Glioblastoma (GBM) is a lethal form of brain cancer with standard surgery and radiation giving a median survival of only 12.6 months (Carlsson et al., 2014). The addition of temozolomide chemotherapy provides only an additional 2.5 months in the small subset of responsive patients (Stupp et al., 2005). Despite extensive characterization and stratification of the bulk primary tumors, no targeted therapies have been successfully developed (Carlsson et al., 2014; von Neubeck et al., 2015). GBM tumors are rooted in self-renewing tumor-initiating cells commonly referred to as glioblastoma stem cells (GSCs) (Venere et al., 2011) that drive disease progression in vivo (Chen et al., 2012; Gallo et al., 2015) and display resistance to chemo- and radiotherapy leading to disease recurrence (Bao et al., 2006). The promise of therapeutically targeting self-renewing tumor-initiating cancer cells depends on our capacity to capture the full range of heterogeneity within this population from individual tumors. Intratumoral heterogeneity within primary GBM has recently been documented through single-cell RNA-seq experiments and revealed a continuum between four cellular states (Neftel et al., 2019): neural-progenitor-like (NPC), oligodendrocyte-progenitor-like (OPC), astrocyte-like (AC), and mesenchymal-like (MES) (Neftel et al., 2019). A subsequent study (Wang et al., 2019) using single-cell gene-centric enrichment analysis placed GBM cells along a single axis of variation from proneural to mesenchymal transcriptional profiles, with cells expressing stem-associated genes lying at the extremes of this axis. Hence, primary GBM consists of distinct states, across which stem-like cells appear to be found. Whether these stem-like cells found across GBM states represent functionally distinct GSC populations with tumor-initiating properties and unique dependencies remains to be established to guide therapeutic progress. To address this issue, we combined single-cell technologies to define GSC composition in primary GBM with functional assays to reveal the unique dependencies across GSCs, reflective of invasive, constructive, and reactive states that relate to patient outcome.

Chromatin accessibility readily discriminates stem from mature cell populations (Stergachis et al., 2013), which can be resolved at the single-cell level through single-cell ATAC-seq (Buenrostro et al., 2018; Corces et al., 2016), taking into account non-gene centric features, such as accessibility of noncoding elements and total amount of accessible DNA sequences. Applying single-cell chromatin accessibility profiling (scATAC-seq) across four primary adult GBM tumors (3797 cells), wild type for both IDH1 and IDH2, revealed seven to nine accessibility modules in each tumor based on unsupervised clustering (Figure 1A). We assigned cells to each of the four scRNA-seq-derived cellular states (Neftel et al., 2019) based on individual cells’ chromatin accessibility enrichment scores for the promoter regions of each state’s signature genes. Across the four tumors, 35–55.2% of the cells were significantly enriched for at least one state’s signature genes (Figure 1—figure supplement 1A). The MES state reported from scRNA-seq (Neftel et al., 2019) dominates the identity of two or more modules reported from chromatin accessibility in every tumor (Figure 1B,C, Figure 1—figure supplement 1B). In contrast, the NPC and OPC states are mixed within the same module defined based on chromatin accessibility, dominating over the other states typically in at least two modules. Cells assigned to the AC state did not preferentially cluster within a single module reported from chromatin accessibility (Figure 1B,C, Figure 1—figure supplement 1B). Collectively, our results suggest that chromatin accessibility reflects a greater stratification of the MES state and detects similarities between the OPC and NPC states and heterogeneity within the AC state.

Figure 1 with 1 supplement see all

Download asset Open asset

To identify putative cancer stem cells within each primary tumor, we next focused on the level of chromatin accessibility at promoters of 19 transcription factors previously associated with self-renewal and tumor-propagating capacity in GBM (Suvà et al., 2014; Figure 1D). Individual cells scoring as putative cancer stem cells were not restricted to a unique module defined by chromatin accessibility but were distributed across a subset of modules, suggesting heterogeneity across cancer stem cells in primary GBM, in agreement with reports relying on single GBM cell labeling (Corces et al., 2016; Gallo et al., 2015; Lan et al., 2017; Liau et al., 2017; Meyer et al., 2015; Miller et al., 2017; Orzan et al., 2017; Park et al., 2017; Rheinbay et al., 2013; Stergachis et al., 2013; Suvà et al., 2014), assessing the heterogeneity of self-renewing tumor-initiating cells. Putative cancer stem cells identified in primary GBM through scATAC-seq were found in modules ascribed to every one of the four cellular states defined by gene expression (Neftel et al., 2019), predominantly within NPC and OPC containing modules and a smaller fraction (<10%) in MES-specific modules across all four tumors (Figure 1E). This suggests that the core transcriptional unit of cancer stem cells in primary GBM (Suvà et al., 2014) is not restricted to a unique population defined by its global transcriptional or chromatin accessibility profile with the resolution achieved with current single-cell technologies.

