© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. in in overcoming therapy resistance caused by intratumoral heterogeneity. in vivo mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that contain glioblastomas both proneural and Using both genetic and inhibition, pharmacologic we found that proneural GSCs are sensitive preferentially to EZH2 disruption, regions expressed BMI1 protein, which promoted cellular survival under stress due to of downregulation the E3 ligase RNF144A. epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the histological features are associated with distinct programs, transcriptional with vascular regions showing a proneural profile, are Glioblastomas lethal cancers defined by angiogenesis and necrosis. pseudopalisading Here, we demonstrate that these Valentim ( Surgery, Medical University Center,Hospitals–Cleveland Cleveland, Ohio, USA. should Correspondence be addressed to J.N.R. ( Houston, Texas, USA. of Case Western Reserve University, Cleveland, Ohio, USA. 6 Western Reserve University, Cleveland, Ohio, USA. Institute and Hospital, Tianjin, P.R. China. 1 with mutations in is which the genes associated encoding (G-CIMP), phenotype methylator island CpG glioma subtypes: distinct several into glioblastoma categorized have samples glioblastoma adult bulk nutrient maintenance GSC promote and restriction, acid hypoxia, as such stressors, microenvironmental interact with that their microenvironment GSCs critically but rather are enriched in and perivascular hypoxic niches, suggesting angiogenesis and brain normal into invasion therapies, conventional to resistance promote they their that observations unresolved, by supported are been has GSCs significance of origin and cells) stem definition cancer the as Whereas known (also GSCs called tumor cells cell–like -initiating stem contain Glioblastomas pathways. molecular single of targeting to respond to unlikely is that complexity cellular tumors within a and (intratumoral and indicating and molecular heterogeneity), cellular heterogeneity) (intertumoral tumors between both heterogeneity, remarkable display Glioblastomas therapeutics. targeted effective into translated not has lesions molecular of ation sent one of the most rigorously characterized solid cancers, yet deline only with offering palliation current therapies tumor, brain primary lethal and prevalent most the is Glioblastoma Ryan C Gimple Xun Jin EZH2 inhibition Targeting glioma stem cells through combined BMI1 and nature medicine nature Received 23 September 2016; accepted 6 September 2017; published online 9 October 2017; [email protected] Division Division of Regenerative Medicine, Department of Medicine, University of San Diego, San Diego, California, USA. Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA. Global transcript profiling and DNA methylation analyses from from analyses methylation DNA and profiling transcript Global , , suggesting that strategies that target simultaneously multiple epigenetic regulators within may glioblastomas be effective 1 – 1 3 , , Qi-gang Zhou

4– 6

. GSCs are not uniformly distributed within tumors tumors within distributed uniformly not are GSCs . , Leo J , Y Leo Kim 9

1 Case Comprehensive Case Cancer Comprehensive Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. , 4 advance online publication online advance – 6 , , Xiuxing Wang ). 1 1 , , Jill S Barnholtz-Sloan , 4 – 3 6 First First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China. , , Qiulian Wu 7 , 9 , 1 0 5 1 . Medical Medical Scientist Training Program, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. , 6 1 , , Stephen C Mack . Glioblastomas . repre Glioblastomas 8 Department Department of Pediatrics, Division of Pediatric Hematology and Oncology, Baylor College of Medicine,

1 , 6 , Lisa , C Lisa Wallace 6– 8 . . Indeed, 9 , , Shideng Bao 2 , 3 1 - - .

, 8

high expression of mesenchymal genes ( genes mesenchymal of expression high show hypoxic) (NR; region necrotic the whereas genes, proneural of at barrier areas showed expression of high blood–brain angiogenesis, the of disruption by defined (ER), region enhancing the from cells by ( sampling glioblastoma multiregional microenvironments image-guided tumor specific within patterns expression subgroups different from cells contain As tumors bulk RESULTS niches. specific their of context in the subsets of different targeting therapeutic differential requiring niches, in reside that different of they suggests GSCs ous distribution biology of a cell-intrinsic late the dominant subtype of the parental tumor, retention suggesting tumor grown recapitu cells stem under conditions cell Nevertheless, plastic are proneural- therapy, cytotoxic toward after transition tendency to-mesenchymal a including groups, transcriptional expression gene subtype-specific divergent with for efforts glioblastoma medicine precision guide to able been not has profiling mesenchymal and proliferative; or classical proneural; ( 2 and 1 dehydrogenases isocitrate

, , Tyler E Miller 1 , , Briana C Prager 1 , 7 doi:10.1038/nm.441 , 9 , , Andrew E Sloan 1 2 , , possibly because the tumors comprise cell populations 1 , 4 , 5 7 , , Ping Huang 17 Cleveland Cleveland Clinic Lerner College of Medicine 1 , , 1 5 4 8 – . We hypothesized that the heterogene the that . We hypothesized 7 , , Tanwarat Sanvoranart 9 , 10 [email protected] IDH1 & Jeremy N Rich 2 Tianjin Medical University Cancer 4 Department Department of Pathology, Case 1 and and Fig. 1 Fig. , Claudia , L Claudia 10 Department Department of Neurological IDH2 b ). An intermediate intermediate An ). 13 s e l c i t r a , 1 ); non-G-CIMP non-G-CIMP ); 4 Fig. Fig. 1 . Glioblastoma Glioblastoma . 1 3 1 , we profiled , we profiled ) ) or A.E.S. 1 . Genomic Genomic . 1 a , ). ). Tumor 6 1 , , 7

, 9 15 , 1 6  - - . © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. -independent mechanism -independent gene can a H3K27me3 expression through silence also noncanonical, PRC1 that suggests evidence recent However, II. polymerase RNA of pausing and compaction chromatin causes on 2A (H2AK119Ub) K119 histone of monoubiquitination then BMI1, contains which PRC1, by recognized then is H3K27me3 (H3K27me3). K27 at residue H3 histone trimethylates EZH2, subunit, chro catalytic to its and binds matin first PRC2 modifications: histone specific through compaction chromatin regulate PRC2 and PRC1 expression. gene global of regulation epigenetic of modifiers chromatin major prise com (PRCs) complexes repressive Polycomb regulation. chromatin 4c Fig. ( features and hypoxic vascular with correlated tures signa transcriptional in variation regional the that demonstrating tiregional patient biopsy samples validated these predictors of unfavorable glioblastoma patient survival as major microenvironments and hypoxic microvascular supporting hypoxic expression patterns fared the worst ( ( survival patient with anticorrelated were hypoxia and signatures lar hypoxia and markers for expressed microvasculature glioblastomas mesenchymal Proneural glioblastomas expressed markers of mature vessels, whereas ogy, features ( molecular and grade defining ( database, and signatures in hypoxia were glioblastoma expressed vascular both glioma–glioblastoma low-grade (TCGA) Atlas Genome ( database GAP and samples the Ivy natures in and glioblastoma were genes analyzed normoxia to hypoxia glioblastoma comparing analyses and (HMVEC)) cells lial endothe and human microvascular (HUVEC) cells endothelial vein umbilical (human sources vascular from data microarray on based ( nature and microvascular proliferative regions expressed a mesenchymal sig perinecrotic whereas a signature, proneural tumor expressed regions from biopsy results, the edge MRI-guided leading and infiltrating ( RNA-seq with microdissected regionally 42 glioblastomas from data contains which database, GAP) (Ivy Project Atlas Glioblastoma Ivy the interrogated we cohort, tumor larger a in observations our date ( hypoxia and vascularity of variance regional the confirmed hypoxia for (CA9) 9 anhydrase carbonic and vessels for (vWF) factor Willebrand von using ( staining hypoxia Immunofluorescence with cells, mesenchymal and regions, vascular markers confirmed that proneural-enriched cells were associated with markers ( YKL40 SOX2 (refs. YKL40 and CD44 ers mark GSC mesenchymal the and OLIG2 and SOX2 markers GSC measured by of immunofluorescence each region using the proneural ( natures sig gene subtype glioblastoma with biopsies patient multiregional ( signatures expression gene proneural and classical comprising signature tional transcrip mixed a displayed EM) margin, (enhanced region tumor A  staining and H3K27me3 H2AK119Ub with activity and PRC2 PRC1 Supplementary Supplementary Figs. 2j Supplementary Figs. Supplementary 2d http://glioblastoma

Regional transcriptional variation may reflect differences in in differences reflect may variation transcriptional Regional signatures gene microenvironment-related constructed we Next, s e l c i t r + , and OLIG2 and + d Fig. 1f Fig. , indicating segregation of proneural and mesenchymal GSC and mesenchymal of proneural segregation , indicating ). Supplementary Fig. 1 Fig. Supplementary Fig. Fig. 1 i. 1 Fig. 20 22 , , – 2 2 c 1 Supplementary Figs. 2c Figs. Supplementary h 3 ). ). Gene expression profiling for and vascular hypoxic ( ( ). Supplementary Figs. 2a Figs. Supplementary Supplementary Figs. 2e Figs. Supplementary b + ). These findings were validated in two other other two in validated were findings These ). , whereas NR GSCs were exclusively CD44 exclusively were GSCs NR whereas , .alleninstitute.org , k , g 2 and 6 and . Given this background, we investigated we investigated background, . this Given 16 , 3g 1 ). GSCs displayed regional variation, variation, regional displayed GSCs ). 3d 9 ). ). Patients with both vascularity and ). GSCs in the ER were exclusively exclusively were ER the in GSCs ). ) ) and associated with tumor histol / ). Confirming our findings findings our Confirming ). , f , i , and and b Supplementary Fig. 4a Supplementary Supplementary Supplementary Fig. 4b , h and and and and in silico 3c 3a , f Fig. 1 Fig. 3e ). In The Cancer Cancer The In ). , Supplementary Supplementary b ). Both vascu Both ). ). Selected sig Selected ). 24 observations, , 2 5 e . . Our mul ). Tovali ). Fig. 1 Fig. + and and d ). ). ). ). ), ), ------

While intertumoral variation was substantial, shared regions con regions shared substantial, was variation intertumoral While regions. different in residing GSCs in function PRC distinct cating ( marks H2AK119Ub or H3K27me3 differential displayed genes target 80% of region-specific the same in anatomic region from that both patients revealed more than H2AK119Ub or H3K27me3 by marked genes region-specific of anatomic particular ( samples a the to between shared were and unique region both were that peaks identified and samples both in peaks overlapping analyzed we peaks, specific region- To determine (ChIP-seq). sequencing by deep followed tion immunoprecipita chromatin using regions necrotic and enhancing (H2AK119Ub) PRC1 from marks of chromatin distribution genome-wide we interrogated 5a ( Fig. GSCs of characteristics functional displayed and H3K27me3 or H2AK119Ub expressed regions ferent CD15 shown). not (data CD133 than sensitive more SSEA1) as known (also CD15 marker, subgroup glioblastoma for specific be Fig. 5a Supplementary (necrotic) and (enhancing) regions, vascular ( respectively tribution of cells positive for H2AK119Ub and H3K27me3 in hypoxic dis dichotomous observing biopsy samples, patient in multiregional PRC1–PRC2 activation portends greater tumor malignancy. tumor greater portends activation PRC1–PRC2 ( signatures or Supplementary Figs. 6e proteins both of expression high with associated was prognosis worst the but prognosis, patient poor with associated were signatures or activation expression BMI1 and EZH2 ( signature microenvironment proneural a with negatively and signatures microenvironment sical or clas mesenchymal with positively correlated signature activation regions neovascular tumor in function EZH2 ( signatures ronment microenvi and classical but with mesenchymal correlated negatively signature vascular mature and proneural with correlated positively microenvironment-related signatures. The EZH2 activation signature and BMI1 EZH2, correlated we subtypes, glioblastoma of tribution ( marker mesenchymal a of CD44, expression high with levels protein marker, BMI1 and high high EZH2 of protein levels with high expression of OLIG2, a proneural correlation the confirmed we microarray, tumor glioblastoma a ( ER the Table 1 Supplementary in H3K27me3 by marked significantly most targets H3K27me3 and SUZ12 substantial EZH2, with identity, gene without in overlap albeit NR, and ER the both in targets opment devel and marked neuronal cellular of with inhibition transcription, associated generally H3K27me3, targets. gene important on verged activation signature (containing 341 genes downregulated upon upon downregulated knockdown) genes BMI1 341 (containing signature activation of a BMI1 enrichment showed profiles transcriptional mesenchymal of partners EZH2) or targets putative 41 (containing signature activation EZH2 an of enrichment showed signatures proneural with tumors Furthermore, PRC1. and PRC2 of functions environment-specific ( factor- growth ing with associated pathways, signaling such mesenchymal as transform encoding those strongly genes marking regions hypoxic the from CD15 in H2AK119Ub with NR, and ER the in targets ferent Fig. 2 Fig. o ses R cnrbto t mconiomn-pcfc dis microenvironment-specific to contribution PRC assess To d 2 – 9 and and c ( ). Using image-guided biopsies from two glioblastomas, glioblastomas, two from biopsies image-guided Using ). Fig. 2 Fig. Supplementary Table 1 Table Supplementary advance online publication online advance e and β 3 (TGF- 0 Supplementary Figs. 6f Figs. Supplementary ( 2 Supplementary Fig. 6g Fig. Supplementary 8 Fig. 2 Fig. ). ). As the GSC and markers CD133 CD44 may or PRC2 (H3K27me3) in CD15 in (H3K27me3) or PRC2 , ). In contrast, H2AK119Ub marked very dif very marked ). In H2AK119Ub contrast, h Fig. 2 Fig. Fig. 3 Fig. , i β , 7e ), nuclear factor- nuclear ), f and and , c a h upeetr Fg. 7f Figs. Supplementary and and ). and Supplementary Fig. 7g Fig. Supplementary Supplementary Table1 Supplementary 8a 1 ), indicating probable micro probable indicating ), 2 i. 2 Fig. 6 7 , d , we employed another GSC GSC another employed we , , which was less specific but but specific less was which , ), ), suggesting that combined a κ ), whereas tumors with tumors ), whereas and and 3 and and 1 B (NF- B Fig. 2 Fig.

