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Supplementary Figure S1: Effect of MRK-003 on medulloblastoma cell line DAOY. A, Hes1 mRNA in response to treatment with MRK-003. B, Western blot and quantification of Hes1 protein after MRK-003 treatment (M). C, MTS assay measuring cell growth after MRK-003 treatment. D, QPCR of other pathway targets in response to MRK-003 treatment. * P <0.05, * P<0.005, using Student’s t-test. Supplementary Figure S2: A, Hes1 and Hes5 expression after treatment with cyclopamine. B, ChIP of Hes1 in JHH-GBM10. C, ChIP of Hes1 measured via quantitative PCR in DAOY. D, Hes5 expression after infection with NICD2 virus. Supplementary Figure S3: A, MTS assay measuring DAOY growth after treatment with vehicle, 0.4 M MRK-003, 10 M Cyclopamine, or both. B, Response of low passage GBM-derived neurosphere lines to MRK-003.

Supplementary Table S1: Primers used for chromatin immunoprecipitation. Supplementary Table S2: List of top genes altered by treatment with MRK-003. Primer Name Forward Reverse GAG GCA ACA GAG CCA CTA GG CAA GTC CAG AAG ACA CTC CTT TCC Gli Nbox0.5 AGA ATG ACC CCA TGC AAT TT CCT GAG GTC AGA GGG TCA AG CTC CAC CAT TAT CTC CCT GGC ATC GAC ACC TTA GGT TGC TGG TTA TAG CAG Gli Nbox1.1 CTC CCC CTC CAC CAT TAT CT GGA CAC CTT AGG TTG CTG GT GTG TAG CCC CAT TTC CTT GGA GGC TCT TC CAG GGG AAG GAG GGA AGT TTG CAA ATG ATC Gli Nbox 1.9 AGC CTG AAG GGG AGA TAT GG GGA GGG AAG TTT GCA AAT GA CCT GAT TTA AAC TAC CCC AGA ATA ACA CC CAC AAG CCA TCT GCC CTC TTA GAC Gli Nbox2.5 AGA TTG TAC ACG GGC TGT CC TGG ACC CTC TAA TTC TTC TTG C CAC GAG AAG GAA GGG GGA ACG CCT CTA ATT CTT CTT GCT GTA ACC CTG G Gli Nbox2.7 ACA CAG ACC ACA CAG GCA AA AGG GGT AAG GGG AAA TGA TG p27 GTG CAA TGG CAT GAT CAT AGC TCA CTG GTC AGA GTC CTT GCC TGT GGG Hes1 CGT GTC TCC TCC TCC CAT T GGC CTC TAT ATA TAT CTG GGA CTG C p63 GAA CAA CAG TAG AGA GGA TGC CCA GC GCT ATC AGA CAT AGA AGT TTA GGC ACT TCT CTC C Adjusted P- LnFC P. Value EntrezID Symbol Gene Name value