To further probe the heterogeneity in chromatin accessibility within the GBM cancer stem cell pool, we derived GSC populations from 27 adult IDH wild-type GBM tumors (Pollard et al., 2009) and profiled their chromatin accessibility by bulk ATAC-seq (Figure 2A). Each patient-derived GSC showed a similar enrichment for accessible chromatin regions in promoters and 5′UTRs, and depletion in introns and distal intergenic regions (Figure 2B). Collectively, we uncovered 92% of the total predicted regions of accessible chromatin (255,891 regions) within GSCs based on a saturation analysis using a self-starting non-linear regression model across the 27 samples (Figure 2C). We next assessed the similarity between these GSCs and the putative cancer stem cells found by scATAC-seq in the four primary GBMs. GSCs were identified within each tumor by calculating the enrichment of accessible chromatin regions shared by a majority of GSCs (>14/27) in each tumor cell (Figure 2D). On average, 11.3% of cells in each primary GBM were labeled as GSCs. Comparing the distribution of GSCs across the seven to nine modules defined by scATAC-seq to that of the 19 transcription factor-derived cancer stem cell signature demonstrates concordance between the two signatures (Figure 2E). Moreover, the enrichment z-scores for both cancer stem signatures (i.e. stem transcription factors signature and GSC chromatin accessibility signature) are significantly correlated across cells in all four tumors (p≤1.6×10⁻⁵) (Figure 2F). The overlap in cells significantly enriched for either stem signatures across the four tumors is also significant (hypergeometric test p-value=8.8×10⁻⁸) Additionally, an average of 91.2% (85.1–100%) of the cells identified by either signature display the hallmark GBM copy number changes at chromosomes 7 and 10, confirming their neoplastic status (Figure 2—figure supplement 1). Collectively, these results demonstrate that the patient-derived GSC populations reflect the chromatin identity of putative cancer stem cells found in primary brain tumors, highlighting the value of these GSCs as models to deepen our understanding of individual cells within primary GBM with features found in self-renewing tumor-initiating cells. Accordingly, spectral clustering of the 27 patient-derived GSC ATAC profiles identifies three distinct states of self-renewing tumor-initiating cells (Figure 3A). Expression profiling of these 27 GSCs by RNA-seq reveals GSCs significantly enriched for the signatures of each of the three TCGA GBM subtypes (proneural, classical, and mesenchymal) (Wang et al., 2017; Figure 3B). However, the assignment of the proneural, classical, and mesenchymal subtypes across GSCs did not match the three clusters identified from ATAC-seq (Figure 3B). Conversely, clustering the GSCs by gene expression, independently of their chromatin accessibility, did largely recapitulate the GSC states defined by chromatin accessibility (Figure 3B). This suggests that the mismatch between the GSC cluster from chromatin accessibility profiles and TCGA expression subtypes is not mainly due to differences between chromatin accessibility and gene expression. Potential alternative causes for this observed mismatch include the absence of a tumor microenvironment in the GSC populations or that given the TCGA subtypes were determined from bulk GBM, they may not fully capture the nature of rarer populations found within a tumor, such as the cancer stem cell populations.

Figure 2 with 1 supplement see all

Download asset Open asset

Figure 3 with 1 supplement see all

Download asset Open asset

Gene set enrichment analysis with GSEA (Gene Set Enrichment Analysis) (Subramanian et al., 2005) and g:profiler (Reimand et al., 2016) using genes exclusively enriched for both expression and promoter chromatin accessibility in each subtype reported significantly enriched terms defining the largest GSC state as a Reactive state, with terms related to immune cells and response (Figure 3C, top panel). A second GSC state was enriched for Constructive gene sets involved in brain, neuron, and glial cell development (Figure 3C, middle panel). The third and smallest GSC state presented an Invasive state characterized by terms relating to the extracellular matrix and angiogenesis (Figure 3C, bottom panel). We next mapped copy number alterations (CNAs) across the 27 GSCs by applying the molecular neuropathology classifier tool (Capper et al., 2018) to DNA methylation data from the GSCs (Figure 3D). While common GBM CNAs, including EGFR gains and CDKN2A/B loss, were observed, the CNA-based classification of GSCs failed to match the three chromatin accessibility-derived states, suggesting the three GSC states are not defined by somatic copy number events (Figure 3D). Further comparison of the accessible chromatin in each GSC state reveals that only a small subset of accessible chromatin regions drives the three GSC states (Figure 3E, Figure 3—figure supplement 1). Our ability to discriminate GSC state-specific regions of accessible chromatin is reflective of the comprehensiveness of our cohort to saturate the detection of accessible regions to 93%, 88%, and 71% across the Reactive (n = 13), Constructive (n = 9), and Invasive (n = 5) state GSCs, respectively (Figure 3F).