. Conversely, a BMI1 BMI1 a Conversely, . nature medicine nature 8b Supplementary Supplementary b , c κ ). Annotation Annotation ). + ), implicating implicating ), B) and WNT WNT and B) + cells in dif in cells Fig. 2 Fig. i. 3 Fig. GSCs from from GSCs and and Fig. Fig. 2 ). Using Using ). + ), indi ), GSCs GSCs d b 8b a and and and and and , c ). ). ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. the Ivy GAP database. The corresponding histological feature for each RNA sample is labeled (top). ( (top). labeled is sample RNA each for feature histological corresponding The database. GAP Ivy the sample (RNA set data Study Structure Anatomic the in sample RNA each for (ssGSEA) analysis enrichment gene-set ( 25 bars Scale (bottom). magnification higher in shown areas indicate (top) numbers and Boxes samples. glioblastoma ( samples. brain human nonmalignant four and samples glioblastoma multiregional three ( hypoxia of markers of bar, 25 Scale samples glioblastoma multiregional in expression (green) YKL40 and ( samples. brain human nonmalignant four and samples glioblastoma multiregional three in ( 2 cm. bars, Scale analysis. immunofluorescence and qPCR for patients) four of (representative 1 Figure nature medicine nature ( profiles transcriptional among distributions f e c ) Heat map showing showing map ) Heat ) Immunofluorescence images (representative of three fields for each region) showing vWF showing region) each for fields three of (representative images ) Immunofluorescence (red) OLIG2 (green), CD44 (red), SOX2 of (bottom) quantification and region) each for fields 10 of representative (top, images ) Immunofluorescence

Anatomical distribution of transcriptional profiles in glioblastoma. ( glioblastoma. in profiles transcriptional of distribution Anatomical d b g a Frequency (%) f 100 20 40 60 80 0 GBM (CW1757) 32 32

( n Normal brain Normal brain z ER =25 MV advance online publication online advance CA9 -scores of each glioblastoma subtype signature, normalized within each patient sample set determined via single-sample single-sample via determined set sample patient each within normalized signature, subtype glioblastoma each of -scores 44 44 ) , , GLUT3 52 52 ( n PSEU =23 53 53 ) , , Low Low GLUT1 Expression level Expression level ( ER ER GBM (CW1757) GBM (CW1757) n =30) ** CT EM EM , , EM g ** ** HIF1A, MCT1 HIF1A, ) and molecular subtype distribution among histological structures ( structures histological among distribution subtype molecular ) and ( n NR NR =24) ** High High IT VEGFB CD34 VEGFR CD31 LDH5 MCT4 MCT1 HIF1A GLUT1 GLUT3 CA9 TGFB1 TIMP1 CHI3L1 CD44 EGFR NES FOXO3 CD133 OLIGO2 DLL3 ACTIN AKT2 ASCL1 Transcriptional subtype Microenvironmental ( n

=19) LE , , 2 MCT4 NR markers markers Vascular Hypoxia Mesenchymal Classical Proneural Classica Proneural Mesenchymal and and l LDH5 a ) and vascular specimen ( specimen vascular ) and ) T1-weighted MRI images of multiregional glioblastoma sampling process process sampling glioblastoma multiregional of images MRI ) T1-weighted h SOX2 c vW e 1 ER Mesenchymal

F Cell population (%) / µ CD44 / CA9 m. ** m. 100 n 20 40 60 80 =49 0 / z DAPI / Proneural Classical Mesenchymal Anatomic regions DAPI -scores were calculated from change in qPCR qPCR in change from calculated were -scores ( CD44 SOX2 P z n EM -scores were calculated from qPCR qPCR from calculated were -scores < 0.001, < 0.001, =90) + ER ER (red) and CA9 and (red) 2 ** b ** ) A heat map of molecular subtype marker expression expression marker subtype molecular of map ) A heat 1 ( Negative SOX2/CD44 n =66) EM ** NR g , χ h CD31 2 2 ) ) ** Classical test. ( test. χ ( n n =37 2 ** =72) NR

test of glioblastoma histological feature feature histological glioblastoma of test Enrichment score + OLIG2 (green) locations in multiregional multiregional in locations (green) , , (z-score) VEGFR2 3 d ER YKL40 OLIG2 ) Heat map showing mRNA expression expression mRNA showing map ) Heat ( n h / 3 –2 2 YKL40 =77) ER ). ** ). EM ** n = 122; patient patient = 122; ** µ , , P / m (top) or 10 10 or m (top) CD34 DAPI ( Negative OLIG2/YKL40 around necrosis Pseudopalisading region region Microvascular proliferative Leading edge Infiltrating tumor Cellular tumor < 0.001. n EM =56) EM ** Proneural n , , =36 ** ACTIN ∆ 4 ( n C =74) NR t NR values. values. NR s e l c i t r a and and 4 µ n ∆ m (bottom). m (bottom). = 10) from from = 10) C t values. values. VEGFB

) ) in

 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. aling pathways were activated in different GSC subtypes. GSC different in activated were pathways aling GSCs ( but BMI1 activation, not and signatures activity correlated with a proneural signature, whereas that confirmed that we generated models GSC derived OLIG2 SOX2 with associated were H3K27me3 and EZH2 whereas in were CD44 increased markedly and H2AK119Ub BMI1 models. cell progenitor and neural GSC mal mesenchy and proneural validated several in expression EZH2 and A  in microenvironment and subtype proneural the and signature tion with Tukey’s method for multiple comparisons. Tukey’smultiple for with method ( in shown ( ChIP-seq. modification histone other the in called also and region anatomic same the within patients both by shared peaks region-specific of fraction the Purple, patients. both from regions necrotic or enhancing in shown peaks specific ( specimens. glioblastoma primary two on experiments ChIP-seq H2AK119Ub or H3K27me3 from derived peaks specific region CD15 in genes binding H2AK119Ub bar, 25 Scale samples. glioblastoma multiregional in (green) H2AK119Ub and (green) H3K27me3 2 Figure n

= 10, nontumor; nontumor; = 10, EZH2 BMI1 investigated we GSCs, regulate BMI1 and EZH2 both As s e l c i t r + d Supplementary Fig. 9 Supplementary proneural GSCs ( GSCs proneural

mRNA expression levels correlated with the EZH2 activa EZH2 the with correlated levels expression mRNA Epigenetic GSC signatures in multiregional primary specimens. ( specimens. primary multiregional in signatures GSC Epigenetic b . . ( P PRC2 complexpathways =0.05 Neuronal development CD15-positive GSCsfromER e , f ) Enrichment levels of EZH2 ( EZH2 of levels ) Enrichment c b a CW2473 FDR corrected n Cellular proliferatio CD15-positive GSCs = 161, proneural; proneural; = 161, CW2451 NR ER 2,498 25 Regionally uniquegenesmarkedbyH3K27me3 b µ that are regionally unique annotated genes marked by H3K27me3 (orange) or H2AK119Ub (blue) and are common between between common are and (blue) H2AK119Ub or (orange) H3K27me3 by marked genes annotated unique regionally are that CD31 from ER m 114 H3K27me3 BMI1 5% target 95% Fig. 3 Fig. n s CW2473 4,892 Angiogenesis ), indicating that ), BMI1 and indicating EZH2 sign H3K27me3 ChIP-seq development epithelial cel P H3K27me3 ChIP-seq mRNA, correlated with mesenchymal development =1.7×10 Regionally sharedgenesmarkedbytheotherhistonemodification Cellula c H3K27me3 ). RNA-seq data from 19 patient- 19 from data RNA-seq ). + + cells derived from primary glioblastoma samples (CW2451 and CW2473). Venn diagrams show overlaps between between overlaps show Venn diagrams CW2473). and (CW2451 samples glioblastoma primary from derived cells and YKL40 l r

& –6 CA9

n CD15-positive GSCs = 209, classical; classical; = 209, CW2451 13,005 Mature neuronal e from NR 16% CD15-positive GSCsfromNR ) or BMI1 ( BMI1 ) or P PRC2 complexpathways =0.01 84% 981 CD15 + CW2473 mesenchymal GSCs, mesenchymal 3,324 FDR corrected signalin BMI1 target TGF- f ) activation signatures in transcriptional subgroups from the TCGA glioblastoma data set set data glioblastoma TCGA the from subgroups transcriptional in signatures ) activation β g n EZH2 d CD15-positive GSCs = 167, mesenchymal). Black bars indicate median values. ** values. median indicate bars Black mesenchymal). = 167, s ) Bubble plots showing gene signatures enriched in the overlapping region-specific peaks peaks region-specific overlapping the in enriched signatures gene showing plots ) Bubble CW2451 Regionally uniquegenesmarkedbyH2AK119Ub 2,384 CD31 Cellular development FGF signalin 4% from ER mRNA P 160 =3.3×10 + 96% H2AK119Ub ChIP-seq and and CW2473 g Hypoxi 7,155 H2AK119Ub –21 - - - a a ) Immunofluorescence staining for CD31 (red), CA9 (red), CD15 (red), (red), CD15 (red), CA9 (red), CD31 for staining ) Immunofluorescence CA9 maintains BMI1 protein stability in GSCs of different subtypes. different of GSCs in stability protein BMI1 maintains ( conditions cystin but weakly accumulated in mesenchymal GSCs under the same in proneural GSCs after treatment with the proteasome inhibitor lacta ( GSCs Fig. 10a proneural in reduced markedly but GSCs mesenchymal in unchanged were levels protein BMI1 that showed post- regulated is BMI1 transcriptionally. but Inhibiting translation with cycloheximide treatment level transcriptional the at regulated ( signature ment microenviron or subtype mesenchymal the or signature activation that find to TCGA tumor samples ( CD15-positive GSCs CW2451 CD15-positive GSCsfromER PRC2 complex 12,760 P development Neuronal pathways =0.05 from NR 7% ). ). Conversely, polyubiquitinated BMI1 was strongly increased 2,376 93% CD15 FDR corrected CW2473 Cell proliferation Fig. 3 Fig. BMI1 6,721 advance online publication online advance Supplementary Fig. 7 Fig. Supplementary mRNA expression did not correlate with a BMI1 BMI1 a with correlate not did expression mRNA d ), suggesting that ubiquitin-mediated proteolysis proteolysis ubiquitin-mediated that suggesting ), µ Angiogenesis Cell development m. ( m. H2AK119Ub ChIP-seq Supplementary Supplementary Fig. 6 P H3K27me3 f e =3.3×10 b targets BMI1 activation signature EZH2 activation signature ) Overlap in region-specific H3K27me3 or H3K27me3 region-specific in ) Overlap enrichment score enrichment score –9 1,000 2,000 3,000 –2,000 2,000 4,000 6,000

