. 3I) and ONS76 brain was approximately 14% (Supplementary Table S1). (Supplementary Fig. S6J). A significant antiproliferative effect We then assessed the distribution of foretinib in different was observed in both medulloblastoma cells (Fig. 3J and Sup- mouse organs using matrix-assisted laser desorption/ionization plementary Fig. S6K) stably expressing cMET shRNA compared with time-of-flight mass spectrometer (MALDI-TOF) imaging. with nonsilencing control cells. Furthermore, a significant inhi- Foretinib produces a strong signal (Fig. 5C and D) and five hours bition of HGF-mediated invasion was observed (Fig. 3K and after oral administration, when it reaches the maximum plasma Supplementary Fig. S6L). Interestingly, the relative inhibition of concentration, the drug is seen in the liver and also in the renal invasion in cMET shRNA expressing Daoy cells mimics the cortex (Fig. 5E). The concentration of foretinib in the brain after treatment of Daoy cells with 500 nmol/L to 1 mmol/L of fore- oral gavage was below the threshold detection of MALDI-TOF tinib (Fig. 3H) imaging. Collectively, these results suggest that foretinib exhibits a To explore the potential use of foretinib for intrathecal significant in vitro inhibitory activity in medulloblastoma cells, therapy in neuro-oncology, we used osmotic pumps to deliver mainly through blockade of the cMET pathway. the drug into the lateral ventricles of mice. After injection of 6 mg/kg of foretinib for 28 days at a rate of 0.25 mL/hour, the Foretinib induces medulloblastoma regression in vivo drug was distributed through the cerebrospinal fluidtothe We sought to investigate the antitumor activity of foretinib in supra- and infratentorial compartments (Fig. 5F; ref. 37). vivo using subcutaneous xenograft models of SHH medullo- Foretinib was detected by MALDI-TOF imaging in the right blastoma. Daoy and ONS76 cells were implanted in the flank of frontal lobe of mice due to its very high concentration at the nude mice, and animals with established tumors were treated drug delivery site (Fig. 5G). The drug was well tolerated and by oral gavage with vehicle (DMSO) or foretinib (60 and 100 the mice did not display any signs of central nervous system mg/kg), once every other day for 9 days. Therapy with foretinib toxicity. significantly induced tumor regression at all drug concentra- Collectively, these studies demonstrate for the first time, to our tions administered. At the end of treatment, tumor volumes knowledge, that foretinib crosses the blood–brain barrier and can were reduced by 37% and 46% with 60 and 100 mg/kg of be safely administered as intrathecal therapy. foretinib, respectively, in Daoy xenografts (Fig. 4A and B) and by 50% in ONS76 xenografts (Supplementary Fig. S7A and S7B). Foretinib was well tolerated as mice body weights were Foretinib reduces SHH medulloblastoma growth and maintained during the treatment period for all cohorts (Fig. 4C dissemination in mouse xenografts and Supplementary Fig. S7C). Having established that foretinib can effectively be delivered in Medulloblastoma tumor growth inhibition in mice treated the brain, we investigated its efficacy in mouse models of dis- with foretinib was associated with concomitant reduction in seminated medulloblastoma. Daoy cells expressing luciferase cMET and PDGFRb pathway activity (Fig. 4D and Supplementary were orthotopically implanted into the fourth ventricle of nude Fig. S7D). Foretinib treatment resulted in a potent inhibition of mice. Animals with a detectable signal by bioluminescence at 3 cMET phosphorylation in all cohorts (Fig. 4E and Supplementary days after inoculation were treated by oral gavage either with Fig. S7E) and a moderate reduction in PDGFRb activation (Fig. vehicle (DMSO) or 60 mg/kg of foretinib once daily, 6 days a 4F). Tumors treated with foretinib also displayed significant dose- week, for 2 weeks. The weekly evaluation of tumor growth and dependent decrease in cell proliferation (Fig. 4G and Supplemen- dissemination by bioluminescence showed a significant

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A C (μmol/L)