Considering that regions of accessible chromatin serve as binding sites for transcription factors engaging in gene expression regulation, we next tested for DNA recognition motif family enrichment across regions exclusively accessible in Reactive, Constructive, or Invasive GSC states (Figure 4A, Figure 4—figure supplement 1A). The most enriched DNA recognition motif families in each state were either depleted or showed low-level enrichment in the other states. Specifically, the DNA recognition motifs for the interferon-regulatory factor (IRF) and Cys2-His2 zinc finger (C2H2 ZF) transcription factor families were enriched in the Reactive state (Figure 4A, top panel). Regulatory factor X (RFX) and basic helix-loop-helix (bHLH) DNA recognition motifs were enriched in the Constructive state (Figure 4A, middle panel), while the Forkhead motif family was enriched in the Invasive state (Figure 4A, bottom panel). Genome-wide CRISPR/Cas9 essentiality screens (Figure 4—figure supplement 1B) in three Reactive, two Constructive and one Invasive GSC (MacLeod et al., 2019) revealed the preferential requirement for expressed transcription factors (Figure 4—figure supplement 1C–E) recognizing the enriched DNA recognition motif in a state-specific manner (Figure 4B). Specifically, the SP1 regulatory network is preferentially essential in the Reactive state GSCs (Figure 4B, top panel), ASCL1, OLIG2, AHR, and NPAS3 are uniquely essential in the Constructive state GSCs (Figure 4B, middle panel) and FOXD1 is essential only in the Invasive state GSC (Figure 4B, bottom panel). Notably, SP1 itself is exclusively essential in only one Reactive GSC (G564). However, of the 36 transcription factors from the Reactive-enriched families (IRF and C2H2 ZF) that were essential in at least one Reactive GSC and not in any of the Constructive or Invasive GSCs, 13 are directly regulated by SP1 (Figure 4—figure supplement 1C), thus suggesting that the SP1 regulatory network as a whole, rather than SP1 on its own, is key in the Reactive GSC state. Notably, all six transcription factors display significantly higher expression in GBM compared to normal brain (Tang et al., 2017; Figure 4—figure supplement 1F), further supporting their function as key regulators of tumor initiation and development. An additional gene set enrichment analysis combining genes exclusively essential to each state with the putative targets of the key transcription factors outlined above identifies additional enriched terms supporting the identities of the three GSC states as Reactive, Constructive, and Invasive (Figure 4—figure supplement 1G).

Figure 4 with 1 supplement see all

Download asset Open asset

Previous work suggests that GBM tumors harbor a heterogeneous population of GSCs (Lan et al., 2017; Meyer et al., 2015; Patel et al., 2014). We therefore quantified the presence of Reactive, Constructive, and Invasive cancer stem cells in our four primary GBM based on their scATAC-seq profiles. The Constructive state was dominant in every primary tumor, ranging from 9 to 21% of all cells captured by scATAC-seq (Figure 4C). The Reactive and Invasive states accounted for only 0–9% of all cells, in varying proportions from one tumor to another (Figure 4C). Collectively, our results further support the heterogeneous nature of cancer stem cells that populate primary tumors.

While various classifications of GBMs and/or their constitutive bulk and stem tumor cells have been reported, with some associating with patient survival (Neftel et al., 2019; Patel et al., 2014; Cancer Genome Atlas Research Network et al., 2010; Wang et al., 2017; Yin et al., 2019; Zuo et al., 2019), molecular signatures in adult GBM stratifying the poorer prognosis IDH wild-type patients by survival are lacking. We performed intracranial xenografts of 37 IDH wild-type GSC populations and classified the transplanted cells by their GSC state to perform a differential survival analysis (Figure 5A). The overall survival times of the transplanted mice grouped by GSC state were significantly different (LogRank test p=0.041), with the Invasive state GSCs leading to the worst prognosis. Relative to the mice injected with Reactive state GSCs, mice with Constructive state GSCs and Invasive state GSCs had hazard ratios of 1.3 (95% confidence interval [CI]: 0.57–2.97) and 3.5 (95% CI: 1.2–10.49), respectively.