Nontumor 0 Nontumor 0 signaling P × 10 =2. In ammation Wnt and NFkB CD15-positive GSCsfromNR –9 4 Proneural Proneural signaling ** TGF- ), suggesting that EZH2 is is EZH2 that suggesting ), P FDR corrected β < 0.01; one-way ANOVA one-way < 0.01; Classical **

), ), but we were surprised Classical ** c targets ) Fraction of region- of ) Fraction BMI1 Mesenchymal ** Mesenchymal ** nature medicine nature ** Supplementary Supplementary Hypoxia Cell death P =9.5×10 –4 1

- -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. mal glioblastomas ( glioblastomas mal mesenchy and ubiquitina proneural by with expressed associated differentially were 22 genes tion, 254 Among activation. BMI1 with inversely correlated expression whose genes ubiquitin-related for database glioblastoma TCGA the interrogated we degradation, mean as are Data conditions. hypoxic and/or low-glucose in or conditions baseline under determined MES738) and MES3565 MES28, (MES20, GSCs mesenchymal and PN3691) and PN1919 ( conditions. hypoxic and/or low-glucose in or conditions baseline under grown MES28) and (MES20 GSCs mesenchymal and PN3691) and (PN1919 GSCs proneural (NPC1), cells progenitor neural in immunoblot by measured SOX2 and OLIG2 ( lysates). (whole-cell input the in immunoblot by analyzed were tubulin and RNF144A BMI1, in as treatment lactacystin of absence or presence the in immunoblotting and immunoprecipitation ubiquitin-specific or BMI1- by examined 3112) shRNF144A- and (shRNF144A-1099 shRNF144A or (shCTRL) control shRNA with transduction after cells PN1919 in RNF144A by polyubiquitination ( 10 h, (5 treatment (lacta) lactacystin of absence or presence the in cells (MES28) mesenchymal and (PN1919) proneural in polyubiquitination BMI1 of analysis (IB) in available are above presented data for immunoblots full-length All cell. progenitor neural NPC, ID. sample indicate (top) Numbers glioblastoma. SOX2) and (OLIG2 proneural and YKL40) and (CD44 mesenchymal P ( microarray tissue a glioblastoma from level expression bar, 500 Scale OLIG2. and CD44 BMI1, EZH2, of expression the 3 Figure nature medicine nature plotted we activity, BMI1 to e values were determined by log-rank test. ( test. log-rank by determined were values ) Rank-ordered list of of list ) Rank-ordered c a On the basis of the apparent region-specific BMI1 proteosomal proteosomal BMI1 region-specific apparent the of basis the On f Input IP: BMI1 2 1 NPCs Proneural Mesenchymal shCTRL

Differential PRC function in glioblastoma subgroups. ( subgroups. glioblastoma in function PRC Differential 1 3 + – EZH2 PN1919 shRNF144A 1099 2 1 Proneural GSCs + – 3 1919 3112 µ

M). Right, quantification of BMI1 polyubiquitination by ImageJ. Data are normalized by input loading controls. Ub, ubiquitin. ubiquitin. Ub, controls. loading input by normalized are Data ImageJ. by polyubiquitination BMI1 of quantification Right, M). Supplementary Fig. 10b Fig. Supplementary 3691 + – advance online publication online advance r Lacta values between ubiquitin ligases and BMI1 activation or inhibition signatures in TCGA glioblastoma samples. ( samples. glioblastoma TCGA in signatures inhibition or activation BMI1 and ligases ubiquitin between values Tubulin RNF144A BMI1 BMI1 Ub 20 Mesenchymal BMI1 3565 GSCs r 28 values for the 22 genes against two two against genes 22 the for values 738 g NPC1 TUBULIN SOX2 OLIG2 H3K27me3 EZH2 YKL40 CD44 H2AK119Ub BMI1 + – –– c CD44 ± ) Immunoblot analysis of BMI1, H2K119Ub, EZH2, H3K27me3 and marker expression in lysates from from lysates in expression marker and H3K27me3 EZH2, H2K119Ub, BMI1, of analysis ) Immunoblot s.e.m. * s.e.m. + + + – PN1919 ). To link these genes genes these link To ). d + – –– Input IP: BMI1 P PN1919 n – + < 0.05, Wilcoxon and Mann–Whitney Mann–Whitney and Wilcoxon < 0.05, = 24, EZH2 = 24, + + + –

PN3691 OLIG2 MES28 + – –– – + a – ) Representative images of glioblastoma tissue microarray samples ( samples microarray tissue glioblastoma of images ) Representative µ m. ( m. + + + Lacta BMI1 Ub Tubulin BMI1 − Supplementary Figure 22 Figure Supplementary BMI1 MES20 b ) Kaplan–Meier curves of patient survival stratified by EZH2 and BMI1 BMI1 and EZH2 by stratified survival patient of curves ) Kaplan–Meier + – –– - -

− ; ; b

+ + + – Ub intensity a BMI1 inhibition signature ( signature inhibition a BMI1 nega with correlated and positively expression activation BMI1 with correlated tively RNF144A ligase, E3 BMI1 a as role potential its protein subunit dependent catalytic DNA-PKcskinase, for DNA- as an ligase E3 described previously was which RNF144A, finger, ring the with associated was activation BMI1 with associated ( signatures BMI1-related n

10,000 20,000 30,000 40,000 Survival proportion (%) = 7, EZH2 = 7, 100 MES28 Lacta 20 40 60 80 0 0 + – –– 5 0 PN1919 + + + – Tubulin YKL40 CD44 OLIG2 SOX2 EZH2 BMI1 Hypoxia Low glucose − + – h BMI1 P P ) Viability NPC1 cells, proneural GSCs (PN11, PN23, PN23, (PN11, GSCs proneural cells, NPC1 ) Viability MES28 <0.01 g <0.01 0 t P . . ( ) Levels of RNF144A, BMI1, EZH2, CD44, YKL40, YKL40, CD44, EZH2, BMI1, RNF144A, of ) Levels EZH2 EZH2 EZH2 EZH2 -test. <0.01 Overall survivaltime(months) d + + – ) Left, immunoprecipitation (IP) and immunoblot immunoblot and (IP) immunoprecipitation ) Left, ; ; n + + – – BMI1 BMI1 BMI1 BMI1 = 8, EZH2 = 8, e Ubiquitin ligase expression vs. 100 + – BMI inhibition signature (r value) + –

P h Low glucose ( –0.1 –0.2 ( ( ( n n n n <0.01 0.0 0.1 0.2 0.3 0.4 0.5 Fig. 3 Fig. Cell viability (%) =24 =8) =7) =8) –0.4 Hypoxia 100 Fig. 3 Fig. 20 40 60 80 P 150 0 Ubiquitin ligaseexpressionvs.BMIactivation ) <0.01 Median survival(95%CI) + BMI1 e RNF144A 19 months(18–21) 18 months(14–28) 28 months(21–46) –0.2 ). The highest highest The ). 5 months(2–14) e NPC ). The results from ChIP-seq for ChIP-seq from results ). The − ; ; 200 n signature 0.0 = 8 EZH2 PN GSCs –– + – 0.2 250 ( r value) r s e l c i t r a n + value negatively negatively value – BMI1 = 96) showing showing = 96) 0.4 f MES GSCs ) BMI1 ) BMI1 3 2 . . Supporting + ). ). ++ + 0.6 *

d 0.8 .

 - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. proneural marker ( marker proneural a SOX2, bound also promoter its and models proneural in stronger tumors proneural for ( mesenchymal specific more in was OLIG2 deposition, whereas models, H3K27ac preferential by showed measured CD44 brain. activation, normal and gliomas other for deposited results to compared then were or signatures proneural mesenchymal with tissues glioblastoma patient primary and glioma GSCs of panel a on (H3K27ac) H3 histone on K27 of acetylation (5 EZH2-i nM), (10 BMI1-i control, vehicle mean bars, Error cells. indicated for ( EZH2-i. or BMI1-i of concentrations increasing with treatment after MES738) and MES3128 MES-MN1, MES3565, MES28, (MES20, GSCs mesenchymal and PN1914.2) and PN1919 PN-MMK1, PN-JK2, (PN11, GSCs proneural (NPC1), progenitors neural of curves ( shBMI1-939 and shBMI1-880 or shEZH2-2450 and shEZH2-950 shCNTRL, with transduced mean are Data point). ( shRNA shBMI1-939) and (shBMI1-880 BMI1-specific or shEZH2-2450) and (shEZH2-950 EZH2-specific (shCNTRL), 4 Figure A  mesenchymal levels, protein BMI1 on RNF144A of role regulatory Supplementary Fig. 10c Fig. Supplementary b a

Fraction of wells Fraction of wells s e l c i t r without tumorspheres without tumorspheres 100 100 Relative cell growth Relative cell growth 20 40 60 80 20 40 60 80 0

0 0 1 2 3 4 0 1 2 3 Differential efficacy of BMI1 and EZH2 inhibitors against glioblastoma subgroups. ( subgroups. glioblastoma against inhibitors EZH2 and BMI1 of efficacy Differential 5 0 0 2 1 2 1 shEZH2-2450 2 1 shEZH2-2450 Cells/well Cells/well PN1919 PN1919 MES20 MES20 Days ** Days ** 10 ** 5 3 4 3 4 3 Supplementary Fig. 10c Fig. Supplementary 15 ± ** ** shCTRL

4 ** s.e.m. ** s.e.m. ** ** shCTRL ** ** ** ** ** ** ** ** 20 ). RNF144A transcriptional regulation was regulation transcriptional ). RNF144A shBMI1-880

Fraction of wells Fraction of wells shBMI1-880 P

< 0.01, one-way ANOVA with Dunnett’s multiple-comparison test. ( test. multiple-comparison Dunnett’s with ANOVA one-way < 0.01, without tumorspheres without tumorspheres

100 100 Relative cell growth ±

Relative cell growth shEZH2-950 20 40 60 80 20 40 60 80 12 16 s.e.m. ( s.e.m. 0 1 2 3 4 5 6 7 0 0 0 4 8 shEZH2-950 5 0 0 2 1 2 1 2 1 µ Cells/well Cells/well M) or both ( both or M) PN3691 PN3691 MES28 MES28 shBMI1-939 e Days Days ) ) 10 ). Supporting a negative a negative Supporting ). shBMI1-939 In vitro In

5 3 ** ** 4 3 ** 4 3 15 * ** 4 ** ** ** ** ** limited dilution assay of PN1919, PN3691, MES738 and MES20 cells after treatment with with treatment after cells MES20 and MES738 PN3691, PN1919, of assay dilution limited ** ** ** 20 n = 15 per group). ** group). per = 15 d c Relative cell viability (%) Relative cell viability (%) 100 BMI1-i IC 50 (nM) 100 20 40 60 80 EZH2-i IC 50 (µM) 20 40 60 80 1,000 0 0 10 10 100 800 200 400 600 20 40 60 80 –1 –2 0 NPC1 0

NPC1 >1,000 >100

out proteolysis inhibition ( inhibition proteolysis out by down knocked or with with levels was at similar persisted RNA (shRNA) hairpin small RNF144A which in cells in protein BMI1 ( expression protein BMI1 biquitinated nonu increased concordantly and inhibition proteolysis after BMI1 polyubiquitinated reduced BMI1 of levels baseline low with GSCs to RNF144A of binding ( BMI1 direct confirmed Immunoprecipitation neural precursors and proneural GSCs ( GSCs proneural and normal precursors neural than protein RNF144A of expression lower showed GSCs 10 53

P 11 699 11 10 Proneural GSCs < 0.01, < 0.01, –1 Proneural GSCs 0

JK2 >100 JK2 MMK1 MMK1 IC 26 Supplementary Fig. 10e Fig. Supplementary EZH2-i BMI1- >1,000 50 10 1919 10 χ =45 1919 1 0 45 2

1914.2 i test. 1914.2 58 a µ M ) Cell growth rates of GSCs transduced with control control with transduced GSCs of rates growth ) Cell advance online publication online advance n 10 10 = 15 per group). ** group). per = 15 20 20 104 2 1 Mesenchymal GSCs >100 28 Mesenchymal GSC 28 63 b

3565 3565 IC ) ) 37 10 10 50 In vitro In =122nM 3 MN1 MN1 2 215 Supplementary Fig. 10f Fig. Supplementary 9 3128 3128 101 75 MES3128 MES3565 MES28 MES20 PN1919 PN11 MES738 MES-MN1 PN1914.2 PN-MMK PN-JK2 NPC MES738 MES3128 MES-MN1 MES3565 MES28 MES20 PN1914.2 PN1919 PN-MMK PN-JK2 PN11 NPC1 limited dilution assay of GSCs GSCs of assay dilution limited ). Depleting RNF144A in proneural proneural in RNF144A Depleting ). >100

738 s 738 213 1 P < 0.01, < 0.01, d ) ) IC 1 1 n = 5 per group and time time and group = 5 per Fig. 3 Fig. Supplementary Fig. 10d Fig. Supplementary 50 e of BMI1-i or EZH2-i EZH2-i or BMI1-i of Fraction of wells Fraction of wells Fraction of wells Fraction of wells χ 2

f without tumorspheres without tumorspheres without tumorspheres without tumorspheres test. ( test.

