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1 Supplementl Figures n Legens Epigeneti trgeting of Hegehog trnsriptionl output through BET romoomin inhiition Yujie Tng 1,2, Shrreh Gholmin 2, Simone Shuert 1,2, Mine I. Willrson 3, Alex Lee 4, Prtiti Bnophyy 5,6,7, Guillme Bergthol 5,6, Srn Msou 1, Brin Nguyen 1, Nujsunusi Vue 1, Brinn Blnsy 1, Furong Yu 1,2, Sekyung Oh 1,2, Pmelyn Woo 1, Spenser Chen 1, Anith Ponnuswmi 1, Mihelle Monje 1, Sott X. Atwoo 4, Rmon J. Whitson 4, Sihrth Mitr 2, Smuel H. Cheshier 2, Jun Qi 8, Rmeen Beroukhim 5,7,8, Jen Y. Tng 4, Ro Wehsler-Rey 9, Anthony E. Oro 4, Brin A. Link 3, Jmes E. Brner 5,7,8, Yoon-Je Cho 1,2,1 * 1Deprtment of Neurology n Neurologil Sienes, Stnfor University Shool of Meiine, Stnfor, CA USA 2Deprtment of Neurosurgery, Stnfor University Shool of Meiine, Stnfor, CA USA 3Deprtment of Cell Biology, Neuroiology n Antomy, Meil College of Wisonsin, Milwukee, WI USA 4Progrm in Epithelil Biology, Stnfor University Shool of Meiine, Stnfor, CA USA 5Deprtment of Cner Biology, Dn-Frer Cner Institute, Boston, MA USA 6Peitri Neuro-onology, Deprtment of Peitri Onology, Dn-Frer Cner Institute n Division of Peitri Hemtology/Onology, Boston Chilren s Hospitl, Boston, MA USA 7Bro Institute of MIT n Hrvr, Cmrige, MA USA 8Deprtment of Meil Onology, Dn-Frer Cner Institute, Boston, MA USA 9Snfor-Burnhm Meil Reserh, L Joll, CA USA 1Stnfor Cner Institute, Stnfor University Meil Center, Stnfor, CA USA *To whom orresponene shoul e resse: Yoon-Je Cho, MD Deprtment of Neurology n Neurologil Sienes Stnfor University Shool of Meiine 121 Welh Ro, MSLS Blg, Rm P213 Stnfor, CA 9435 Ph: Fx: Emil: yjho1@stnfor.eu Nture Meiine: oi:1.138/nm.3613
2 Supplementl Figure 1. Quntittive RT-PCR showing mrna levels in Hh-Light2 ells trete with Hh lign (Shh-N CM or SAG) lone or in omintion with inresing oses of. Dt represent men of triplites ± SD. Supplementl Figure 2. ()Quntittive RT-PCR of,, Br4 n in wil-type (WT) MEFs n Sufu MEFs. Dt represent men of triplites ± SD. () Quntittive RT- PCR of mrna in Sufu MEFs in response to inresing oses of. Dt represent men of triplites ± SD. () Quntittive RT-PCR of,, Br2 n Br3 mrnas in Sufu MEFs expressing shbr2s, shbr3s or shctrl. Dt represent men of triplites ± SD. () Quntittive RT-PCR showing Pth1 mrna levels in Hh-Light2 ells trnsiently trnsfete with HA--FL or My-GLI2-DN n their response to, GDC-449, or SANT-1. Dt represent men of triplites ± SD. (e)quntittive RT-PCR of n mrnas showing the time ourse of erese n mrna levels in response to (1 M). Dt represent men of triplites ± SD. Supplementl Figure 3. In WT-MB, Me1-MB n ASZ1 ells, () Quntittive RT- PCR of mrna levels in response