Figure 5 with 2 supplements see all

Download asset Open asset

In light of this finding, we sought to evaluate the invasive properties of the Invasive GSC state population. To that end, we conducted an invasion assay (Restall et al., 2018) wherein neurospheres from representative GSCs of each state (four Reactive, three Constructive, and three Invasive; three independent biological replicates for each GSC) are embedded in a collagen matrix and invasion is monitored in real time (Figure 5B,C, Figure 5—figure supplement 1A). GSCs of the Invasive state invaded strikingly faster and further into the collagen matrix and overall displayed a three times greater invasive ability than that of the Reactive and Constructive states. This invasiveness was further confirmed in vivo through human-specific staining of mouse brains injected with Invasive-state GSCs (Figure 5—figure supplement 1B–C).

Next, we investigated the prognostic value of the GSC states using the TCGA GBM cohort (IDH1 and IDH2 wild type, n = 144). When classified by dominant GSC state, TCGA tumors display the same trend as the xenografts with Invasive state-dominated tumors showing the lowest survival (Figure 5—figure supplement 2A). However, with only two tumors classified as Invasive-dominant, the difference in survival between the three patient groups was not statistically significant (p=0.3) (Figure 5—figure supplement 2A). We proceeded to rank the TCGA tumors solely by their concordance to Invasive GSCs and classified the patient tumors into Invasive-low (z-score < 1), Invasive-mid (z-score = 1–1.65), and Invasive-high (z-score ≥ 1.65) groups (Figure 5D). With this stratification method, median patient survival per group not only decreased with increasing Invasive GSC score, but we also identified an Invasive-high subset of tumors with significantly lower survival (p=0.019, HR (Hazard Ratio) = 2.8, 95% CI: [1.3–5.81]) (Figure 5E). This was further validated using an additional cohort of TCGA samples with microarray gene expression (Figure 5—figure supplement 2D–E). These results show that cancer stem cell states defined based on the chromatin accessibility in GSCs can identify transcriptional programs associated with poor prognosis and can serve as a signature to identify high-risk patients in IDH wild-type GBM.

Defining the nature of self-renewing tumor-initiating cells in primary GBM is required to identify vulnerabilities for therapeutic intervention. Quantifying their heterogeneity within tumors can guide treatment strategies and assist in predicting the course of disease progression. Here we show that chromatin accessibility assays capture a heterogeneity across self-renewing tumor-initiating cells in primary GBM that extends beyond their genetic diversity and underlies the heterogeneity in bulk progeny (Meyer et al., 2015). This heterogeneity aligns with diversity in the three-dimensional genome organization of GSCs (Johnston et al., 2019) and agrees with how the three-dimensional genome organization instructs cis-regulatory plexuses underlying gene regulation (Bailey et al., 2016; Kim et al., 2016; Sallari et al., 2017; Schmitt et al., 2016; Zheng and Xie, 2019). While the chromatin accessibility signature derived from GSCs robustly identified cancer stem cells across the four primary GBM tumors used in this study, a larger cohort will be required to establish whether this signature is always sufficient to capture all cancer stem cells in primary GBMs.

We further reveal a specific cancer stem state that is significantly predictive of patient survival and can be used as a signature to identify high-risk patients. Specifically, the Invasive GSC signature draws from a population with enhanced ability to invade and spread compared to other GSC states. Collectively, our results argue for distinct GSC populations whose composition in tumors impacts survival.

Our results also highlight dependencies unique to each cancer stem state. Specifically, the Reactive GSC state-associated transcription factor SP1 and its regulatory partners are involved in cellular differentiation and growth, apoptosis, response to DNA damage, chromatin remodeling (O'Connor et al., 2016), stimulation of TERT expression in cancer stem cells (Liu et al., 2016), and increased stemness and invasion in GBM (Lee et al., 2014). In contrast, the Constructive GSC state relies on transcription factors including OLIG2, a known GSC marker (Trépant et al., 2015); AHR involved in tumor microenvironment responses and metabolic adaptation (Gabriely et al., 2017); NPAS3, a regulator of Notch signaling and neurogenesis (Michaelson et al., 2017); and ASCL1, a critical regulator of GSC differentiation and marker of sensitivity to Notch inhibition in GSCs (Park et al., 2017; Rajakulendran et al., 2019). Finally, the Invasive GSC state relies on FOXD1, a pluripotency regulator and determinant of tumorigenicity in GSCs regulating expression of the aldehyde dehydrogenase ALDH1A3, a functional marker for invasive GSCs (Cheng et al., 2016; Koga et al., 2014). These results support developing combination therapy using targeting agents against each GSC state, such as Notch inhibitors (Park et al., 2017) and small molecule inhibitors of ALDH (Cheng et al., 2016), to eradicate self-renewing tumor-initiating cells with the hope to cure GBM patients.