). Nonubiquitinated Nonubiquitinated ). 100 100 100 100 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 nature medicine nature 0 0 0 0 1 0 5 0 5 0 5 0 c ), highlighting highlighting ), Combination EZH2-i BMI1-i Vehicle ) Cell viability viability ) Cell PN1919 Cells/well Cells/well Cells/well Cells/well MES20 MES738 PN3691 4 2 10 10 ** ** ** 10 ** 5 3 15 15 ** ** ** 15 ** ** 20 20 20 ). ). - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ronmental transcriptional signatures ( signatures transcriptional ronmental ( low Tumors with tion. 1p and co-dele 19q chromosome with association and tion a weaker strong inverse correlation with glioblastoma histology and ( tions; chromosome 1p and 19q as co-deletion) well as patient survival ( features molecular common and grade ogy, of expression the Fig. 11a oma (LGG) and inversely ( related to patient survival gli low-grade than glioblastoma in lower was expression RNF144A in and patient tissues databases. and its survival regulation examined for node degradation. regulatory protein BMI1 a as polyubiquitination RNF144A-mediated test. log-rank by determined ( weekly thrice EZH2-i mg/kg 350 and week per once BMI1-i mg/kg 10 of treatment combined after MES20) group). ( PN1919 bearing mice for n control, Vehicle samples. tumor intermediate the in population tumor total and (green) MES20 and (red) PN1919 by occupied of area relative of quantification Right, 48). (day samples tumor intermediate the in population ( comparisons. Tukey’smultiple for with ANOVA method one-way ( mouse each for intensity signaling 10 day to normalized were Signals cells. MES20 and photons/s/cm in measured is bioluminescence populations; GSC mixed of injection intracranial after days Timeindicate points growth. tumor on weekly thrice EZH2-i mg/kg 350 and week per once BMI1-i mg/kg 10 of treatment combined of effect the showing cells, MES20 and PN1919 luciferase-expressing from ( GSCs. (MES20-GFP) mesenchymal and ( 5 Figure nature medicine nature of the determine prognostic significance a RNF144A Supplementary Fig. 12a Fig. Supplementary = 10. * = 10. b a d PN1919-tdTomat ) Experimental design to assess assess to design ) Experimental To determine the clinical relevance of RNF144A in gliomas, we we gliomas, in RNF144A of relevance clinical the determine To Day

Survival proportion (%) 45 38 31 24 17 MES20-GF 100 Combinatio BMI1- EZH2-i Vehicl 20 40 60 80 0 P

– values were determined by log-rank test. ( test. log-rank by determined were values P 1 0 In vivo In d < 0.01; ** < 0.01; Vehicle i e ). ). Using the LGG–glioblastoma TCGA data set, we mapped low 1 P 1,000 Median survival(95%CI EZH2 2 0 P P n o Survival time(d

2 1,000 Vehicl therapeutic efficacy of combined pharmacological inhibition of BMI1 and EZH2 on subtype-mixed glioblastoma model. model. glioblastoma subtype-mixed on EZH2 and BMI1 of inhibition pharmacological combined of efficacy therapeutic <0.01 <0.01 2 /sr. Right, quantification of bioluminescence signals during 45 d of treatment in mice implanted with luciferase-expressing PN1919 PN1919 luciferase-expressing with implanted mice in treatment d of 45 during signals bioluminescence of quantification /sr. Right, P P P PN1919 45 d(45–45) 33 d(32–33) 39 d(36–42) 32 d(25–32) RNF144A <0.01 <0.01 <0.01 3 0

3 e high P advance online publication online advance < 0.01, one-way ANOVA with Tukey’s method for multiple comparisons. Scale bars, 4 mm. ( 4 mm. bars, Scale comparisons. Tukey’smultiple for with ANOVA method one-way < 0.01, 4 EZH2-i 4 0 ) were enriched with unfavorable microenvi unfavorable with enriched were ) RNF144A d ) ) or MES20 ( MES20 ) or ). ). 5 5 0 and and RNF144A 6 ) in vivo in 0 EZH2 and high high and Day 10 7 e Bioluminescence (×10 Survival proportion (%) BMI1- Combinatio BMI1- EZH2- Vehicl effects of BMI1-i (PTC596) and EZH2-i (EPZ6438) on xenograft of mixed proneural (PN1919-tdTomato) (PN1919-tdTomato) proneural mixed of xenograft on (EPZ6438) EZH2-i and (PTC596) BMI1-i of effects 100 b GW120918 100mg/kg4hprior EZH2-i 350mg/kg EZH2- e 20 40 60 80 mRNA expression showed a showed expression mRNA Supplementary Fig. 12b Fig. Supplementary ) Left, bioluminescence images of mice bearing mixed proneural and mesenchymal xenografts derived derived xenografts mesenchymal and proneural mixed bearing mice of images bioluminescence ) Left, mRNA against tumor histol tumor against mRNA ) orthotopic tumors with combined treatment of 12.5 mg/kg BMI1-i and 350 mg/kg EZH2-i ( EZH2-i mg/kg 350 and BMI1-i mg/kg 12.5 of treatment combined with tumors ) orthotopic 0 i RNF144A Day 17 i e 1 0 i i P EZH2 P P <0.01 Median survival(95%CI <0.01 <0.01 n IDH1 2 0 Combinatio P P Survival time(d f 43 d(42–43) 40 d(37–40) 36 d(32–37) 26 d(18–27) Day 24 <0.01 ) Kaplan–Meier survival curves for mice bearing subtype-mixed orthotopic tumors (PN1919 and and (PN1919 tumors orthotopic subtype-mixed bearing mice for curves survival ) Kaplan–Meier =0.04 mRNA expression expression mRNA and MES20 Bioluminescence imaging 3 0 or or 4 Supplementary Supplementary )

n Day 31 EZH2 4 0 ATRX IDH1 ) c 5 0 ) Left, whole-brain images showing distribution of PN1919 (red) and MES20 (green) (green) MES20 and (red) PN1919 of distribution showing images whole-brain ) Left, BMI1- mRNA muta Day 38 muta BMI1-i 10mg/kg ) Relative bioluminescnece ). To ). 100,000 150,000 200,000 n 0 i 50,000 = 10; EZH2-i alone, alone, EZH2-i = 10; f Combinatio BMI1- EZH2- Vehicl ------Survival proportion (%) Day 45 0 Time aftertumorimplantation(d 100 1 0 20 40 60 80 i e BMI1 expression and increases in expression of mesenchymalin markerschanges no showed stress under GSCs mesenchymal contrast, In BMI1 lost ( markers GSC of proneural loss with GSCs expression, and EZH2 proneural and precursors Neural restriction. ent nutri and hypoxia including conditions, stressful under precursors and neural in GSCs expression and BMI1 EZH2 we examined GSCs, a malignancy. tumor in role negative supporting outcome, patient and grade tumor with associated is Table 3 glioma of grades all ( across survival overall for factors prognostic RNF144A and grade tumor age, patient sidering ( lowest survival vival trends, which showed that sur derived and targets for each levels expression mapped we levels, 0 i upeetr Tbe 2 Table Supplementary 2 0 To determine a functional role for differential PRC utilization by utilization PRC differential for role functional a Todetermine Median survival(95%CI Combinatio Vehicl EZH2- BMI1- 2 0 n Drug holiday P P P 68 d(66–69) 60 d(58–66) 60 d(59–63) 52 d(47–54) Random sampling for tissuestaining ). Collectively, these results show that suppression of RNF144A =0.075 =0.015 <0.01 4 0 Survival time(d 3 0 I e I PN1919–MES20 P P Combinatio <0.01 <0.01 mRNA and BMI1 activation signatures as independent independent as signatures activation BMI1 and mRNA n = 10 per group and time point). * point). time and group per = 10 4 0 n 6 0 n = 15; BMI1-i alone, alone, BMI1-i = 15; 5 0 Supplementary Fig. 12c Supplementary 8 0 n ) 0 ) ) 0 * ** 100 bt o gibatm ( glioblastoma not but ) c n

= 8 per group). group). = 8 per EZH2-i

RNF144A

– EZH2- + BMI1- Proportion of tumor in brain n d 10 20 30 = 14; combined treatment, treatment, combined = 14; , 0 e i i ) Kaplan–Meier survival curves curves survival ) Kaplan–Meier IDH + – – – ** ). Multivariate analysis ). con Multivariate analysis – low + + – P mutation status revealed revealed status mutation ** EZH2 + < 0.05; ** < 0.05; P values in in values BMI1- + – – – ** + + high s e l c i t r a – * ** i Supplementary Supplementary n + tumors had the = 5 per = 5 per

P e < 0.01, < 0.01, and and + – – – + + + * ** – Fig. 3 Fig. f + were were

g ). ).  - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. completely blocked tumorsphere formation and eradicated GSCs GSCs eradicated and formation tumorsphere blocked completely inhibitors EZH2 and BMI1 of concentrations low, of achievable combination clinically the interaction, synergistic a observe not did ( GSC cultures patient-derived mesenchymal two and proneural two against inhibitor each of tions concentra of range a of effects the interrogated we efficacy, natorial ( respectively GSCs, IC mean the comparing by confirmed was sensitivity Differential treatment. ( line precursor neural a and GSCs proneural in than GSCs enchymal (IC concentration inhibitory the half-maximal to treatment; BMI1-i sensitivity played preferential dis GSCs Mesenchymal inhibitor. EZH2 an (EZH2-i), EPZ6438 or inhibitor, BMI1 a (BMI1-i), PTC596 to subtypes GSC of sensitivity prognosis. poor associated and adaptation niche specific subtype- to divergent of may PRCs contribute utilization differential role results these support a for that context-specific BMI1, suggesting Collectively, subtypes. molecular on based depletion PRC to sitivity ( formation tumorsphere and growth cell GSC mesenchymal reduced preferentially depletion ( GSCs of proneural generation sphere and growth cell tumor reduced shRNAs preferentially EZH2 against EZH2 in proneural cells, RNA interference using two nonoverlapping fitness in the hypoxic niche. In line with the preferential expression of ( CA9 to localized ( GFP to compared as progression, disease ural GSCs ( mCherry overexpressing BMI1- of co-implantation orthotopic with experiment cell-mixing an performed we growth, tumor regional mimicking growth 14d Fig. ( stress of absence the in formation tumorsphere or proneural in cell under viability GSCs stress increased without cell altering growth expression BMI1 forced findings, these support ther growth harsh To stress. under is that EZH2 dispensable fur to suggesting conditions, GSCs sensitize not did depletion EZH2 contrast, ( GSCs proneural on effects no or est decreased cell viability of mesenchymal GSCs under stress, with mod modifications (H2K119Ub and H3K27me3). BMI1 depletion potently respective their by measured immunoblot, by confirmed was effects Fig. 14a mesen ( GSC two cultures patient-derived chymal early passage, and proneural two in target each for shRNAs specific nonoverlapping two using BMI1 or EZH2 either depleted we stress, survival cell ( increased stress under GSCs proneural in expression of stress ( absence the in essential not is BMI1 that suggesting self-renewal, or proliferation cell alter not did but expression BMI1 increased GSCs proneural in depletion RNF144A stress, low Under protein. BMI1 expressing cells glioma for mesenchymal may select regions necrotic Thus, stress conditions similar to those found in the pseudopalisading whereas almost all neural precursors and proneural GSCs died ( ( A  ( Fig. 4c Fig. Supplementary Fig. 14i Fig. Supplementary Supplementary Fig. 14j Fig. Supplementary Fig. 4 Fig. Fig. 3 Fig. Supplementary Fig. 13c Fig. Supplementary