Control 500 nmol/L 1 μmol/L 2.5 μmol/L

(μmol/L)

(μmol/L) (μmol/L) D

(1 μmol/L)

B β-Actin (μmol/L)

E , (μmol/L) μmol/L ,

μ ( mol/L) (μmol/L) F G H

500 nmol/L 1 μmol/L 2.5 μmol/L 500 nmol/L 1 μmol/L 2.5 μmol/L

Daoy Daoy NS control shRNA I J Daoy K + HGF 150 *** 15,000 NS control shRNA *** MET shRNA 100 NS controlMET shRNA shRNAMET 1 shRNA 2 10,000 MET shRNA 2 ** MET +HGF Cell number Cell 50 β-Actin 5,000

100 (490 nm) Absorbance 80 0 60 40 0 050100150200 20 - HGF + HGF - HGF + HGF Percent (%) Percent 0 Time (hours) NS control shRNA MET shRNA 2

Figure 3. cMET targeting by foretinib in vitro. A and B, foretinib inhibits cMET pathway activation and downstream signaling as determined by Western blot and chemiluminescence densitometric analysis. Densitometric analysis represents the phosphorylated proteins compared with total proteins. C, proliferation of Daoy cells treated with increasing doses of foretinib for 4 days. D and E, representative Western blot and cleaved caspase 3/7 assays demonstrating induction of apoptosis after foretinib treatment in Daoy cells. RFU, relative ﬂuorescence units. F, representative images of a radial migration assay (magniﬁcation, 2.5). G and H, foretinib inhibits HGF-dependent cell migration (G) and invasion (H) in Daoy cells. (Continued on the following page.)

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A B C

) 120 3 Day 25 Before treatment

) After treatment 100 3 120 30 100 80 *** 80 *** 20 60 60 *** Control 40 10 40 60 mg/kg Foretinib treatment 20 Average weight (g) Average Tumor volume (mm 100 mg/kg 20 Tumor volume (mm 0 0 262422201816 Control 60 100 Control 60 100 Foretinib (mg/kg) Foretinib (mg/kg) Figure 4. Time (days post-surgery) Foretinib induces in vivo regression of medulloblastoma. A and B, nude mice D Control 60 mg/kg 100 mg/kg E with Daoy hindﬂank xenografts were 50 treated with vehicle control (10% DMSO, 40 n ¼ 6) or foretinib (60 and 100 mg/kg; 30 n ¼ 6 per group) once every other day 20 * H&E ** for 9 days. Effect of foretinib on tumor 10 growth was compared during treatment positive % p-cMET 0 Control 60 100 (A) and in the end of treatment (B). Foretinib (mg/kg) Data represent group means SEM F

( , P < 0.001). C, average animal body H&E 50 weight before and after therapy. Data 40 represent group mean SEM. D, ﬂank positive 30 * * tumors treated with foretinib show 20 b inhibition of p-cMET and p-PDGFR , 10 decreased cell proliferation (Ki-67

% p-PDGFR 0 staining), and increased apoptosis p-cMET Control 60 100 Foretinib (mg/kg) (cleaved caspase 3 staining). Scale bar, m – G 100 m. E H, immunohistochemical 50 ﬁ quanti cation of p-cMET (E), 40 b p-PDGFR (F), Ki-67 (G), and cleaved 30 *

caspase 3 (H). Data represent p-PDGFR β 20 *** mean SEM ( , P < 0.05; , P < 0.01; 10 % Ki-67 positive , P < 0.001). 0 Control 60 100 Foretinib (mg/kg) Ki-67 H 50 40 ** *** 30 20 positive