The GSCs are available upon reasonable request from PBD and SW. The GSC ATAC-seq and DNA methylation data have been deposited at GEO (GSE109399). The scATAC-seq data has been deposited at GEO (GSE139136). RNA-seq data are available at EGA (EGAS00001003070).

1. 1. Guilhamon P
   2. Kushida MM
   3. Dirks PB
   4. Lupien M

   (2019) NCBI Gene Expression Omnibus

   ID GSE109399. Epigenetic characterization of glioblastoma stem cells.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109399
2. 1. Nikolic A
   2. Ellestad K
   3. Singhal D
   4. Gallo M

   (2021) NCBI Gene Expression Omnibus

   ID GSE139136. Single-cell ATAC-Seq of Adult GBM.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139136
3. 1. Guilhamon P
   2. Kushida MM
   3. Cavalli FMG
   4. Dirks PB
   5. Lupien M

   (2018) EGA

   ID EGAS00001003070. RNA-seq of Glioblastoma stem cells.

   https://ega-archive.org/search-results.php?query=EGAS00001003070

1. 1. Anders S
   2. Huber W

   (2010) Differential expression analysis for sequence count data

   Genome Biology 11:R106.

   https://doi.org/10.1186/gb-2010-11-10-r106

   • PubMed
   • Google Scholar
2. 1. Bailey SD
   2. Desai K
   3. Kron KJ
   4. Mazrooei P
   5. Sinnott-Armstrong NA
   6. Treloar AE
   7. Dowar M
   8. Thu KL
   9. Cescon DW
   10. Silvester J
   11. Yang SY
   12. Wu X
   13. Pezo RC
   14. Haibe-Kains B
   15. Mak TW
   16. Bedard PL
   17. Pugh TJ
   18. Sallari RC
   19. Lupien M

   (2016) Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast Cancer

   Nature Genetics 48:1260–1266.

   https://doi.org/10.1038/ng.3650

   • PubMed
   • Google Scholar
3. 1. Bao S
   2. Wu Q
   3. McLendon RE
   4. Hao Y
   5. Shi Q
   6. Hjelmeland AB
   7. Dewhirst MW
   8. Bigner DD
   9. Rich JN

   (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response

   Nature 444:756–760.

   https://doi.org/10.1038/nature05236

   • PubMed
   • Google Scholar
4. 1. Buenrostro JD
   2. Giresi PG
   3. Zaba LC
   4. Chang HY
   5. Greenleaf WJ

   (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

   Nature Methods 10:1213–1218.

   https://doi.org/10.1038/nmeth.2688

   • PubMed
   • Google Scholar
5. 1. Buenrostro JD
   2. Corces MR
   3. Lareau CA
   4. Wu B
   5. Schep AN
   6. Aryee MJ
   7. Majeti R
   8. Chang HY
   9. Greenleaf WJ

   (2018) Integrated Single-Cell analysis maps the continuous regulatory landscape of human hematopoietic differentiation

   Cell 173:1535–1548.

   https://doi.org/10.1016/j.cell.2018.03.074

   • PubMed
   • Google Scholar
6. 1. Cancer Genome Atlas Research Network
   2. Verhaak RG
   3. Hoadley KA
   4. Purdom E
   5. Wang V
   6. Qi Y
   7. Wilkerson MD
   8. Miller CR
   9. Ding L
   10. Golub T
   11. Mesirov JP
   12. Alexe G
   13. Lawrence M
   14. O'Kelly M
   15. Tamayo P
   16. Weir BA
   17. Gabriel S
   18. Winckler W
   19. Gupta S
   20. Jakkula L
   21. Feiler HS
   22. Hodgson JG
   23. James CD
   24. Sarkaria JN
   25. Brennan C
   26. Kahn A
   27. Spellman PT
   28. Wilson RK
   29. Speed TP
   30. Gray JW
   31. Meyerson M
   32. Getz G
   33. Perou CM
   34. Hayes DN

   (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1

   Cancer Cell 17:98–110.