To leverage our findings for clinical application, we examined the the examined we application, clinical for findings our Toleverage To determine the potential role of BMI1 in mediating s e l c i t r e g , ). As all drugs may have off-target effects, we validated the the validated we effects, off-target have may drugs all As ). , ). Some mesenchymal GSCs remained viable under stress, stress, under viable remained GSCs mesenchymal Some ). d – b 50 Supplementary Supplementary Fig. 13a ), proneural GSCs were generally more sensitive to EZH2-i to EZH2-i more sensitive were generally GSCs ), proneural g ). ). Targeting of BMI1 or EZH2 protein and levels chromatin of BMI1-i and EZH2-i in proneural and mesenchymal mesenchymal and proneural in EZH2-i and BMI1-i of ). Supplementary Supplementary Fig. 14h + hypoxic regions in the subsequent brain tumors tumors brain subsequent the in regions hypoxic Supplementary Fig. 15a Fig. Supplementary + Fig. 4a Fig. ). BMI1-overexpressing cells preferentially preferentially cells BMI1-overexpressing ). ), suggesting that BMI1 confers tumor cell cell tumor confers BMI1 that suggesting ), proneural GSCs and control GFP control and GSCs proneural ). To further assess PRC function under under function PRC assess further To ). 50 ) ) of was BMI1-i fourfold lower in mes Supplementary Fig. 15b Supplementary , b , b ), demonstrating differential sen differential demonstrating ), ). ). In contrast, targeting RNF144A ). ). BMI1 Fig. 4a Fig. Supplementary Fig. 14c Fig. Supplementary + + control proneural GSCs GSCs proneural control cells showed accelerated ). To determine combi To). determine , b ). Reciprocally, BMI1 ). Reciprocally, Supplementary Supplementary Supplementary Supplementary in vivo , c ). While ). we While + Fig. 3 prone in vivo in tumor ). In ). h ). ------

a fraction of proliferating cells that express stem cell programs cell stem express that cells proliferating of fraction a with profiles transcription in variation gliomas of intratumoral RNA-seq demonstrated Single-cell microenvironment. and state cell Tumor from epigenetic in arises variation cell genetics, heterogeneity DISCUSSION glioblastoma. subtype microenvironment-dependent for heterogeneous strategy treatment effective an is EZH2 and BMI1 of inhibition combinatorial the that ( treatment combined with survival superior of ( superiority treatment and combined subtype tumor specific the of inhibitor each of activity preferential confirming therapy, each after population cell tumor labeled fluorescently each of number residual the quantified show changes substantial with any of the therapies tested. Further, we cence, as measured by senescence-associated partments. Cell apoptosis, as measured by active caspase-3, and senes com both in marker on each effects had inhibitors EZH2 and BMI1 the of combination The population. mesenchymal the ablated ment treat BMI1-i contrast, In changes. modest more much showed cells mesenchymal whereas Ki67, marker proliferation cell the and CD15 the number of cells with the PRC2 mark the H3K27me3, GSC marker In mCherry tumors ( the and analyzed in combination), or (alone or inhibitors control the vehicle with treated GSCs, chymal ( tumors orthotopic from obtained tissues tumor in H3K27me3 and H2K119Ub attenuated specifically inhibitors EZH2 and BMI1 that confirmed course time a over models intracranial against EPZ6438 16d Figs. an ent with 80% consist GSCs, subtype different four on studies efficacy binational com the against brain the in measured respectively) EPZ6438, and we plotted the concentrations of BMI1 and EZH2 inhibitors (PTC596 efficacious, were brain the in achievable concentrations the whether blood in ( brain BMI1-i the into penetrates the of 20% least at that indicated treatment after (Elacridar) GW120918 inhibitor ABCG2 and ABCCB1 dual the with bination the of penetration brain a com using approach developed a the previously we leveraged EZH2-i, augment To barrier. a by protected tumor of regions have usually barrier, blood–tumor disrupted a ing represent gliomas, delivery, as contrast-enhancing even intracranial ( inhibitor) EZH2 (another DZNep and inhibitor) BMI1 (another PTC209 tors: inhibi unrelated structurally with ability tumorsphere-forming and on viability cell inhibitors of and EZH2 BMI1 effect subtype-specific xenografted a xenografted combination of mCherry in vivo tive ( inhibition BMI1 with showed combined therapy the more being opposite effect, effec whereas cells, mesenchymal against than cells effective more proneural were inhibition EZH2 of effects antitumor that confirmed imaging bioluminescence of limit the near detection, 18a Fig. ( both Supplementary of mixture a or GSCs mesenchymal GSCs, ural prone from derived tumors intracranial bearing mice on EPZ6438 and PTC596 of effects therapeutic the examined we brain, the into Supplementary Fig. 17c Fig. Supplementary Systemically delivered drugs against brain tumors must achieve achieve must tumors brain against drugs delivered Systemically Leveraging evidence that the BMI1 and EZH2 inhibitors penetrate that penetrate and the BMI1 inhibitors EZH2 evidence Leveraging Fig. Fig. 5 cellular effects of the EZH2 or BMI1 inhibitor treatments, we treatments, inhibitor or BMI1 EZH2 of the effects cellular and and b 3 Supplementary Fig. 16 Fig. Supplementary 3 + and . Measurement of the plasma and brain levels of BMI1-i of BMI1-i levels and brain of plasma the . Measurement proneural cells, EZH2-i treatment specifically reduced reduced specifically treatment EZH2-i cells, proneural 17a advance online publication online advance in in vitro Supplementary Figs. 18b Supplementary , b ). Pharmacodynamics analysis of PTC596 and and PTC596 of analysis Pharmacodynamics ). Fig. 5 Fig. inhibitory concentration inhibitory ). Though the bioluminescent signal was was signal bioluminescent the Though ). – Supplementary Fig. 17a Fig. Supplementary e ). c ). These These ). ). in vivo in + proneural and GFP – Supplementary Fig. 20 Fig. Supplementary e β and results translated into into translated results -galactosidase, did not -galactosidase, Fig. 5d Fig.

3 nature medicine nature 19 3 ( , b ). ). To the address Supplementary Supplementary ). To). determine – f ), indicating indicating ), Fig. 5 Fig. + mesen a and and 1 3 ). ). ------. © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. mutation, and the TCGA glioblastoma data included one patient patient one included data glioblastoma TCGA the and mutation, had a glioma missense not (data One low-grade with patient shown). lung and uterine breast, prostate, including amplifications, or tions have shows Portal cancer types database that several DNA-PKcs for ligase E3 an is RNF144A therapeutics. cancer to adaptation limit and heterogeneity intratumoral collapse could that approach treatment of a combinatorial development the may inform Thus, differential region-specific dependency on epigenetic modifiers ( and EZH2 of BMI1 utilization differential with niches different in present are GSCs which in model a support cell state under different conditions. microenvironmental Our results of plasticity permit may but interchangeable biochemically not are EZH2 and BMI1 of roles the that suggests PRC2 and PRC1 between gliomas pediat and high-grade adult ric against benefit therapeutic potential studies suggesting numerous with target, therapeutic cancer a as investigated cells stem cancer against effects with types, cancer of number a in efficacy preclinical shown have BMI1 against phenotype mesenchymal a to linked strongly TGF- including genes, of numerous promoters the lines glioma stem-like human and and growth in a engineered murine genetically model of glioblastoma neoplasia to maintain epigenome on converge the pathways as oncogenic targets, effective particularly transformation and methylation DNA in alterations to lead which histones, variant in mutations by ing harbor gliomas of growth tumor attenuate methylation inhibitors IDH1 Mutant DNA of modulation through astrocytes evolution. transform tumor accelerate to plasticity cellular permitting rather suppressors, tumor and oncogenes into mutations of dichotomization simple a to adhere not does likely but targeting, therapeutic to amenable be and neoplasia induce may dysregulation BMI1 including and regulators, epigenetic EZH2 (ref. in mutations harbor glioblastomas of 40% that observations genetic microenvironments. stressful in reside to GSCs empower may mechanism regulatory RNF144A–BMI1 the tenance, main GSC promote restriction nutrient and stress acidic hypoxia, features other and angiogenesis of induction through environment their remodel actively they cues, these of recipients passive their being than rather from but microenvironment, cues maintenance receive GSCs cells, stability. stem protein neural BMI1 Like of increase an in results which RNF144A, ligase, E3 of a BMI1 expression by repressing GSCs survival of mesenchymal the promotes hypoxia which in model a support findings these Collectively, availability. nutrient and oxygen low of areas in survival cellular promoting PRC1, of component core a BMI1, with on PRCs, dependencies for different may select responses stress that epigenetic regulation could extend to DNA methylation. Here we find donors methyl deplete that yltransferases meth of induction hypoxic and methionine of with availability reduced associated hypomethylation DNA show GSCs mesenchymal that reported Werecently therapies. targeted to signa response and tures transcriptional specific with GSCs to defined, features spatially radiographic linking by observations patient these single refine a results from Our derived be can clones GSC Multiple nature medicine nature nonsense a with one and amplification an with one deletion, a with Little is known about RNF144A beyond its enzymatic function. function. enzymatic its beyond RNF144A about known is Little by supported is tumors in diversity epigenetic of significance The IDH1 mutations

4 advance online publication online advance 0 . Pediatric glioblastomas are commonly driven driven are commonly glioblastomas . Pediatric 51– 5 6 . The dynamic interchange of elements elements of interchange dynamic The . 3 7 41– 4 . As we and others have found that that found have others and we As . 4 . BMI1 regulates tumor initiation initiation tumor regulates . BMI1 45– 4 3 . Epigenetic regulators may be be may regulators Epigenetic . 4 8 . BMI1 binds and regulates regulates and binds BMI1 . 3 3 3 . Interrogation of the cBio the of Interrogation . 6 , suggesting that regional regional that , suggesting Supplementary Fig. 21 Fig. Supplementary 5 0 4 . EZH2 has also been been also has EZH2 . 9 . Targeted therapies therapies Targeted . β IDH1 , which has been been has , which RNF144A

3 8 ). ). Epigenetic mutations mutations 14 muta , 34 , 3 3 5 9 ). ). ------. .

