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Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1–4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5–8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES +KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

Legends describing all supplemental figures.

A. ChIP-qPCR on H3.3K27M-GFP, and H3.3WT-GFP (n=3 each) expressing murine tumor lines for DNMT1 localization to p16 promoter region. B. Pyrosequencing analysis of DNA methylation at the p16 promoter after 7days of 1uM GSK343 treatment (n=3 for both groups). C. Pyrosequencing DNA methylation analysis of p16 and p19 promoters on human DIPG lines (SF8628, DIPG IV, and DIPG 007) treated with 72hr decitabine or vehicle at 10uM (*= p< 0.05, n=3). D. Western blot analysis of human DIPG lines treated with 72hr decitabine or vehicle and blotted for p16 (n=2) E. BrdU incorporation IC50 analysis of decitabine and azacitidine DNA methyltransferase inhibitors on mouse tumor cells (top 2 panels). Decitabine IC50 on human DIPG lines (bottom 3 panels). IC50''s for human lines were as follows: SF6828 (8μM), (0.5μM), DIPG IV, and DIPG 007 (2μM).

A. Positive (Hoxa11) and negative (actin) promoter control loci for H3K27me3 ChIP-qPCR. B. H3K27me3 ChIP-qPCR analysis of murine tumor cells on downstream p16 exon 1α and exon 2. C. Histone extraction western blot of H3.3K27M-GFP and H3.3WT-GFP expressing murine tumor lines treated with 1μM EPZ-6438 for 7 days. D. H3K4me3 ChIP-qPCR analyzing the p16 promoter region with or without 7 day GSK343 treatment (p= 0.035 by paired t-test) E. 21 day treatment with EZH2 inhibitor GSK343 followed by qPCR for p16 expression (p=0.036) F. BrdU incorporation on H3.3K27M-GFP and H3.3WT-GFP expressing murine tumor lines with EZH2 inhibitors GSK343 (p= 0.012), EPZ-6438, and GSK126 (p= 0.002) treatments incubated for 72hrs.

Genes displaying more than 2-fold change among IDH1LSL:R132H/WT ad-GFP versus ad-Cre NSCs were selected for pathway analysis by DAVID.

list of all viral infection combinations performed on mice and the median survival (days) of each model.

Sequences of primers used for genotyping of animals and for q-RT-PCR.

Supplementary Figure S1: p53 is upregulated in response to mutant IDH1 induction. Supplementary Figure S2: Hierarchical clustering of top 1% variant genes. Supplementary Figure S3: Further characterization of NSC and brain tumor cell lines. Supplementary Figure S4: E18.5 tamoxifen treatment of pregnant dams promotes robust recombination in embryos. Supplementary Figure S5: D-2HG detection in brain tumor cell lines.

Affymetrix GeneChip Mouse Gene 2.0ST Array used for gene expression profiling. Gene list displays those genes with more than 2-fold change when comparing IDH1LSL:R132H/WT ad-GFP transduced NSCs compared to IDH1LSL:R132H/WT ad-Cre transduced NSCs.

Percent DNA methylation and p16 expression changes with or without DNA methytransferase inhibition.

lists of antibodies and primer information used in the manuscript.

Materials and Methods used for gene expression profiling.

List of all significantly differentially expressed genes in empty vector vs H3.3K27M tumor RNA-Seq analysis.

A. Gene set enrichment analysis of differentially upregulated genes from RNA-Seq of high grade tumor model with or without H3.3K27M-HA expression shows high association with known PRC2-regulated H3K27me3 target genes in mouse embryonic, and neuronal progenitor cells (1). B. GSEA comparing genes losing H3K27me3 (UPS) and gaining H3K27me3 (DOWN) from Chan et al to our mouse tumor RNA-Seq. C. qPCR analysis of both p16 and p19 alternate transcripts from ex-vivo infected neuronal progenitor cells isolated from neonatal Nestin-tv-a pups and infected with either H3.3K27M-HA, H3.3WT-HA, or empty vector 2 weeks prior to collection.

A. Analysis of tumor volume of PDGF driven, p53 WT models with expression of H3.3K27M (n= 6), H3.3WT (n= 6), or empty vector (n= 7) shows trend for larger tumors with mutation compared to H3.3WT (p= 0.25) or empty vector (p= 0.28) controls. B. Kaplan-Meier survival curve of murine tumors driven by co-injection of nestin tv-a; p53 floxed mice with RCAS-PDGFB; RCAS-Cre; and either RCAS-H3.3K27M-GFP (n=12) or RCAS-H3.3WT-GFP (n= 18). C. In vivo proliferation quantification of H3.3K27M or H3.3WT expressing tumors driven by PDGFB overexpression and p53 knockout using pH3 (H3.3K27M n= 5, H3.3WT n=4) or D. EdU incorporation IF quantification (n=3 each) E. Histone extraction followed by western blot using total H3 antibody comparing murine tumor cells infected with empty vector, H3.3WT, or H3.3K27M to determine the amount of H3 overexpression.

Confirmation of calculation strategy of evaluating immune infiltration between two MB subtypes via staining of different APC markers. Same differential expression in Ptch1 tumor model observed.

Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control. Supplementary Figure 2. (A) Crystal violet staining images for acquired TMZ-resistant (TR) cell lines after treatment. (B) The heatmap of quantification results of the crystal violet images of TR cells. % relative to control. (C) Senescence assay for DBTRG-05MG, M059K and H4 cells following THIO treatment at 1 µM for 7 days, and then fixed and stained using Senescence β-Galactosidase Staining Kit and taken images by microscope. (D-F) Quantification results of senescence assay for DBTRG-05MG, M059K and H4 cells. 7 fields were counted for each group. (G-J) Neurosphere growth curves for T98G, U87MG, 13.0302 and TS-603 treated with or without THIO at 5 µM.