   https://doi.org/10.1016/j.ccr.2009.12.020

   • PubMed
   • Google Scholar
7. 1. Capper D
   2. Jones DTW
   3. Sill M
   4. Hovestadt V
   5. Schrimpf D
   6. Sturm D
   7. Koelsche C
   8. Sahm F
   9. Chavez L
   10. Reuss DE
   11. Kratz A
   12. Wefers AK
   13. Huang K
   14. Pajtler KW
   15. Schweizer L
   16. Stichel D
   17. Olar A
   18. Engel NW
   19. Lindenberg K
   20. Harter PN
   21. Braczynski AK
   22. Plate KH
   23. Dohmen H
   24. Garvalov BK
   25. Coras R
   26. Hölsken A
   27. Hewer E
   28. Bewerunge-Hudler M
   29. Schick M
   30. Fischer R
   31. Beschorner R
   32. Schittenhelm J
   33. Staszewski O
   34. Wani K
   35. Varlet P
   36. Pages M
   37. Temming P
   38. Lohmann D
   39. Selt F
   40. Witt H
   41. Milde T
   42. Witt O
   43. Aronica E
   44. Giangaspero F
   45. Rushing E
   46. Scheurlen W
   47. Geisenberger C
   48. Rodriguez FJ
   49. Becker A
   50. Preusser M
   51. Haberler C
   52. Bjerkvig R
   53. Cryan J
   54. Farrell M
   55. Deckert M
   56. Hench J
   57. Frank S
   58. Serrano J
   59. Kannan K
   60. Tsirigos A
   61. Brück W
   62. Hofer S
   63. Brehmer S
   64. Seiz-Rosenhagen M
   65. Hänggi D
   66. Hans V
   67. Rozsnoki S
   68. Hansford JR
   69. Kohlhof P
   70. Kristensen BW
   71. Lechner M
   72. Lopes B
   73. Mawrin C
   74. Ketter R
   75. Kulozik A
   76. Khatib Z
   77. Heppner F
   78. Koch A
   79. Jouvet A
   80. Keohane C
   81. Mühleisen H
   82. Mueller W
   83. Pohl U
   84. Prinz M
   85. Benner A
   86. Zapatka M
   87. Gottardo NG
   88. Driever PH
   89. Kramm CM
   90. Müller HL
   91. Rutkowski S
   92. von Hoff K
   93. Frühwald MC
   94. Gnekow A
   95. Fleischhack G
   96. Tippelt S
   97. Calaminus G
   98. Monoranu CM
   99. Perry A
   100. Jones C
   101. Jacques TS
   102. Radlwimmer B
   103. Gessi M
   104. Pietsch T
   105. Schramm J
   106. Schackert G
   107. Westphal M
   108. Reifenberger G
   109. Wesseling P
   110. Weller M
   111. Collins VP
   112. Blümcke I
   113. Bendszus M
   114. Debus J
   115. Huang A
   116. Jabado N
   117. Northcott PA
   118. Paulus W
   119. Gajjar A
   120. Robinson GW
   121. Taylor MD
   122. Jaunmuktane Z
   123. Ryzhova M
   124. Platten M
   125. Unterberg A
   126. Wick W
   127. Karajannis MA
   128. Mittelbronn M
   129. Acker T
   130. Hartmann C
   131. Aldape K
   132. Schüller U
   133. Buslei R
   134. Lichter P
   135. Kool M
   136. Herold-Mende C
   137. Ellison DW
   138. Hasselblatt M
   139. Snuderl M
   140. Brandner S
   141. Korshunov A
   142. von Deimling A
   143. Pfister SM

   (2018) DNA methylation-based classification of central nervous system tumours

   Nature 555:469–474.

   https://doi.org/10.1038/nature26000

   • PubMed
   • Google Scholar
8. 1. Carlsson SK
   2. Brothers SP
   3. Wahlestedt C

   (2014) Emerging treatment strategies for glioblastoma multiforme

   EMBO Molecular Medicine 6:1359–1370.

   https://doi.org/10.15252/emmm.201302627

   • PubMed
   • Google Scholar
9. 1. Chen J
   2. Li Y
   3. Yu TS
   4. McKay RM
   5. Burns DK
   6. Kernie SG
   7. Parada LF

   (2012) A restricted cell population propagates glioblastoma growth after chemotherapy

   Nature 488:522–526.

   https://doi.org/10.1038/nature11287

   • PubMed
   • Google Scholar
10. 1. Cheng P
    2. Wang J
    3. Waghmare I
    4. Sartini S
    5. Coviello V
    6. Zhang Z
    7. Kim SH
    8. Mohyeldin A
    9. Pavlyukov MS
    10. Minata M
    11. Valentim CL
    12. Chhipa RR
    13. Bhat KP
    14. Dasgupta B
    15. La Motta C
    16. Kango-Singh M
    17. Nakano I

    (2016) FOXD1-ALDH1A3 signaling is a determinant for the Self-Renewal and tumorigenicity of mesenchymal glioma stem cells

    Cancer Research 76:7219–7230.

    https://doi.org/10.1158/0008-5472.CAN-15-2860

    • PubMed
    • Google Scholar
11. 1. Corces MR
    2. Buenrostro JD
    3. Wu B
    4. Greenside PG
    5. Chan SM
    6. Koenig JL
    7. Snyder MP
    8. Pritchard JK
    9. Kundaje A
    10. Greenleaf WJ
    11. Majeti R
    12. Chang HY