likely to be more effective in combination than alone. Both BMI1 BMI1 Both alone. than combination in effective more be to likely are tumors heterogeneous against therapies epigenetic that suggest findings GSCs Our of therapies. targeted to pools resistance adaptive support may different between interconversion how determine to interesting be It would treatments. inhibitor EZH2 and BMI1 by between effects different to lead may that microenvironment tumor different of influence the under transitions subtype molecular undergo can cells con Glioblastoma tumor trol. effective most the achieved tumors mesenchymal and proneural both against therapy combinatorial of the limits detection, near growth tumor hindered PRC component either against therapy mono Though absolute. not was efficacy the but EZH2, and BMI1 inhibitors. BMI1 for development biomarker inform signatures activation BMI1 and RNF144A the of relevance clinical the Moreover, discovery. target BMI1 of transcriptional a limitation and reveals of in glioblastoma regulation mechanism unknown previously a is BMI1 of regulation post-translational RNF144A tumors. proneural than tumors chymal BMI1 with correlate not does activity BMI1 that demonstrate results but our tumors, proneural in expression increased support expression investigating reports Prior not shown). (data mutation R.C.G., and S.C.M. performed bioinformatics analysis of published expression expression of published analysis bioinformatics performed S.C.M. and R.C.G., X.J., L.J.Y.K., B.C.P., experiments. animal and/or culture cell Q.Z. and performed X.J., L.J.Y.K.,manuscript. L.C.W., T.S.,T.E.M., Q.W., S.C.M., P.H., X.W., C.L.V., the wrote and data, analyzed experiments, overall the designed X.J. J.N.R. and (X.J.); Canadian Institutes of Health Research Banting Fellowship (S.C.M.). General Program of the National Natural Science Foundation of China (81572891) Foundation, the Jerry Kaufman GBM Research Fund, and CA217956 (A.E.S.); the (J.N.R.); the Peter D. Cristal Chair, the Kimble Family Foundation, the Ferry (S.B.); CA197718, CA154130, CA169117, CA171652, NS087913 and NS089272 (B.C.P.); CA043703 (J.S.B.-S.); CA169117, CA184090, NS091080 and NS099175 grants CA203101 (L.K.); CA183510 (T.E.M.); CA217065 (R.C.G.); CA217066 Finally, we would like to thank our funding sources: the National Institutes of Health service teams. We also thank members of J.N.R.’s lab for input about the manuscript. as the Cleveland Clinic Lerner Research Institute imaging core and proteomics core and MES-MN1). We would like to thank N. DeWitt for editorial assistance, as well subtype-characterized GSCs (PN11, PN23, PN-JK2, PN-MMK1, MES20, MES28 Cancer Center) and A.W. Boyd (Queensland Institute of Medical Research) for performing measurement of drug levels. We thank E.P. Sulman (MD Anderson We thank PTC Therapeutics for providing PTC209 and PTC596, as well as online ve Note: Any Supplementary Information and Source Data files are available in the pape the the in available are references, and codes accession statements of including Methods, and data availability any associated M pools. GSC also but state transitions, cell of plasticity the prevent may which processes, of epigenetic targeting combined only not considering advocate we GSCs, of alone target either in than EZH2 significance and prognostic BMI1 negative of greater have may tumor bulk expression combined the findings, nostic therapies com in conventional with useful bination be may strategy targeting dual our that suggesting chemotherapy, and radiation to resistance to contribute EZH2 and AUTH A ckn ethods Our results demonstrate the efficacy of combined inhibition of of inhibition combined of efficacy the demonstrate results Our mRNA levels and that BMI1 protein levels are higher in mesen o O rsion of the pape wle R R C r . O d NTRIBUTI gments 5 9 r . As single tumors may contain different pools pools different contain may tumors single As . . O NS in vitro in 54 , 57 and and , 5 8 . Similarly to our prog our to Similarly . in vivo in s e l c i t r a online version of version online experiments experiments BMI1 mRNA  - - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 16. 15. 14. 13. 12. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional reprints at online available is information permissions and Reprints version of The authors declare competing interests:financial details are available in the manuscript. final approved the and edited input, scientific provided authors All tissues. patient input and intellectual provided A.E.S. and S.B., J.S.B.-S., sets. data A 1 C 0 O

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MPETING FINANCIAL INTERESTS FINANCIAL MPETING ht K.P.L. Bhat, J. Halliday, Meyer, M. A.P. Patel, Y.T.Oh, Son, M.J., Woolard, K., Nam, D.-H., Lee, J. & Fine, H.A. SSEA-1 is an enrichmentTavares,L. marker Begg, A.C., Stewart, F.A. & Vens, C. Strategies to improve radiotherapy with targeted T.G.Graeber, Masouyé, I. M. Gynther, Marotta, D. T.S. Park, D. Hägerstrand, K.M. Joo, Mao, P. R.G.W. Verhaak, Flavahan, W.A. A.B. Hjelmeland, C. Calabrese, Z. Li, S. Bao, S. Bao, Jin, X. R. Galli, S.K. Singh, Wen, P.Y. & Kesari, S. Malignant gliomas in adults. augmented capacity for regenerating ischemic retinal vasculature. retinal ischemic regenerating for capacity augmented profile. sphere-forming glioblastoma cells with a distinct tyrosine kinase inhibitor sensitivity glioblastomas Rep. human of biology and histopathology the recapitulate 110 1A3. dehydrogenase aldehyde involving metabolism glioblastoma. in resistance radiation shift. USA Sci. Acad. proneural–mesenchymal Natl. and program transcriptional p53 protective andgenomic heterogeneity. glioblastoma. primary glioblastoma. of characteristics genetic and in abnormalities by characterized glioblastoma of subtypes uptake. glucose preferential Differ. Death 11 fortumor-initiating cells humanin glioblastoma. H3K27me3. and PRC2 of independently sites target drugs. tumours. solid in potential protein. acid–binding fatty prodrugs. via markerEF5andlaser-capture microdissection. (2014). 359–372 cells. factor. growth endothelial vascular response. damage DNA the of Lett. p27 of inhibition ID3-mediated abolishing by glioblastoma. human from precursors (2004). 396–401 (2008). , 69–82 (2007). 69–82 , /index.htm , 8644–8649 (2013). 8644–8649 , NF1

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. CAG-3 TGGCGTAGTA-3 5 5 reverse CD44 CATGACCAC-3 CAAGCCACAAGTGTTCC-3 EGFR GAAATATGGGAAG-3 reverse 5 AGTT-3 CTACCTAAGGACAA-3 5 AATG-3 TCCAATCATCAGC-3 5 3 CTGACAAGAAAG-3 5 GG-3 GCGCGCTGAATGTC-3 5 reverse and follows: to (SA as normalized Mastermix were primers SYBR-green values an using Expression on cycler Biosciences). performed 7900HT was cDNA PCR Biosystems aScript Real-time the Applied using Biosciences). cDNA (Quanta into SuperMix reverse-transcribed and (Qiagen) RT-PCR. Quantitative factor (20 ng/ml). A, Invitrogen), basic fibroblast growth factor (20 ng/ml) and epidermal growth (withoutvitamin B27 with medium neurobasal werein maintained GSCs and progenitor cells (NPC1 (NHP1), NPC2 and(NPC16357), NPC3 (NPC17893)) Normalexpressionneural arrayorRNA by either sequencing. performed was subtyping Molecular testing. mycoplasma and analysis (STR) repeat tandem invivo prospective enrichment of stem markercell expression, sphere formation, and and functionally validated as previously described xenografts or specimens surgical from isolated were MES3128) and MES738, Cultures(LDA). enrichedassay or dilution depleted limited for GSCsby (PN1919, validated PN3691, PN1914.2,functionally MES3565,ability, self-renewal their and 130-046-601), Miltenyi Biotec, microbeads, (MACS,CD15 columns magnetic with isolated were specimens primary glioblastoma CW2473 and condition. culture and Cells below. indicated as analyzed and separately processed then from anatomic regions different of specimens each tumor were distinct These of at the procedure. the beginning techniques areas stereotactic using distinct these from regions sampled then surgeon the Stealth), (BrainLab accurate be surgical strategy. In his the operating to room, after co-registration according was confirmed to sample to tissue invading and distinct necrotic enhancing, of pre-selected areas and patient the of imaging volumetric obtained geon in indicated as respectively region necrotic avascular and margin, enhancing region, necrotic of (enhancing tumor the center in the regions and cells; tumor infiltrating contain to thought and ing imag FLAIR on hyperintense but gadolinium with MRI weighted T1 on ing non-enhanc were which regions, peritumor tumor; enhancing of quantities significant with tumors only selected surgeon the At UH-CMC, (UH-CMC). Center Medical Hospitals-Cleveland University Cleveland or University, from Duke protocols Clinic, Board Review Institutional approved with ance accord in obtained was consent informed whom from patients glioblastoma specimens. glioblastoma Human ONLINE METHODS doi: TIMP1 ACATCCTA-3 CTAATAGGGCCAGCCTC-3 ′ ′ ′ ′ ′ ; ; -GCCACCATCCCTTGTGAG-3 -GCCAGATCCTGTCCAAGCTG-3 -ACCCATTGGCATTCTCTTTG-3 -CCTGAGGCTTTTCGGAGC-3 CD133 10.1038/nm.4415 forward 5 forward ′ forward 5 forward ; ; tumor formation. Cellular contamination was ruled out by serial short short out byserial contaminationruled was tumor formation. Cellular ′ owr 5 forward n rvre 5 reverse and ′ OLIG2 ′ ′ -AAGGACAAGCGGACAAAATG-3 ; ; ; ; owr 5 forward ′ MBP -CTGAAAAGGGCTTCCAGTCC-3 PDGFA ′ - AGTTAGCATGCCAGAGTCTC-3 - ′ owr 5 forward n rvre 5 reverse and owr 5 forward ′ ′ -TGACACTGTCCAAAGGTTTTC-3 -CTCCGTTTCTTCTTTGCCCAG-3 ′ n rvre 5 reverse and ′ owr 5 forward ; ; ′ -TTTTGGATTCATATGCCTTCTGT-3 18S ′ ′ CA9 n rvre 5 reverse and -TGGTAACTCTTTATTTCATTGTCCG-3 ′ ′ and reverse 5 reverse and Total RNA was isolated with the RNeasy kit kit RNeasy the with isolated was RNA Total n rvre 5 reverse and ′ ′ owr 5 forward n rvre 5 reverse and n rvre 5 reverse and ′ -CAGCCAGGCAGGAATTCAGC-3 owr 5 forward ′ ′ -AGGTCTCGTTCCGTGCTG-3 -CTGGCGTCCGAGTCCAT-3 Fresh CD15 Fresh ′ ′ ; ; ; ; ′ -GACCGATCCTCAAGCATCTC-3 YKL40 AKT2 Excess tumor tissues were collected from from collected were tissues tumor Excess ′ -GAAGGGGAAGTAGGATAGGGG-3 ′ ; ; ′ ′ ; ; -CTCTGCAAAGAGGGCATCAG-3 GABRB2 ′ ′ ′ ′ ; ; -TGCATGGCCGTTCTTAGTTG-3 -TCTCATGGCTCTGGTTTTCC-3 n rvre 5 reverse and ASCL1 ′ owr 5 forward -GGAGCTTCTCGACTTCACCA- owr 5 forward FOXO3 ′ + -ACCTGGTGACTCTCGGCTA ′ Fig. Fig. GSCs from CW2451, CW2472, CW2472, CW2451, from GSCs -AGTTTGCAACTTAAATCTC ′ ′ -CATCGAAACCTGGAGAGA -CGAGCTATAGACACCCTG ′ ; ; owr 5 forward owr 5 forward 5– NES 1 owr 5 forward 7 ′ ). Prior to surgery, the sur the surgery, to Prior ). . Functional assays included ; and and ; ′ forward 5 ; ; ′ DLL3 ′ -ACATCATCTCGTA -CCAAGGAGCCAA ′ ′ and reverse 5 reverse and and reverse 5 reverse and ′ 18S -GGTGACCTCCT ′ TGF forward 5 forward GGAACAATA - . Gene-specific Gene-specific . ′ ′ ′ -CAACGCCA and reverse reverse and -GGTGAATT ′ ′ ′ and reverse reverse and - - GCAGCAG β and reverse reverse and 1 ′ forward forward ; ; GLUT3 ′ ′ and and ′ ′ ′ -GCA -TCA -CCT and and ′ ′ ′ - - - - ′ ; ; ;