Cleaved 10 caspase 3

% Cleaved caspase 3 0 Control 60 100 Foretinib (mg/kg)

reduction in tumor size and metastases in the animals treated with Foretinib is effective against the primary and the metastatic foretinib (Fig. 6A and B), as confirmed by the smaller increase in compartments in a transgenic model of metastatic SHH the total photon flux from luciferase expressing Daoy xenografts medulloblastoma (Fig. 6C). Histologic examination [hematoxylin and eosin (H&E) We then tested the efficacy of foretinib in a recently published stain] of brains and spinal cords of animals in the control group metastatic mouse model of SHH medulloblastoma (2). The showed a higher number of metastases in the ventricles and authors used the SB transposon system where random insertion þ leptomeningeal spaces, as well as surrounding the spinal cord events in the cerebellar progenitor cells of Ptch / mice induce a and the nerve roots, when compared with foretinib-treated ani- very aggressive form of medulloblastoma with high incidence of mals (Fig. 6D and E). leptomeningeal dissemination by 10 weeks of age (2). SB medul- These results demonstrate that foretinib is effective in loblastomas express high levels of cMET, HGF, and other cMET reducing tumor growth and treating established medulloblas- pathway activators (Fig. 7A and B). We asked whether early and toma metastases in orthotopic mouse models of disseminated continuous treatment with foretinib could prevent the formation medulloblastoma. of metastases and improve survival in this mouse model. Osmotic

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

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A B Plasma Brain 16,000 30 mg/kg 2,500 30 mg/kg 60 mg/kg 14,000 60 mg/kg 100 mg/kg 12,000 2,000 100 mg/kg 10,000 1,500 8,000 6,000 1,000 4,000 500 2,000 Foretinib concentration (ng/mL) concentration Foretinib 0 (ng/mL) concentration Foretinib 0 Figure 5. 5 h 10 h 5 d 5 h 10 h 5 d Pharmacokinetics of foretinib in Time Time preclinical mouse models. A and B, concentration-time proﬁle of C D E foretinib determined by high- performance LC/MS/MS method in Mass-spectrometry MALDI-TOF PC(38:6) + H Foretinib Merge the plasma (A) and in the brain (B) of 10,000 Foretinib mice after oral administration of

8,000 gavage

rev foretinib. Data represent mean of oral iL quadruplicates SEM. C and D, 6,000

identiﬁcation of foretinib by after 4,000

5hmg/kg,100 MALDI-TOF imaging. m/z, mass-to- Foretinib Intensity, cps charge. E, distribution of foretinib in 2,000 the liver and kidney of mice 5 hours 632 633 634 635 636 637 yend after oral administration of 100 m/z iK mg/kg. F, comparison of foretinib F concentration (ng/mL) in different

10,000 areas of the brain after intrathecal Foretinib, 8,000 therapy (osmotic pumps) versus oral gavage. Data represent mean of 6,000 G triplicates SEM. G, foretinib 4,000 PC(32:0) + Na Foretinib Merge imaging with MALTI-TOF in the

2,500 mouse brain after local delivery by

no lortnoC 2,000 itatna osmotic pump. 1,500 1,000 lpm

500 ip Foretinib concentration (ng/mL)

0 m

ptsop b Plasma ini

Left brain

62 t

Right brain

roF ya Cerebellum-30Cerebellum-60 Left cerebellum Cerebellum-100

Right cerebellum D Osmotic pump (Day 26) Oral, 5 days (mg/kg)