    (2016) Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution

    Nature Genetics 48:1193–1203.

    https://doi.org/10.1038/ng.3646

    • PubMed
    • Google Scholar
12. 1. Corces MR
    2. Trevino AE
    3. Hamilton EG
    4. Greenside PG
    5. Sinnott-Armstrong NA
    6. Vesuna S
    7. Satpathy AT
    8. Rubin AJ
    9. Montine KS
    10. Wu B
    11. Kathiria A
    12. Cho SW
    13. Mumbach MR
    14. Carter AC
    15. Kasowski M
    16. Orloff LA
    17. Risca VI
    18. Kundaje A
    19. Khavari PA
    20. Montine TJ
    21. Greenleaf WJ
    22. Chang HY

    (2017) An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

    Nature Methods 14:959–962.

    https://doi.org/10.1038/nmeth.4396

    • PubMed
    • Google Scholar
13. 1. Dobin A
    2. Davis CA
    3. Schlesinger F
    4. Drenkow J
    5. Zaleski C
    6. Jha S
    7. Batut P
    8. Chaisson M
    9. Gingeras TR

    (2013) STAR: ultrafast universal RNA-seq aligner

    Bioinformatics 29:15–21.

    https://doi.org/10.1093/bioinformatics/bts635

    • Google Scholar
14. 1. Gabriely G
    2. Wheeler MA
    3. Takenaka MC
    4. Quintana FJ

    (2017) Role of AHR and HIF-1α in glioblastoma metabolism

    Trends in Endocrinology & Metabolism 28:428–436.

    https://doi.org/10.1016/j.tem.2017.02.009

    • PubMed
    • Google Scholar
15. 1. Gallo M
    2. Coutinho FJ
    3. Vanner RJ
    4. Gayden T
    5. Mack SC
    6. Murison A
    7. Remke M
    8. Li R
    9. Takayama N
    10. Desai K
    11. Lee L
    12. Lan X
    13. Park NI
    14. Barsyte-Lovejoy D
    15. Smil D
    16. Sturm D
    17. Kushida MM
    18. Head R
    19. Cusimano MD
    20. Bernstein M
    21. Clarke ID
    22. Dick JE
    23. Pfister SM
    24. Rich JN
    25. Arrowsmith CH
    26. Taylor MD
    27. Jabado N
    28. Bazett-Jones DP
    29. Lupien M
    30. Dirks PB

    (2015) MLL5 orchestrates a Cancer Self-Renewal state by repressing the histone variant H3.3 and Globally Reorganizing Chromatin

    Cancer Cell 28:715–729.

    https://doi.org/10.1016/j.ccell.2015.10.005

    • PubMed
    • Google Scholar
16. 1. Hart T
    2. Chandrashekhar M
    3. Aregger M
    4. Steinhart Z
    5. Brown KR
    6. MacLeod G
    7. Mis M
    8. Zimmermann M
    9. Fradet-Turcotte A
    10. Sun S
    11. Mero P
    12. Dirks P
    13. Sidhu S
    14. Roth FP
    15. Rissland OS
    16. Durocher D
    17. Angers S
    18. Moffat J

    (2015) High-Resolution CRISPR screens reveal fitness genes and Genotype-Specific Cancer liabilities

    Cell 163:1515–1526.

    https://doi.org/10.1016/j.cell.2015.11.015

    • PubMed
    • Google Scholar
17. 1. Hart T
    2. Tong AHY
    3. Chan K
    4. Van Leeuwen J
    5. Seetharaman A
    6. Aregger M
    7. Chandrashekhar M
    8. Hustedt N
    9. Seth S
    10. Noonan A
    11. Habsid A
    12. Sizova O
    13. Nedyalkova L
    14. Climie R
    15. Tworzyanski L
    16. Lawson K
    17. Sartori MA
    18. Alibeh S
    19. Tieu D
    20. Masud S
    21. Mero P
    22. Weiss A
    23. Brown KR
    24. Usaj M
    25. Billmann M
    26. Rahman M
    27. Costanzo M
    28. Myers CL
    29. Andrews BJ
    30. Boone C
    31. Durocher D
    32. Moffat J

    (2017) Evaluation and design of Genome-Wide CRISPR/SpCas9 knockout screens

    G3: Genes, Genomes, Genetics 7:2719–2727.