5 signaling, #8240S), CD44 (1:1,000, BD Biosciences, 550392), YKL40 (1:1,000, (1:1,000, YKL40 550392), Biosciences, BD (1:1,000, CD44 #8240S), signaling, Cell (1:1,000, H2K119Ub ab126783), Abcam, (1:1,000, BMI1 against bodies anti primary with Tween-20 probed + andvol/vol) PBS (0.5% in nonfatmilk (wt/vol) 5% with blocked Membraneswere (Millipore). membranes PVDF to separated by 12% SDS–PAGE (NuPAGE Bis-Tris gel, Invitrogen) phatase inhibitorand and cocktail (Roche) protease inhibitor andcocktail (Sigma) transferred phos phosSTOP containing Scientific) (Thermo buffer Lysis IP in lysed and assay. immunoprecipitation and analysis Western blot TGCAAGTAAGCATCTTACA-3VEGFB CTGGCACCACACC-3 5 reverse CTCTGAATGATTATTA-3 VEGFR2 GATGCACATCC-3 5 reverse CCTGC-3 GTGCTC-3 GGCAATTTTGCTAAGAATG-3 5 forward ACTCTTC-3 ATGGTGGCATAGATGGGCTCTTGA-3 5 forward Millipore, 07-689), and secondary HRP-conjugated antibodies. Nuclei in in Nuclei antibodies. HRP-conjugated secondary (1:1,000, and 07-689), anti-H3K27me3 Millipore, 8240S), Signaling, Cell (1:100, anti-H2K119Ub with stained PFAand 4% in fixed were tissues brain xenografted inhibitors) non- to EZH2 or BMI1 with compared treatment without or (+) (with PN3691 controls. positive neoplastic or (−) negative as scored was TMA on ing stain Overall Scanner. Slide SCN400 by a were taken Leica TMA images The antibodies. HRP-conjugated secondary and AF2418), system, R&D (1:100, 612667), Biosciences, anti-CD44 (1:100, BD 550392), Biosciences, anti-OLIG2 staining with istry anti-BMI1 (1:50, Abcam, anti-EZH2 BD (1:100, ab126783), and/or immunohistochem on after from the TMA analysis crushed were eliminated small too Tissues glioblastomas. 27 IV grade cases, 38 and glioma gliomas, II III grade grade 16 dysplasias), (cortical controls non-neoplastic 15 including block, TMA the on arrayed were cases 96 of total A Approval. Board Ohio State from after obtaining glioma University Review Institutional (TMAs) were microarrays tissue constructed paraffin-embedded deidentified (TMA) and Tissue immunohistochemistry. microarray were captured by DM4000Leica B microscope. in tumorcells senescent tophase-contrast the detect tissues, used light images Scanner. A senescence Slide SCN400 Leica a or Microscope Confocal SP5 TCS Leica bya weretaken antibodies. Nuclei were counterstained with DAPI. Immunofluorescence images CASPASE3 (1:100, Cell Signaling, 9661S) and the secondary fluorescence-labeled 8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active Signaling, Cell (1:500, H2AK119Ub 07-689), Millipore, (1:1,000, H3K27me3 GTX70020), Genetex, (1:100, CA9 AB7356), Millipore, (1:500, vWF 550392), antibodies to SOX2 (1:500, R&D system, AF2018), CD44 (1:100, BD primary using Biosciences, immunolabeled and paraformaldehyde 4% in fixed were tissue brain frozen xenografted (VEC/BMI1) PN3691 and images) MRI weighted T1- according CW1757 patient (glioblastoma samples multiregional frozen and Immunofluorescence biquitination was quantified by ImageJ. with Lactacystin (5 Forkit Co-IP (Thermo Scientific). the ubiquitination assays, cells were treated and IP magnetic Crosslink Pierce by detected were polyubiquitination BMI1 ( ( tubulin sc-9133), system, AF2018), RNF144A (1:500, Abcam, ab89260), Ub (1:5,000, Santa Cruz, Millipore, 07-689), OLIG2 (1:1,000, R&D system, AF2418), SOX2 (1:1,000, R&D Abcam, ab86428), EZH2 (1:1,000, BD Biosciences, 612667), H3K27Me3 (1:1,000, β ′ -GGGTCATCACTGGCTTGGGT-3 -actin,1:10,000, Sigma-Aldrich, A1978). BMI1-RNF144A interaction and interaction BMI1-RNF144A A1978). Sigma-Aldrich, -actin,1:10,000, forward 5 owr 5 forward ′ ′ -ATCCCAGGATGTGACTCACTG-3 ′ -TCATAGCCCAGATCAGCTC-3 ; ′ ′ -CCTATGTAGTTGTGGAAGTTTTGC-3 n reverse and 5 ′ -AGCTCTCTGGGATCAATGCTGTGT-3 ′ n rvre 5 reverse and LDH5 ′ -CCATCTCTTTTATCAGGGTTGG-3 ′ µ α n rvre 5 reverse and owr 5 forward ′ M or 10 -TAGCATGTCTTATAGTCATT-3 tbln11,0, im-lrc, 67) ad ACTIN and T6074), Sigma-Aldrich, -tubulin,1:10,000, ′ β n rvre 5 reverse and -galactosidase staining kit (Cell Signaling, 9860S) was 9860S) staining Signaling, kit (Cell -galactosidase ′ ′ ; -AACCTACTTCTTTCCCCCATC-3 CD34 b µ -gal staining. -gal M; Sigma) for 5 h before collection. BMI1 polyu ′ . ′ ′ -GATGAAGACGTAGGGACCAC-3 ; forward 5 ′ -TGCTGTACGTACTGCATTTGC-3 ′ MCT1 -TTCTACCCAACATTAACTTAGCAGG-3 ′ n rvre 5 reverse and ′ ; ′ GLUT1 AGAGGCGTACAGGGATAGCA-3- owr 5 forward ′ ′ 10- ; -CCGTCATTGAAACCAGG-3 ′ ACTIN ; µ forward 5 CD31 m thick slides of deidentified deidentified of slides thick m ′ n rvre 5 reverse and owr 5 forward ′ owr 5 forward ′ ′ -TGTAATCTACCAGTG -GGAACACGGGACTG n rvre 5 reverse and ′ n reverse and 5 ′ nature medicine nature -TCATCGTGGCTGA Cells were collected collected were Cells ′ 5- n rvre 5 reverse and µ ′ m m thick slides of ; MCT4 ′ ′ -AAGGCCA -AGAAAAT ′ ; ′ -CTCTG forward ′ ′ -CACT -ACTA ′ HIF1 and ′ and α ′ ′ ′ ------; ;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 158.6–793 nM (1/3 of 475.8–2,379 nM) for the BMI1 inhibitor). BMI1 the for nM) 475.8–2,379 of (1/3 nM 158.6–793 (51.3–366.7 two-dimen assays concentration drug from sional concentrations IC80 in detected benefit we combinational those having with as consistent concentrations achieved agent each for at administered 10 mg/kg nM. as Collectively, 475.8–2,379 calculations, we estimated the brain concentration range of the BMI1 inhibitor concentration inhibitor EZH2 the for used we that approach same the Using 1 achieved treatment inhibitor in and the 10 plasma brain. BMI1 mg/kg EPZ6438 inhibitor EZH2 350 mg/kg ment with 10 mg/kg or and (MW PTC596 420.34) 12.5 mg/kg BMI1 inhibitor 51–366 of 4 the the inhibitor BMI1 concentration h treat combined after determined of range concentration brain calculated a into translating 29–210 of brain concentration calculated a to leading dose), higher 140-fold example, (for EPZ6438 0.21 = 0.35)/2.5 × (1.5 as centration con brain minimum the calculated we blood, by measurements brain the of mg/kg 100 and contamination likely the EPZ6438 and observations mg/kg these of basis 2.5 the On GW120918. with treatment after h 1 brain the in 1.5 was concentration EPZ6438 the that (Elacridar) inhibitor, and GW120918 ABCG2 the with ABCB1 concentrations. reported the pharmacokinetics drug of the EZH2 inhibitor, achievable EPZ6438, in of combination Calculation pCMV-Gag-Pol helper plasmids (Addgene). pCMV-VSVGwith cells 293T toin produce were retroviralused particles and stem cell medium. Retroviral plasmids PMSC-BMI1 and PMSC-VEC (control) produced with PAX2 cells in 293T and helper (Addgene)plasmids PMD2G in were used to label the BTIC cells in L-2-T and L-2-G, pGIPZ-GFP,PRSC-mCherry, plasmids Lentiviral Aldrich. Sigma- from purchased were shCNTRL) for for (SCH002 sequence control cific and (TRCN0000004414 swhBMI1-880, RNF144A shRNF144A-1099, for and forTRCN0000004416 shRNF144A-3112) and nonspe shBMI1-939), for (TRCN0000020158 TRCN0000020157 BMI1 shEZH2-2450), for TRCN0000293738 and shEZH2-950, for (TRCN0000040077 EZH2 targeting transduction. retroviral or lentiviral and Plasmid ( software analysis Dilution Limiting Extreme by analyzed and measured were or without BMI1 or/and EZH2 inhibitors per 3 d. After 14 d, the tumorspheres or/and inhibitors, EZH2 20 and added plates containing 100 96-well in wereseeded well) per cells (rangeof40–1 well per ofcells numbers pathway,different signaling EZH2 and BMI1 by change frequency cell stem vitro In 1 × 10 seeding after (Promega) kit Assay Viability Cell Luminescent CellTiter-Glo EPZ6438. Relative cell growth was measured over a four-day time course using and PTC596 of doses different with treatment combined different after GSCs four subtype on performed was assay titration Two-dimensional DZNep. and PTC-209, with cells PN528-mCherry and MES83-GFP co-cultured the of GFP proportion the inhibitors, of and assay EZH2 BMI1 response For competitive software. Prism GraphPad with calculated were inhibitors EZH2 or BMI1 of seeded for drug response under BMI1 or EZH2 inhibitor treatment. IC 10 × 2 conditions, hypoxic or/and low-glucose under viability cell Medchemexpress LLC; DZNep, SELLECKCHEM). 1 × 10 PTC therapeutics; PTC209, PTC therapeutics), and EZH2 inhibitor (EPZ6438, (0.45 g/l glucose) -glucose low including conditions different under d 3 exposing after er’smanufactur instructions the to according (Promega) Assay Viability Cell Luminescent viability Cell and growth assays. Profiler. by IHC analyzed were and Microscope, DM4000B Leica a by taken were images nohistochemistry immu The hematoxylin. with counterstained were immunohistochemistry nature medicine nature h t t p : / 3 / + cells. b iiig iuin uoshr frain assay. formation tumorsphere dilution limiting or mCherry i o i n f . w e h i . e d + u µ cells was determined by flow cytometry after treatment treatment after by cytometry flow was determined cells . l l completed neurobasal medium with or without BMI1 a u µ / 1 s g/ml. The molecular weight of EPZ6438 is 572.74, 572.74, is EPZ6438 of weight molecular The g/ml. 0 o , , hypoxia (1% oxygen) f t w a µ r Cell viability was measured by CellTiter-Glo e g/ml in plasma and 2 mg/kg in the brain. brain. the in mg/kg 2 and plasma in g/ml in vivo / e l d µ a µ g/ml. Our studies used 350 mg/kg of of mg/kg 350 used studies Our g/ml. ). l l completed medium with neurobasal experiments. Lentiviral particles were µ g/ml in the plasma and 0.35 mg/kg mg/kg 0.35 and plasma the in g/ml µ M for the EZH2 inhibitor and inhibitor EZH2 M for the 7 , , BMI1 inhibitor (PTC596, Zhang and co-workers co-workers and Zhang Lentiviral plasmids for plasmids Lentiviral 6 cells were seeded for in in vivo o dtcin of detection For 3 3 . They . found They drug ranges 3 cells were were cells µ 50 M. We M. values ------