pumps loaded with 6 mg/kg of foretinib or vehicle (10% cremo- outcome in pediatric patients. Targeting the cMET receptor with phor) were implanted in 4 to 5 weeks old mice and delivered the foretinib, a kinase inhibitor that crosses the blood–brain barrier, drug for 28 days, at a rate of 0.25 mL/hour. Strikingly, foretinib- significantly reduces primary medulloblastoma growth and inva- treated animals showed a significant increase in survival (Fig. 7C) sion, diminishes the incidence of metastases, and increases sur- and displayed medulloblastomas in the cerebellum with a less vival in disseminated mouse models of SHH medulloblastoma. invasive phenotype when compared with controls (Fig. 7D Our study identifies for the first time a subgroup-specific targeted and E). Moreover, treatment with foretinib reduced the incidence drug with promising efficacy against both medulloblastoma of metastases by 36% (Fig. 7F) and effectively blocked cMET primary tumors and metastases. activation (Fig. 7G and H). Mice tolerated intrathecal adminis- We demonstrate that cMET is highly expressed in SHH tration of foretinib, although 4 animals presented with intratu- medulloblastomas although the mechanisms leading to cMET moral hemorrhage in the cerebellum (2 animals in the vehicle up-regulation in this subgroup remain unclear. Of 332 medul- control group and 2 animals in the foretinib-treated group). loblastomas recently sequenced, there were no recurrent muta- Taken together, these results demonstrate that foretinib is tions or amplifications of the cMET gene (32, 38–40). These effective both against the primary and the metastatic compart- results suggest that the high expression of cMET is not driven by ments in medulloblastoma, by reducing primary tumor invasion CNAs but may relate to the specific signaling events or cell of and preventing leptomeningeal dissemination in an aggressive origin of the SHH subgroup. Interestingly, our previous finding mouse model of metastatic SHH medulloblastoma. that SPINT2, an inhibitor of cMET, is silenced by promoter methylation (10) and the recent publication that cMET is among Discussion the most significantly hypermethylated genes in group 4 tumors We show here that activation of cMET signaling is a hallmark (41) suggest that cMET regulation in medulloblastomas may be feature of SHH medulloblastomas and correlates with poor driven by epigenetic factors.

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Foretinib Treatment in Metastatic SHH Medulloblastoma

AB Week 1 Week 2 Week 1 Week 2 1.2

1.0

0.8 x107 0.6

Mouse #1 Mouse #1 0.4

0.2

Figure 6. p/sec/cm2/sr Control

Foretinib decreases tumor growth and Foretinib metastases in medulloblastoma xenografts. A and B, representative bioluminescence imaging of nude mice intraventricular xenografts Mouse #2 treated with vehicle control (10% Mouse #2 DMSO, n ¼ 11; A) or foretinib (60 mg/kg, n ¼ 11; B) orally once daily for 2 weeks. C, foretinib decreases medulloblastoma growth as denoted by a smaller change in total photon C D ﬂux. Data represent group mean 40 8 Control (n = 11) Control SEM. D, foretinib-treated Foretinib (n = 11) Foretinib intraventricular xenografts show 30 6 signiﬁcant reduction in the total number of metastases. Data represent 20 4 ** group mean SEM (, P < 0.01). E, representative H&E analysis of brain 10 2 and spinal cord samples in the control P < 0.05 group and the foretinib-treated % Change in total flux

0 Number of mets / animal 0 group. Arrows denote metastatic Week 1 Week 2 Control Foretinib deposits in a representative animal from the control group, whereas E Posterior fossa Brain Thoracic spinal cord Lumbar spinal cord arrowheads denote metastases, smaller in number and size of a representative animal from the foretinib-treated group. Scale bar, 1,000 mm. Foretinib Control

Examination of molecular pathways characterizing SHH We identify a gene signature of cMET activators, including cMET tumors with high and low cMET expression revealed distinct and GAB1, unique to SHH-driven tumors. Our results are sup- patterns of alteration. Although low cMET SHH medulloblas- ported by a previous publication where immunoreactivity for tomas were associated with deregulation of biologic processes GAB1 was reported to be a surrogate marker for SHH medullo- involved in neurogenesis and neurotransmission, high cMET blastomas (43). Furthermore, we demonstrate that the cMET SHH medulloblastomas were characterized by alterations in a pathway activation status can segregate patients with SHH medul- number of cancer-related networks, namely cell migration, loblastomas into distinct prognostic outcomes. In pediatric cell cycle, DNA repair, and transcription. The activation of patients with SHH tumors, activation of cMET is correlated with distinct biologic pathways in high cMET SHH medulloblas- an increased rate of relapse and a shorter progression-free survival. tomas supports the existence of a different cell of origin for Notably, this association is not seen in adult SHH patients these tumors, in keeping with a recent study that has shown conﬁrming the previously reported clinical and molecular dis- that a speciﬁc population of Nestin-expressing neuronal pro- tinction between adult and pediatric SHH medulloblastomas genitors in the cerebellum can give rise to SHH-driven medul- (44). Furthermore, p-cMET status does not correlate with the loblastomas (42). presence of metastases or TP53 mutations, two known markers of

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Faria et al.