1.053 5.87E-17 9.46E-15 19 ABCA1 ATP-binding cassette sub-family A (ABC1) member 1 -0.626 1.05E-19 2.92E-17 39 ACAT2 acetyl-Coenzyme A acetyltransferase 2 -0.3997 3.15E-17 5.46E-15 79611 ACSS3 acyl-CoA synthetase short-chain family member 3 -0.4334 1.61E-17 2.94E-15 9465 AKAP7 A kinase (PRKA) anchor protein 7 aldo-keto reductase family 1 member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid -0.4227 9.55E-21 3.45E-18 1645 AKR1C1 dehydrogenase) -0.4444 5.52E-19 1.32E-16 9068 ANGPTL1 angiopoietin-like 1 -0.5592 1.87E-20 6.14E-18 307 ANXA4 annexin A4 apolipoprotein B mRNA editing enzyme catalytic -0.4814 3.73E-19 9.24E-17 60489 APOBEC3G polypeptide-like 3G 0.4908 4.73E-17 7.89E-15 50649 ARHGEF4 Rho guanine nucleotide exchange factor (GEF) 4 -0.9672 2.68E-22 1.81E-19 9823 ARMCX2 armadillo repeat containing X-linked 2 -0.4179 2.93E-21 1.22E-18 140462 ASB9 ankyrin repeat and SOCS box-containing 9 -0.8313 2.63E-21 1.11E-18 10409 BASP1 brain abundant membrane attached signal protein 1 0.7588 3.72E-19 9.24E-17 27113 BBC3 BCL2 binding component 3 butyrobetaine (gamma) 2-oxoglutarate dioxygenase -0.6208 4.42E-23 3.99E-20 8424 BBOX1 (gamma-butyrobetaine hydroxylase) 1 0.4289 4.05E-20 1.21E-17 605 BCL7A B-cell CLL/lymphoma 7A -0.5798 1.47E-18 3.31E-16 79365 BHLHE41 basic helix-loop-helix family member e41 -0.4236 2.14E-17 3.79E-15 642938 C10orf141 chromosome 10 open reading frame 141 -0.8444 4.68E-24 6.55E-21 715 C1R complement component 1 r subcomponent -0.5754 1.14E-20 4.02E-18 716 C1S complement component 1 s subcomponent -0.498 7.86E-22 4.16E-19 24141 C20orf103 chromosome 20 open reading frame 103 -0.4197 1.50E-17 2.80E-15 51313 C4orf18 chromosome 4 open reading frame 18 -0.4496 3.65E-20 1.10E-17 414328 C9orf103 chromosome 9 open reading frame 103 -0.4505 3.31E-18 7.19E-16 84302 C9orf125 chromosome 9 open reading frame 125 -0.4422 1.54E-17 2.87E-15 375759 C9orf50 chromosome 9 open reading frame 50 -0.4112 1.56E-17 2.90E-15 11092 C9orf9 chromosome 9 open reading frame 9 chaperone ABC1 activity of bc1 complex homolog (S. 0.4816 3.44E-17 5.88E-15 56997 CABC1 pombe) -0.5531 9.98E-18 1.95E-15 837 CASP4 caspase 4 apoptosis-related cysteine peptidase -0.3801 4.98E-17 8.20E-15 831 CAST calpastatin -0.9424 1.92E-23 2.01E-20 857 CAV1 caveolin 1 caveolae protein 22kDa -1.108 1.37E-20 4.73E-18 6347 CCL2 chemokine (C-C motif) ligand 2 0.4739 1.97E-19 5.18E-17 595 CCND1 cyclin D1 0.3758 4.04E-18 8.47E-16 64405 CDH22 cadherin-like 22 -0.379 6.52E-18 1.30E-15 1069 CETN2 centrin EF-hand protein 2 -0.595 4.90E-19 1.19E-16 3426 CFI complement factor I

-0.7333 1.10E-17 2.11E-15 9435 CHST2 carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 -0.4991 1.18E-17 2.25E-15 1266 CNN3 calponin 3 acidic -0.4646 2.74E-20 8.79E-18 1268 CNR1 cannabinoid receptor 1 (brain) -0.5089 1.76E-20 5.82E-18 1278 COL1A2 collagen type I alpha 2 -0.479 2.29E-19 5.90E-17 1280 COL2A1 collagen type II alpha 1 -0.4314 1.62E-17 2.94E-15 1363 CPE carboxypeptidase E 0.4355 1.57E-18 3.50E-16 10815 CPLX1 complexin 1 -0.4502 3.03E-20 9.46E-18 1368 CPM carboxypeptidase M -1.199 4.66E-32 8.85E-28 57699 CPNE5 copine V -0.7984 1.04E-21 5.18E-19 126129 CPT1C carnitine palmitoyltransferase 1C -0.485 9.48E-19 2.18E-16 54504 CPVL carboxypeptidase vitellogenic-like 0.5076 4.88E-17 8.11E-15 23418 CRB1 crumbs homolog 1 (Drosophila) -0.608 2.67E-18 5.81E-16 115908 CTHRC1 collagen triple helix repeat containing 1 -0.5363 6.42E-19 1.51E-16 51700 CYB5R2 cytochrome b5 reductase 2 -0.5833 1.04E-21 5.18E-19 1573 CYP2J2 cytochrome P450 family 2 subfamily J polypeptide 2