blinded to the group allocation and study outcome assessments of all mice. monitored by bioluminescence channel of IVIS Spectrum. The investigatorsGW120918 werewere administered orally by gavage. The size of orthotopicand 10 d tumorfor subtype-mixed was model), the model, BMI1 inhibitor subtype or single EZH2 for inhibitor d with 4 (after detectable were cells tumor from signals toma model were implanted intracranially into NSG mice. When the luciferase labeled PN1919 and 1,000 GFP-labeled MES20 cells for subtype-mixed glioblas PN1919 or MES20 cells for single subtype glioblastoma model, and 1,000 RFP- testing, For gavage. by inhibitor orally administered EZH2 were GW120918 or with (EPZ6438) (PTC596) inhibitor BMI1 the week, 1 After mice. NSG 10 × inhibitors, 5 EZH2 and enhanceuptake EPZ6438 into brain to LCC) Medchemexpress inhibitor; ABCG2 and ABCCB1 (dual GW120918 oral after administration of spectrometry BMI1 inhibitor mass (PTC596) or EZH2 HPLC-coupled inhibitor (EPZ6438) by with determined was brain and For pharmacokinetic study, BMI1 inhibitor (PTC596) concentration in plasma frontal right lobes of 3-week-old NOD the SCID- into implanted were cells control) (VEC/VEC PN3691-GFP-VEC mCherry-BMI1 and 2.5 × 10 appropriate treatment groups. For the co-implantationto assigned study,randomly were were mice 2.5All analysis. × sizes power 10 priori a an by sample calculated Required protocols. approved IACUC Clinic Cleveland experiments. Animal tcga/-J were downloaded from TCGA ( anatomicsamplespatientand ofinformation RNA-seq, data, survival, patient In silico -G (known genes only) option. FPKM values were quantile normalized. genes were calculated using v2.0.2 Cufflinks provided with the GTF file via the known for values FPKM strand. first to set option type library the with v2.0.6 Genomics Core Facility. Reads were to aligned the hg19 genome using TopHatUniversity Reserve Western Case the at instrument 2500 HiSeq Illumina an nomes.htm from (retrieved build genome hg19 the to ForGenomics. geneBeckman-Coulter expression analysis, reads were aligned by platform 2500 HiSeq Illumina the on sequenced were libraries RNA-seq Kit. Prep Library RNATotal Stranded TruSeq Illumina the using Genomics Beckman-Coulter by sequencing for prepared was RNA Total (Qiagen). kit rated using Phase Lock Gel tubes (5 Prime), and purified using the miRNAeasy sequencing. RNA software ( Map Enrichment Lab Bader the and v3.5 Cytoscape using created were plots js molecular signatures database at Pathway analysis was performed using the gene-set enrichment analysis (GSEA) ping peaks were defined using the BEDtools intersect function. Gene Ontology specific peaks were identified using the BEDTools subtract function and overlapfollowing settings: -F 2, -P 0.01, -L 2, -LP 0.01, the -minDist with inputdata ChIP versus sets 5000data H2AK119ub and –size H3K27me3 the in 1000. Region- cse.ucsc Sequencing reads were usingaligned toBowtie2 the hg19 ( sequencing. single-end 100-bp 2000 HiSeq Illumina using Children) Sick for Technologies) and sequenced at(Agilent the CenterBioanalyzer for using Applied assessed Genomics kb), (the Hospital (<1 selected size (1.0%) gel agarose turer recommendations. Following library amplification, DNA fragments were manufac to according Genomics) (Rubicon kit preparation DNA-seq library (Invitrogen) and ChIP libraries were amplified and barcoded using the Thruplex experiment (Abcam, AB4729). Enriched DNA was quantified using Picogreen 39155), and H2AK119Ub (10 mg, Signaling, Cell #8240S) antibodies per ChIP Activemg;Motif, (5 Active H3K27me3 mg;Motif, H3K27Ac (5 39133), using glioblastomaprimary tumors and 1 million fresh CD15 Chromatin immunoprecipitation (ChIP) assay. in vivo p and using the HOMER annotatePeaks function. Molecular Signature bubble inhibition effect of BMI1 and EZH2 inhibitors, 1 × 10 C .edu analyses. ) ) and Ivy ( GAP http l ) ) using TopHat v2.0.6. Paired-end 125-bp reads were generated on / ) genome using default settings. HOMER was used for calling peaks ://www.baderlab.org/ Collected Total RNA was extracted with TRIzol (Invitrogen), sepa (Invitrogen), TRIzol with extracted was RNATotal All animal studies were performed in accordance with with accordance in performed were studies animal All ht tp://glioblastoma.al in silico 5 5 PN3691-GFP-VEC cells (BMI1/VEC), or 5 × 10 PN3691 cells were implantedintowereintracranially cells PN3691 https://tcga-data.n http://softwar Software/EnrichmentM resources, including transcript microarray 3 3 . For. pharmacodynamic testing of BMI1 γ (NSG) mice as previously described http://cufflin leninstitute.or e.broadinstitute.org ChIP of 5–10 mg of flash-frozen ci.nih.gov/docs/publ + ks.cbcb.umd.edu/ige GSCs was performed 5 doi: luciferase-expressing http:/ a g p 10.1038/nm.4415 ) portal websites. ) portal ). /hgdownload. /gsea/index. 5 PN3691- ications/ 7 5 - - - - - .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. between groups was analyzed by one-way ANOVA or Student’sANOVAor one-way by analyzed was groups between Statistical analysis. analyzed by Excel, and patient was analyzed survival with GraphPad Prism. ment in TCGA and Ivy GAP data sets. Correlation of genes and signatures was 3a ( regions normoxia comparedregionsto hypoxic in lated hypoxia inhibition signature (HYPOXIA_IN), 23 genes significantly downregu ( region normoxia comparedregionsto hypoxic in twofoldupregulated significantly genes (HYPOXIA_AC),signature nine tion ( HUVEC to compared HMEC in ascular signature (MICRO_VAS), eight genes significantly upregulated twofold ( (HMEC) cells endothelial microvascular fold in human umbilical vein endothelial cells ( (HUVEC) compared to human vascular signature (MATURE_VAS), nine genes significantly upregulated two publications EZH2 and BMI1 activation or inhibition signatures were informed by previous mesenchymal), and classical, proneural, (including subtypes molecular GBM doi: , b 10.1038/nm.4415 ). Single-sample GSEA (ssGSEA) was used to analyze gene signature enrich 29 , 30 , 5 9 . Microenvironment-related gene signatures included: mature- All grouped data are presented as mean Supplementary Fig. 2a Fig. Supplementary Supplementary Fig. 2a Fig. Supplementary Supplementary Fig. 3a Fig. Supplementary Supplementary Fig. Fig. Supplementary ± , b s.e.m. Significance ); hypoxia activa hypoxia ); , t b -test using -test ); microv ); , b ); ); - - - - - Gene Gene Expression Omnibus (GEO Data availability. allocation during the experiment and/or when assessing the outcome. mice into different experimental arms. The investigator was blinded to the group mental setup. This study complies with randomization in grouping age-matched power calculation formula in JMP (Version appropriate12, the using SAS determined was mutant).size InstituteSampleversus (wild-type Inc.) for each experi co-deletionversuschromosome(co-deletednot)and19qand 1p droglioma,orglioblastoma), ableswere(astrocytoma,bytumor histologyoligoastrocytoma, type oligoden MultivariateIndependent3.2.4. variregressionR usinglinearperformed was cal duplicates (patient-derived xenograft cell models) with technical triplicates. biologi least at presentedin specimen each in repeatedwere experiments All ated by GraphPad Prism, and log-rank coefficients were calculated by Excel. Kaplan–Meier survival curves were gener Life Sciences Reporting Summary GraphPad Prism. The All raw ChIP-seq and RNA-seq data are available in the NCBI F -values and DF for each ANOVA test can be found in the IDH1 GSE10336 associated with this manuscript. Correlation mutation statusversus mutant),(wild-type P value was used to determine significance. 6 ). nature medicine nature ATRX status - - - - - nature research | life sciences reporting summary Corresponding Author: Jeremy N. Rich Date: August 31, 2017

Life Sciences Reporting Summary Nature Research wishes to improve the reproducibility of the work we publish. This form is published with all life science papers and is intended to promote consistency and transparency in reporting. All life sciences submissions use this form; while some list items might not apply to an individual manuscript, all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

` Experimental design 1. Sample size Describe how sample size was determined. The number of animals per arm is based upon the following calculation: N=1+2C(s/d) 2 where: N = number of animals per arm C = 7.85 when alpha=0.05 and 1-beta=0.8 (significance level of 5% with a power of 80%) s = standard deviation, and d = difference to be detected. These parameters are derived from prior experience with similar studies, leading to a final sample size of at least 5 animals per arm. 2. Data exclusions Describe any data exclusions. No piece of data from experiments presented in this manuscript was excluded. 3. Replication Describe whether the experimental findings were reliably reproduced. All experimental findings were repeated using multiple patient-derived models as cell line replicates (biological replicates) with technical replicates in each experiment. 4. Randomization Describe how samples/organisms/participants were allocated into All animal experiments underwent randomization at entry. age- and experimental groups. gender-matched mice were randomly allocated to each experimental arm through blinding the experimenter to animals.Animal subjects were randomly assigned to treatment groups and during analysis. 5. Blinding Describe whether the investigators were blinded to group allocation For bioluminescent imaging experiments of tumor-bearing mice treated during data collection and/or analysis. with monotherapy or combinatorial therapy, the experimenter was blinded to the mice selected for imaging on IVIS imager. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used. June 2017

1 Nature Medicine: doi:10.1038/nm.4415 6. Statistical parameters nature research | life sciences reporting summary For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or the Methods section if additional space is needed).

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly.

A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons

The test results (e.g. p values) given as exact values whenever possible and with confidence intervals noted A summary of the descriptive statistics, including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range) Clearly defined error bars

See the web collection on statistics for biologists for further resources and guidance.

` Software Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this study. Microsoft Excel, Matlab R2016a, R v3.3.0, GraphPad Prism, HOMER, TopHat2, BedTools, Cufflinks For all studies, we encourage code deposition in a community repository (e.g. GitHub). Authors must make computer code available to editors and reviewers upon request. The Nature Methods guidance for providing algorithms and software for publication may be useful for any submission.

` Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of unique - All cell lines and constructs are available to anyone in the community materials or if these materials are only available for distribution by a through appropriate material transfer agreements. for-profit company. - BMI1 inhibitors PTC596 and PTC209 are available for distribution through PTC Therapeutics (South Plainfiled, NJ, USA). 9. Antibodies Describe the antibodies used and how they were validated for use in BMI1 (Abcam Ab126783) the system under study (i.e. assay and species). H2AK119Ub (Cell Signaling #8240S) CD44 (BD Biosciences 550392) YKL40 (Abcam ab86428) EZH2 (BD Biosciences 612667) H3K27Me3 (Western, IF) (Millipore 07-689) H3K27Me3 (ChIP) (Active Motif 39155) OLIG2 (R&D system AF2418) SOX2 (R&D system AF2018) RNF144A (Abcam ab89260) Ub (Santa Cruz sc-9133) TUBULIN (Sigma-Aldrich T6074) ACTIN (Sigma-Aldrich A1978) vWF (Millipore AB7356) CA9 (Genetex GTX70020) CD15 (Millipore MAB4301) Ki67 (Dako M7240) active CASPASE3 (Cell signaling 9661S)

H3K27ac (Active Motif 39133) June 2017

2 Nature Medicine: doi:10.1038/nm.4415 10. Eukaryotic cell lines nature research | life sciences reporting summary a. State the source of each eukaryotic cell line used. - All GSC models were derived from disassociation of patient specimens. -293T cells used to produce virus for transduction were obtained from the CCF/LRI cell culture core.

b. Describe the method of cell line authentication used. All GSC models were verified for purity using STR analysis.

c. Report whether the cell lines were tested for mycoplasma All cell lines were regularly tested for mycoplasma contamination. contamination.

d. If any of the cell lines used in the paper are listed in the database N/A of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use.

` Animals and human research participants Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived materials used in For all animal experiments were performed with 3-4 week-old male and the study. female NSG mice

Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population characteristics of the All patients whose excess tissue specimens were analyzed in this study human research participants. were informed and consented according to respective IRB protocols approved by the Cleveland Clinic Foundation, University Hospitals, and The Ohio State University Medical Center. June 2017

3 Nature Medicine: doi:10.1038/nm.4415