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Supplementary Tables

supplementary figures and legends

Summary of adverse events with possible relation to study.

Supplementary Figure Legends - PDF file 88K, Supplementary Figure Legends 1-9

Organoid stem cell gradients are stable over time.

Supplementary Table 1:Tumors maintained expression of subgroup genes previously identified as unique to corresponding human MB subtypes, SHH and Group 3 tumors, respectively. Supplementary Table 2: Flow cytometric antibody panel.

Supplementary Figure Legends

Orthotopically transplanted tumors are uniformly lethal at determined minimum tumorigenic dose. (A) Tumors arising from Patched1 heterozygous (Ptch1+/-) mice and immune deficient NOD scid gamma (NSG) mice implanted with retrovirally transduced cerebellar neural stem cells (NSC) were serially passaged via stereotactic implantation in the cerebellum of immunocompetent C57BL/6 hosts. Animals were implanted with varying numbers of tumor cells to determine the minimum dose required to induce 100% lethality in tumor-bearing mice within one month. Stereotactic implantation of (B) 1 x 103 NSC MB tumor cells and (C) 1.25 x 105 Ptch1 MB cells were determined as the minimum tumorigenic dose carried forth in subsequent animal studies.

Purpose:To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2′-deoxyguanosine (THIO) in gliomas both in vitro and in vivo.Experimental Design:A panel of human and mouse glioma cell lines was used to test therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO.Results:THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model.Conclusions:The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.

Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control.

PDF file - 182K, Tumor reduction in 3 patients who achieved a partial response following (A) 21 days, (B) 15 months, and (C) 18 months of treatment

Ex vivo analysis of CMV pp65-specific CD8+ T cells in circulation before and after immunotherapy.

Inactivating mutations in the transcriptional repression factor Capicua (CIC) occur in approximately 50% of human oligodendrogliomas, but mechanistic links to pathogenesis are unclear. To address this question, we generated Cic-deficient mice and human oligodendroglioma cell models. Genetic deficiency in mice resulted in a partially penetrant embryonic or perinatal lethal phenotype, with the production of an aberrant proliferative neural population in surviving animals. In vitro cultured neural stem cells derived from Cic conditional knockout mice bypassed an EGF requirement for proliferation and displayed a defect in their potential for oligodendrocyte differentiation. Cic is known to participate in gene suppression that can be relieved by EGFR signal, but we found that cic also activated expression of a broad range of EGFR-independent genes. In an orthotopic mouse model of glioma, we found that Cic loss potentiated the formation and reduced the latency in tumor development. Collectively, our results define an important role for Cic in regulating neural cell proliferation and lineage specification, and suggest mechanistic explanations for how CIC mutations may impact the pathogenesis and therapeutic targeting of oligodendroglioma. Cancer Res; 77(22); 6097–108. ©2017 AACR.

Gating strategy for NSC MB Tumors.

Microarray profile of HOG isogenic cell lines

Gating strategy for Ptch1 MB tumors.

Fig S3, proliferation assay. Fig S4 and S5, differentiation assay of Cic+/+ and Cic-/- NSCs.

Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2+, OLIG2+, and TLX+ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465–77. ©2016 AACR.

Supplemental Figures 1 through 10.

Purpose: Increased mitogenic signaling and angiogenesis, frequently facilitated by somatic activation of EGF receptor (EGFR; ErbB1) and/or loss of PTEN, and VEGF overexpression, respectively, drive malignant glioma growth. We hypothesized that patients with recurrent glioblastoma would exhibit differential antitumor benefit based on tumor PTEN/EGFRvIII status when treated with the antiangiogenic agent pazopanib and the ErbB inhibitor lapatinib.Experimental Design: A phase II study evaluated the antitumor activity of pazopanib 400 mg/d plus lapatinib 1,000 mg/d in patients with grade 4 malignant glioma and known PTEN/EGFRvIII status not receiving enzyme-inducing anticonvulsants (EIAC). The phase II study used a two-stage Green–Dahlberg design for futility. An independent, parallel phase I component determined the maximum-tolerated regimen (MTR) of pazopanib and lapatinib in patients with grade 3/4 glioma receiving EIACs.Results: The six-month progression-free survival (PFS) rates in phase II (n = 41) were 0% and 15% in the PTEN/EGFRvIII-positive and PTEN/EGFRvIII-negative cohorts, respectively, leading to early termination. Two patients (5%) had a partial response and 14 patients (34%) had stable disease lasting 8 or more weeks. In phase I (n = 34), the MTR was not reached. On the basis of pharmacokinetic and safety review, a regimen of pazopanib 600 mg plus lapatinib 1,000 mg, each twice daily, was considered safe. Concomitant EIACs reduced exposure to pazopanib and lapatinib.Conclusions: The antitumor activity of this combination at the phase II dose tested was limited. Pharmacokinetic data indicated that exposure to lapatinib was subtherapeutic in the phase II evaluation. Evaluation of intratumoral drug delivery and activity may be essential for hypothesis-testing trials with targeted agents in malignant glioma. Clin Cancer Res; 19(4); 900–8. ©2012 AACR.

T cell infiltration within the NSC MB tumor microenvironment does not correlate with DC infiltration.