-0.5624 7.71E-18 1.52E-15 1595 CYP51A1 cytochrome P450 family 51 subfamily A polypeptide 1 -0.5446 4.43E-17 7.41E-15 79132 DHX58 DEXH (Asp-Glu-X-His) box polypeptide 58 0.8281 1.15E-21 5.56E-19 28514 DLL1 delta-like 1 (Drosophila) 0.8111 3.15E-26 9.75E-23 10683 DLL3 delta-like 3 (Drosophila) -0.4879 9.86E-20 2.81E-17 81704 DOCK8 dedicator of cytokinesis 8 0.5644 1.44E-21 6.66E-19 1826 DSCAM Down syndrome cell adhesion molecule 0.8077 5.36E-23 4.65E-20 253738 EBF3 early B-cell factor 3

-0.6784 4.89E-24 6.61E-21 2202 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 0.585 8.01E-24 9.66E-21 9086 EIF1AY eukaryotic translation initiation factor 1A Y-linked ELAV (embryonic lethal abnormal vision Drosophila)-like 4 0.5655 1.36E-17 2.57E-15 1996 ELAVL4 (Hu antigen D) -1.281 6.71E-21 2.57E-18 2012 EMP1 epithelial membrane protein 1 0.6575 3.25E-19 8.14E-17 2018 EMX2 empty spiracles homeobox 2 0.5784 3.97E-22 2.43E-19 196047 EMX2OS EMX2 opposite strand (non-protein coding) -0.59 5.72E-22 3.22E-19 55556 ENOSF1 enolase superfamily member 1

v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 -0.6474 3.06E-17 5.33E-15 2064 ERBB2 neuro/glioblastoma derived oncogene homolog (avian) -0.6945 9.63E-22 4.97E-19 63901 FAM111A family with sequence similarity 111 member A -0.7939 5.33E-24 6.61E-21 116496 FAM129A family with sequence similarity 129 member A -0.5455 1.24E-19 3.40E-17 2258 FGF13 fibroblast growth factor 13 0.3892 1.62E-17 2.94E-15 1E+08 FLJ25694 hypothetical protein FLJ25694 -0.7746 1.67E-21 7.46E-19 2331 FMOD fibromodulin -0.3966 5.77E-17 9.34E-15 2335 FN1 fibronectin 1 -0.3082 3.58E-17 6.09E-15 257019 FRMD3 FERM domain containing 3 -0.3763 4.29E-17 7.23E-15 84978 FRMD5 FERM domain containing 5 -0.4947 1.52E-21 6.95E-19 2487 FRZB frizzled-related protein -0.5582 9.88E-22 5.04E-19 8325 FZD8 frizzled homolog 8 (Drosophila) -1.026 3.33E-23 3.14E-20 9568 GABBR2 gamma-aminobutyric acid (GABA) B receptor 2 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- -0.6138 1.02E-20 3.66E-18 11227 GALNT5 acetylgalactosaminyltransferase 5 (GalNAc-T5) -0.5602 2.19E-18 4.88E-16 2596 GAP43 growth associated protein 43 glycine amidinotransferase (L-arginine:glycine -0.6034 1.84E-17 3.31E-15 2628 GATM amidinotransferase) H19 imprinted maternally expressed transcript (non- 0.988 6.31E-18 1.27E-15 283120 H19 protein coding) -1.105 5.18E-24 6.61E-21 3280 HES1 hairy and enhancer of split 1 (Drosophila) -3.006 1.44E-26 5.68E-23 388585 HES5 hairy and enhancer of split 5 (Drosophila) -1.517 1.66E-33 7.22E-29 23462 HEY1 hairy/enhancer-of-split related with YRPW motif 1 -0.572 1.27E-19 3.47E-17 23493 HEY2 hairy/enhancer-of-split related with YRPW motif 2 -0.5145 3.08E-20 9.47E-18 85236 HIST1H2BK histone cluster 1 H2bk -0.9026 6.76E-17 1.09E-14 3106 HLA-B major histocompatibility complex class I B -0.8094 2.45E-17 4.30E-15 3107 HLA-C major histocompatibility complex class I C -0.5084 3.89E-18 8.24E-16 3127 HLA-DRB5 major histocompatibility complex class II DR beta 5 -0.5544 4.97E-21 1.98E-18 3133 HLA-E major histocompatibility complex class I E -0.5295 2.78E-19 7.02E-17 221883 HOXA11AS HOXA11 antisense RNA (non-protein coding) -0.4899 1.16E-19 3.19E-17 3201 HOXA4 homeobox A4 -0.5047 2.83E-20 8.92E-18 3202 HOXA5 homeobox A5 -0.7886 7.70E-21 2.83E-18 3204 HOXA7 homeobox A7 -0.6412 1.73E-20 5.77E-18 3205 HOXA9 homeobox A9 -0.5775 7.82E-18 1.53E-15 3225 HOXC9 homeobox C9 -0.6238 1.37E-17 2.57E-15 51478 HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7