+/- +/- Ptch /SB11/T2Onc A CB Ptch /SB11/T2Onc C Treatment

EGFR 1.00 GAB1 HGF

cMET 0.75

5.06 6.12 7.19 8.26 9.35 10.45 10.99

B 0.50 cMET (P = 0.02) HGF (P = 0.002) 7.0

7.5 0.25 6.5 7.0 Probability of OS (%) P = 0.016 6.5 6.0 0.00 6.0 0 50 100 150 200 250

expression 5.5 expression Survival (days) 2 2 5.5 Number at risk log 5.0 log 5.0 Control 18 18 12 3 0 0 Treatment 16 16 14 8 2 1 4.5 4.5 Control Foretinib n = 20) CB (n = 4) CB (n = 4)

E Primary MB invasion Ptch+/-/SB11/T2Onc (n = 20) Ptch+/-/SB11/T2Onc ( 100 80 Control Foretinib D 60 40 20 Percentage (%) P < 0.01 0 Control Foretinib > 75% 25-75% < 25%

F Metastases 100 80 N 60 T 40 20 T Percentage (%) P < 0.0001 N 0 Control Foretinib Metastases No metastases

G Control Foretinib H 20

p-cMET 5 * P = 0.04 % Cells p-cMET positive positive % Cells p-cMET 0 Control Foretinib

Figure 7. Foretinib prevents metastases formation and increases survival in a transgenic mouse model of metastatic SHH medulloblastoma. A, heatmap illustrating aberrant overexpression of cMET activators in medulloblastomas from Ptchþ/ mice with SB transposition (Ptchþ//SB11/T2Onc). B, cMET and HGF are highly þ expressed in medulloblastomas from Ptch / /SB11/T2Onc mice when compared with normal cerebellum (CB). C, Kaplan–Meier survival curves demonstrate that þ Ptch / /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) and the normal (N) cerebellum in H&E sections, and by the quantiﬁcation (E) of the percent tumor invasion. F, foretinib decreases the incidence of metastases in an aggressive model of metastatic medulloblastoma. Data represent group mean SEM. G and H, foretinib pump infusion inhibits cMET activation in mice bearing SB medulloblastomas. Scale bar, 5 mm.

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Foretinib Treatment in Metastatic SHH Medulloblastoma