to inresing oses of, t represent men of triplites ± SD. () Quntittive RT-PCR showing time ourse of n mrna levels in response to tretment (1 M), t represent men of triplites ± SD. (,) ChIPqPCR nlysis of Br4 n PolII oupnies on n promoter regions, t represent men of triplites ± SD. Supplementl Figure 4. () Comprison of ose-response mesuring ell viility in Hh-riven tumor ells (WT-MB n Me1-MB) n norml ells (Hh-Light2 n Sufu MEF). Dt represent group mens ± SD. ose-response urves mesuring ell viility from y1 to y3 in WT-MB () n Me1-MB () ells, t represent group mens ± SD. Annexin V/DAPI stining FACS nlysis mesuring ell poptosis in response to or SMO inhiitors in WT-MB () n Me1-MB (e) ells. (f) In Me1-MB ells, PI stining FACS nlysis mesuring ell yle istriution in response to. (g) Comintion tretment using inresing oses of n GDC-449 in WT-MB ells to test the rug Nture Meiine: oi:1.138/nm.3613
3 synergy. Dt represent group mens ± SD. (h) Quntittive RT-PCR nlysis of My n Myn mrna levels in response to (1 M) or GDC-449 (.1 M) tretment in WT- MB ells. Dt represent men of triplites ± SD. Supplementl Figure 5. () Hierrhil lustering of smples n ifferentilly regulte genes following 6-hour tretment of WT MB ells with, (1 M), or GDC-449 (.1 M). Re inites inrese gene expression n lue reue gene expression n isplye s row normlize t. Venn igrm n sttistil nlysis of the overlpping ifferentilly expresse genes () or enrihe gene sets () etween - n GDC-449- trete WT MB ells. Chi-squre test n Fisher s ext test were use for sttistil nlysis. () GSEA nlysis of -trete WT-MB ells using LEE_GLI1 gene sets. Supplementl Figure 6. In D477G-MB, RCMB25 n CHB_ATRT1 ells, () prolifertive inex ws mesure y EU inorportion in response to, GDC-449 or. () My/MYC n Myn/MYCN expressions were mesure y qrt-qpcr in response to or SMO inhiitors. Dt represent group mens ± SD. Supplementl Figure 7. () Tumor size of or vehile trete Me1-MB flnk llogrfts t y 21 post tretment. Quntittive RT-PCR of,, Br4 n in Me1-MB (), WT-MB (), D477G (), SMOi-nïve mbcc (e) n SMOi-resistnt mbcc (f) llogrfts fter tretment. Dt represent group mens ± S.E.M. Stuent s t-test ws use for sttistil nlysis. * P <.5, ** P <.1, *** P <.1. Nture Meiine: oi:1.138/nm.3613
4 Supplementry Figure 1 Tng et l. 214 Reltive mrna level Hh-Light2 Shh-N SAG Shh-N/SAG - + (μm) Nture Meiine: oi:1.138/nm.3613
5 Supplementry Figure 2 Tng et l Br4 WT MEF Reltive mrna levels 1..5 (μm) e shctrl shbr2-1 shbr shbr3-1 shbr Br2/Br3 h 3 h 6 h 24 h Reltive Pth1 mrna levels DN/FL - + GDC-449 SANT-1 My-GLI2-DN HA--FL Nture Meiine: oi:1.138/nm.3613