-0.5427 6.21E-23 5.18E-20 158160 HSD17B7P2 hydroxysteroid (17-beta) dehydrogenase 7 pseudogene 2 -0.6634 2.17E-24 3.77E-21 259217 HSPA12A heat shock 70kDa protein 12A -0.5057 3.36E-22 2.11E-19 10553 HTATIP2 HIV-1 Tat interactive protein 2 30kDa isoamyl acetate-hydrolyzing esterase 1 homolog (S. -0.9695 6.12E-32 8.85E-28 285148 IAH1 cerevisiae) -0.5422 2.72E-22 1.81E-19 10561 IFI44 interferon-induced protein 44

-0.6659 1.13E-21 5.50E-19 3434 IFIT1 interferon-induced protein with tetratricopeptide repeats 1 -0.8956 3.81E-23 3.51E-20 3490 IGFBP7 insulin-like growth factor binding protein 7 -0.4833 2.60E-19 6.60E-17 3598 IL13RA2 interleukin 13 receptor alpha 2 -0.4856 6.09E-21 2.36E-18 3556 IL1RAP interleukin 1 receptor accessory protein potassium voltage-gated channel Shal-related subfamily 0.48 6.47E-18 1.30E-15 3751 KCND2 member 2 0.5723 4.99E-17 8.20E-15 8645 KCNK5 potassium channel subfamily K member 5 potassium voltage-gated channel KQT-like subfamily 0.6624 2.99E-21 1.24E-18 3785 KCNQ2 member 2 -0.4932 2.05E-17 3.64E-15 9865 KIAA0644 KIAA0644 gene product -0.4375 4.96E-20 1.45E-17 126823 KLHDC9 kelch domain containing 9 -0.7132 5.31E-22 3.03E-19 3910 LAMA4 laminin alpha 4 leucine-rich repeat-containing G protein-coupled receptor 0.361 1.09E-17 2.10E-15 59352 LGR6 6 -0.6749 7.06E-22 3.83E-19 9388 LIPG lipase endothelial -0.5636 5.76E-19 1.36E-16 29995 LMCD1 LIM and cysteine-rich domains 1 -0.608 1.26E-24 2.49E-21 4005 LMO2 LIM domain only 2 (rhombotin-like 1) 0.4611 1.12E-21 5.50E-19 153346 LOC153346 hypothetical protein LOC153346 -0.5509 1.41E-21 6.56E-19 284998 LOC284998 hypothetical protein LOC284998 -0.5323 1.33E-22 9.97E-20 399959 LOC399959 hypothetical LOC399959 -0.3619 8.00E-17 1.26E-14 400043 LOC400043 hypothetical LOC400043 -0.433 4.13E-19 1.01E-16 728855 LOC728855 hypothetical LOC728855