poor prognosis across medulloblastoma subgroups (45) and Disclosure of Potential Conflicts of Interest within the SHH subgroup (46), respectively. We show that a No potential conflicts of interest were disclosed. simple immunohistochemical analysis of activated cMET pro- vides a high-quality clinical trial biomarker for identification of Authors' Contributions patients who could benefit from targeted therapy using cMET Conception and design: C.C. Faria, B.J. Golbourn, A.M. Dubuc, C.A. Smith, inhibitors. M.D. Taylor, J.T. Rutka Foretinib had dramatic therapeutic effect in SHH medullo- Development of methodology: C.C. Faria, B.J. Golbourn, R.J. Diaz, M. Leadley, D. Reynaud, L. Ermini, M. Post, C.A. Smith, J.T. Rutka blastoma, both in vitro and in vivo.Furthermore,ourpharma- Acquisition of data (provided animals, acquired and managed patients, cokinetic studies demonstrate for the first time that foretinib provided facilities, etc.): C.C. Faria, M. Remke, A. Luck, N. Sabha, X. Wu, penetrates the blood–brain barrier and is well tolerated and L. Garzia, X. Wang, M. Leadley, D. Reynaud, L. Ermini, M. Post, S.M. Pfister, distributed through intrathecal administration. Foretinib S.E. Croul, A. Korshunov, M.D. Taylor reduces primary medulloblastoma growth and invasion, and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, has also a potent activity against medulloblastoma metastases. 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, Administration of foretinib to an aggressive metastatic mouse M.D. Taylor, J.T. Rutka model of SHH medulloblastoma increases survival by 45% and Writing, review, and/or revision of the manuscript: C.C. Faria, A.M. Dubuc, diminishes the incidence of metastases by 36%. These tumors M. Remke, S. Agnihotri, S.C. Mack, X. Wang, M. Leadley, P.A. Northcott, express high levels of HGF, suggesting an autocrine signaling S.M. Pfister, M. Kool, C.A. Smith, M.D. Taylor, J.T. Rutka loop that maintains cMET pathway activation. Previous studies Administrative, technical, or material support (i.e., reporting or organizing on glioblastoma have shown a correlation between HGF auto- data, constructing databases): C.C. Faria, A. Luck, P.A. Northcott, C.A. Smith, J.T. Rutka crine expression by tumor cells and an increased sensitivity to Study supervision: J.T. Rutka cMET inhibition, which may explain the efficacy of foretinib Other (analytical method development and sample analysis—LC/MS/MS (47). Interestingly, we show that foretinib also decreases acti- and MALDI-imaging): M. Leadley vation of AKT, a downstream effector of both cMET and PDGFRb signaling, and recently identified as a key contributor Acknowledgments to leptomeningeal dissemination in medulloblastoma (2, 3). Mass Spectrometry was performed at the Analytical Facility For Bioactive Therefore, the antimetastatic properties of foretinib may be Molecules (AFBM). The AFBM is part of the Centre for the Study of Complex Childhood Diseases (CSCCD) at the Hospital for Sick Children, Toronto, related to its unique ability to target three key drivers in Canada. medulloblastoma dissemination, namely cMET, PDGFRb and indirectly, PI3K. Grant Support Given the high expression of cMET in SHH-driven medulloblas- C.C. Faria was supported by a fellowship from The Hospital for Sick tomas, the therapeutic regimen that may offer maximum benefit Children Research Training Centre and the Garron Family Cancer Centre, in this subset of patients is the combination of foretinib with an and by the Programme for Advanced Medical Education, sponsored by Fundaç~ao Calouste Gulbenkian, Fundaç~ao Champalimaud, Ministerio da antagonist of SHH signaling. A significant antitumor effect was Saude e Fundaç~ao para a Ciência e Tecnologia, Portugal. This study was reported in a patient with metastatic medulloblastoma after inhi- supported by the Canadian Cancer Society Research Institute (grant #2011- bition of smoothened (SMO), a critical component of SHH path- 70051), the Pediatric Brain Tumor Foundation of the United States, the way, although the response was transient due to the emergence of Brain Tumour Foundation of Canada, b.r.a.i.n.child, Meagan's Walk and acquired resistance (48). Thus, we anticipate that combining fore- the Wiley Fund at the Hospital for Sick Children. The CSCCD was supported tinib with a SMO inhibitor may have a synergistic and an anti- by the Canadian Foundation for Innovation (CFI). The costs of publication of this article were defrayed in part by the resistance effect treating cMET-dependent SHH medulloblastomas. payment of page charges. This article must therefore be hereby marked In summary, we show that the drug foretinib is effective treat- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate ment in preclinical murine models of metastatic SHH medullo- this fact. blastoma. Given the dismal outcome and lack of options for these patients, our results provide strong rationale for repurposing Received December 18, 2013; revised September 22, 2014; accepted October foretinib as a targeted agent in SHH-driven medulloblastoma. 12, 2014; published OnlineFirst November 12, 2014.

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146 Cancer Res; 75(1) January 1, 2015 Cancer Research

Foretinib Is Effective Therapy for Metastatic Sonic Hedgehog Medulloblastoma

Claudia C. Faria, Brian J. Golbourn, Adrian M. Dubuc, et al.

Cancer Res 2015;75:134-146. Published OnlineFirst November 12, 2014.

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