6 Supplementry Figure 3 Tng et l. 214 SMOWT-MB ells Me1-MB ells ASZ1 ells Reltive mrna levels μm.1 μm 1 μm GDC μm GDC μm GDC μm.1 μm.1 μm 1 μm Ctrl 3 h 6 h 24h GDC h GDC h GDC h Reltive mrna levels 1..5 (μm) h 3 h 7.5 h 24 h Reltive mrna levels 1..5 (μm) h 2.5 h 6 h 24 h Ak1 Reltive DNA levels TSS-1 -TSS-2 Reltive DNA levels TSS-1 -TSS-2 Reltive DNA levels TSS-1 -TSS-2 Anti-Br4 Anti-Pol II Anti-Br4 Anti-Pol II Anti-Br4 Anti-Pol II Reltive DNA levels TSS-1 -TSS-2 Reltive DNA levels TSS-1 -TSS-2 Reltive DNA levels TSS-1 -TSS-2 Anti-Br4 Anti-Pol II Anti-Br4 Anti-Pol II Anti-Br4 Anti-Pol II Nture Meiine: oi:1.138/nm.3613
7 Supplementry Figure 4 Tng et l. 214 Reltvie ell viility Hh-Light2 Me1-MB WT-MB Reltvie ell viility Dy 1 Dy 2 Dy 3 Reltvie ell viility Dy 1 Dy 2 Dy (μm) e (μm) (μm) g GDC-449 +GDC-449 AV DAPI GDC-449 f AV DAPI Perentge of totl ells %1 %8 %6 %4 %2 % G1 S G2/M (nm) GDC-449 (nm) Synergisti - + h GDC My Myn Nture Meiine: oi:1.138/nm.3613
8 Enrihment sore (ES) Supplementry Figure 5 Tng et l. 214 SMOwt-MB 1 SMOwt-MB 2 SMOwt-MB_GDC449_1 SMOwt-MB_GDC449_2 SMOwt-MB 1 SMOwt-MB 2 CCND2 BMP7 CCND1 CDK6 GLI2 PTCH1 HHIP Differentilly expresse genes Differentilly expresse gene sets GDC449 * * 38 *p <.1 Chi-squre test *p <.1 Fisher s ext test LEE_GLI1_MB gene set (Genes ssoite with MB ChIP-hip peks) Gene set NOM p-vl FDR q-vl FWER p-vl LEE_GLI1_MB LEE_GLI1_MB_ONLY LEE_GLI1_GNP_MB_OVERLAP LEE_GLI1_GNP Nture Meiine: oi:1.138/nm.3613
9 Supplementry Figure 6 Tng et l D477G-MB.4 RCMB25 (SUFU mutte) 4 CHB_ATRT1 (SMARCB1/SNF5 mutte) EU + % EU + / DAPI EU + % GDC-449 GDC-449 GDC-449 e f 1..5 My Myn GDC MYC MYCN GDC MYC MYCN GDC-449 Nture Meiine: oi:1.138/nm.3613
10 Supplementry Figure 7 Tng et l. 214 Tumor size (m 3 ) Me1-MB flnk llogrft P = Ctrl (n = 5) (n = 5) 1..5 Me1-MB flnk llogrft ** * ** Br4 Ctrl (n = 4) (n = 4) WT-MB flnk llogrft D477G-MB flnk llogrft Ctrl (n = 4) 1.6 (n = 4) *** ** ** *** *** ***.8.4 Ctrl (n = 4) (n = 4) Br4 Br4 e f SMOi-nive mbcc llogrft *** *** * Ctrl (n = 3) (n = 3) SMOi-resistnt mbcc llogrft *** ** *** Ctrl (n = 2) (n = 4) Br4 Br4 Nture Meiine: oi:1.138/nm.3613
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{ OI: 1.138/n31 Srifie n nlyze APs on week 1 s of iet 1 4 6 High-ft iet BrU High-ft iet BrU 4 High-ft iet BrU 6 High-ft iet BrU Lin - Lin - : C34 + : C9 + 1 1 3 1 4 1 5 C45 1 C34 1 1 1 1 3 1 4 1 5 S-1
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Copy Number ID2 MYCN ID2 MYCN. Copy Number MYCN DDX1 ID2 KIDINS220 MBOAT2 ID2
Copy Numer Copy Numer Copy Numer Copy Numer DIPG38 DIPG49 ID2 MYCN ID2 MYCN c DIPG01 d DIPG29 ID2 MYCN ID2 MYCN e STNG2 f MYCN DIPG01 Chr. 2 DIPG29 Chr. 1 MYCN DDX1 Chr. 2 ID2 KIDINS220 MBOAT2 ID2 Supplementry
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