-0.5965 5.18E-21 2.04E-18 731884 LOC731884 similar to programmed cell death 6 interacting protein -0.4792 2.18E-19 5.70E-17 1902 LPAR1 lysophosphatidic acid receptor 1 -0.5168 1.07E-18 2.43E-16 23643 LY96 lymphocyte antigen 96 MFNG O-fucosylpeptide 3-beta-N- 0.8516 5.89E-18 1.20E-15 4242 MFNG acetylglucosaminyltransferase -0.4994 2.36E-18 5.20E-16 4276 MICA MHC class I polypeptide-related sequence A

-0.4887 1.47E-18 3.31E-16 326625 MMAB methylmalonic aciduria (cobalamin deficiency) cblB type -0.4265 2.99E-17 5.23E-15 4482 MSRA methionine sulfoxide reductase A v-myc myelocytomatosis viral related oncogene 1.604 2.33E-19 5.98E-17 4613 MYCN neuroblastoma derived (avian) -0.7061 4.17E-25 9.52E-22 25791 NGEF neuronal guanine nucleotide exchange factor -0.4127 5.09E-19 1.23E-16 84628 NTNG2 netrin G2 -0.7279 8.28E-19 1.92E-16 169611 OLFML2A olfactomedin-like 2A -0.4545 5.18E-18 1.07E-15 9060 PAPSS2 3'-phosphoadenosine 5'-phosphosulfate synthase 2 -0.6131 7.19E-18 1.43E-15 79668 PARP8 poly (ADP-ribose) polymerase family member 8 -0.5465 2.35E-21 1.01E-18 56133 PCDHB2 protocadherin beta 2 -0.5089 1.77E-23 1.92E-20 56129 PCDHB7 protocadherin beta 7 -0.9422 4.03E-24 6.03E-21 56103 PCDHGB2 protocadherin gamma subfamily B 2

-0.9037 1.28E-23 1.43E-20 5156 PDGFRA platelet-derived growth factor receptor alpha polypeptide -0.6429 1.39E-20 4.75E-18 10630 PDPN podoplanin 0.4787 1.01E-17 1.96E-15 5178 PEG3 paternally expressed 3 -0.4959 4.81E-18 9.93E-16 64065 PERP PERP TP53 apoptosis effector -0.6586 3.75E-28 3.25E-24 10404 PGCP plasma glutamate carboxypeptidase -0.5797 6.62E-19 1.54E-16 54103 PION pigeon homolog (Drosophila) -0.5364 5.13E-17 8.36E-15 5569 PKIA protein kinase (cAMP-dependent catalytic) inhibitor alpha -0.7446 4.60E-24 6.55E-21 5327 PLAT plasminogen activator tissue -0.4105 3.38E-18 7.30E-16 91584 PLXNA4 plexin A4 -0.7416 4.35E-27 2.10E-23 5480 PPIC peptidylprolyl isomerase C (cyclophilin C)

-0.5471 4.78E-18 9.93E-16 79660 PPP1R3B protein phosphatase 1 regulatory (inhibitor) subunit 3B protein phosphatase 2 (formerly 2A) regulatory subunit B -0.5753 2.01E-20 6.51E-18 5521 PPP2R2B beta isoform -0.3828 5.06E-17 8.29E-15 59336 PRDM13 PR domain containing 13 -0.6636 1.37E-17 2.57E-15 112464 PRKCDBP protein kinase C delta binding protein 0.4669 1.81E-19 4.84E-17 5613 PRKX protein kinase X-linked -0.6204 6.07E-21 2.36E-18 5396 PRRX1 paired related homeobox 1 -1.053 3.59E-22 2.22E-19 7837 PXDN peroxidasin homolog (Drosophila) -0.773 5.10E-24 6.61E-21 5925 RB1 retinoblastoma 1 -0.3937 4.04E-17 6.85E-15 201299 RDM1 RAD52 motif 1 -0.5807 3.43E-18 7.37E-16 9628 RGS6 regulator of G-protein signaling 6 -0.5745 7.34E-21 2.73E-18 116832 RPL39L ribosomal protein L39-like 0.6605 9.14E-19 2.11E-16 140032 RPS4Y2 ribosomal protein S4 Y-linked 2 -0.7556 1.84E-26 6.66E-23 6285 S100B S100 calcium binding protein B 0.4142 1.92E-17 3.43E-15 26278 SACS spastic ataxia of Charlevoix-Saguenay (sacsin) -0.5625 9.59E-19 2.19E-16 219285 SAMD9L sterile alpha motif domain containing 9-like -0.9112 5.53E-28 4.00E-24 6307 SC4MOL sterol-C4-methyl oxidase-like -0.4749 3.46E-18 7.40E-16 6383 SDC2 syndecan 2 -0.8005 4.20E-19 1.02E-16 113675 SDSL serine dehydratase-like stress-associated endoplasmic reticulum protein family -0.6709 1.12E-24 2.31E-21 387923 SERP2 member 2

serpin peptidase inhibitor clade F (alpha-2 antiplasmin 0.9688 1.86E-19 4.94E-17 5176 SERPINF1 pigment epithelium derived factor) member 1 solute carrier family 2 (facilitated glucose transporter) 0.458 1.11E-20 3.94E-18 6517 SLC2A4 member 4 solute carrier organic anion transporter family member 0.8007 2.51E-21 1.07E-18 28231 SLCO4A1 4A1 SWI/SNF related matrix associated actin dependent -0.6208 1.29E-17 2.46E-15 6595 SMARCA2 regulator of chromatin subfamily a member 2 -0.5919 7.37E-21 2.73E-18 6649 SOD3 superoxide dismutase 3 extracellular -0.4993 5.51E-18 1.13E-15 22986 SORCS3 sortilin-related VPS10 domain containing receptor 3 -0.4691 3.11E-20 9.49E-18 347689 SOX2OT SOX2 overlapping transcript (non-protein coding) -1.121 9.69E-29 1.05E-24 6696 SPP1 secreted phosphoprotein 1 -0.7958 4.20E-21 1.69E-18 58472 SQRDL sulfide quinone reductase-like (yeast) -0.7803 3.43E-24 5.72E-21 8082 SSPN sarcospan (Kras oncogene-associated gene) -0.4346 2.44E-17 4.30E-15 6840 SVIL supervillin -0.6918 9.71E-23 7.80E-20 56849 TCEAL7 transcription elongation factor A (SII)-like 7

-0.3886 5.56E-17 9.03E-15 83439 TCF7L1 transcription factor 7-like 1 (T-cell specific HMG-box) 0.7177 1.92E-24 3.46E-21 54997 TESC tescalcin -0.6098 4.01E-18 8.44E-16 7045 TGFBI transforming growth factor beta-induced 68kDa -0.7447 1.15E-25 2.93E-22 7079 TIMP4 TIMP metallopeptidase inhibitor 4 -0.5832 3.63E-18 7.73E-16 84866 TMEM25 transmembrane protein 25 -0.552 5.73E-23 4.87E-20 7130 TNFAIP6 tumor necrosis factor alpha-induced protein 6

-0.4255 2.27E-19 5.89E-17 8742 TNFSF12 tumor necrosis factor (ligand) superfamily member 12 -0.5719 7.47E-17 1.19E-14 23321 TRIM2 tripartite motif-containing 2 -0.6589 2.31E-18 5.11E-16 7205 TRIP6 thyroid hormone receptor interactor 6 -0.6719 1.36E-20 4.71E-18 57616 TSHZ3 teashirt zinc finger homeobox 3

-0.4914 2.84E-20 8.92E-18 85416 ZIC5 Zic family member 5 (odd-paired homolog Drosophila) -0.5758 3.08E-20 9.47E-18 79891 ZNF671 zinc finger protein 671