    https://doi.org/10.1534/g3.117.041277

    • Google Scholar
18. 1. Johnston MJ
    2. Nikolic A
    3. Ninkovic N
    4. Guilhamon P
    5. Cavalli FMG
    6. Seaman S
    7. Zemp FJ
    8. Lee J
    9. Abdelkareem A
    10. Ellestad K
    11. Murison A
    12. Kushida MM
    13. Coutinho FJ
    14. Ma Y
    15. Mungall AJ
    16. Moore R
    17. Marra MA
    18. Taylor MD
    19. Dirks PB
    20. Pugh TJ
    21. Morrissy S
    22. St Croix B
    23. Mahoney DJ
    24. Lupien M
    25. Gallo M

    (2019) High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma

    Genome Research 29:1211–1222.

    https://doi.org/10.1101/gr.246520.118

    • PubMed
    • Google Scholar
19. 1. Kim K
    2. Jang K
    3. Yang W
    4. Choi EY
    5. Park SM
    6. Bae M
    7. Kim YJ
    8. Choi JK

    (2016) Chromatin structure-based prediction of recurrent noncoding mutations in Cancer

    Nature Genetics 48:1321–1326.

    https://doi.org/10.1038/ng.3682

    • PubMed
    • Google Scholar
20. 1. Koga M
    2. Matsuda M
    3. Kawamura T
    4. Sogo T
    5. Shigeno A
    6. Nishida E
    7. Ebisuya M

    (2014) Foxd1 is a mediator and Indicator of the cell reprogramming process

    Nature Communications 5:3197.

    https://doi.org/10.1038/ncomms4197

    • PubMed
    • Google Scholar
21. 1. Lan X
    2. Jörg DJ
    3. Cavalli FMG
    4. Richards LM
    5. Nguyen LV
    6. Vanner RJ
    7. Guilhamon P
    8. Lee L
    9. Kushida MM
    10. Pellacani D
    11. Park NI
    12. Coutinho FJ
    13. Whetstone H
    14. Selvadurai HJ
    15. Che C
    16. Luu B
    17. Carles A
    18. Moksa M
    19. Rastegar N
    20. Head R
    21. Dolma S
    22. Prinos P
    23. Cusimano MD
    24. Das S
    25. Bernstein M
    26. Arrowsmith CH
    27. Mungall AJ
    28. Moore RA
    29. Ma Y
    30. Gallo M
    31. Lupien M
    32. Pugh TJ
    33. Taylor MD
    34. Hirst M
    35. Eaves CJ
    36. Simons BD
    37. Dirks PB

    (2017) Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

    Nature 549:227–232.

    https://doi.org/10.1038/nature23666

    • PubMed
    • Google Scholar
22. 1. Lee WS
    2. Kwon J
    3. Yun DH
    4. Lee YN
    5. Woo EY
    6. Park M-J
    7. Lee J-S
    8. Han Y-H
    9. Bae IH

    (2014) Specificity protein 1 expression contributes to Bcl-w-Induced aggressiveness in glioblastoma multiforme

    Molecules and Cells 37:17–23.

    https://doi.org/10.14348/molcells.2014.2161

    • Google Scholar
23. 1. Li W
    2. Xu H
    3. Xiao T
    4. Cong L
    5. Love MI
    6. Zhang F
    7. Irizarry RA
    8. Liu JS
    9. Brown M
    10. Liu XS

    (2014) MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens

    Genome Biology 15:554.

    https://doi.org/10.1186/s13059-014-0554-4

    • PubMed
    • Google Scholar
24. 1. Li B
    2. Dewey CN

    (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome

    BMC Bioinformatics 12:323.

    https://doi.org/10.1186/1471-2105-12-323

    • PubMed
    • Google Scholar
25. 1. Liau BB
    2. Sievers C
    3. Donohue LK
    4. Gillespie SM
    5. Flavahan WA
    6. Miller TE
    7. Venteicher AS
    8. Hebert CH
    9. Carey CD
    10. Rodig SJ
    11. Shareef SJ
    12. Najm FJ
    13. van Galen P
    14. Wakimoto H
    15. Cahill DP
    16. Rich JN
    17. Aster JC
    18. Suvà ML
    19. Patel AP
    20. Bernstein BE

    (2017) Adaptive chromatin remodeling drives glioblastoma stem cell plasticity and drug tolerance

    Cell Stem Cell 20:233–246.

    https://doi.org/10.1016/j.stem.2016.11.003

    • PubMed
    • Google Scholar
26. 1. Liu T
    2. Yuan X
    3. Xu D

    (2016) Cancer-Specific telomerase reverse transcriptase (TERT) Promoter mutations: biological and clinical implications

    Genes 7:38.

    https://doi.org/10.3390/genes7070038

    • Google Scholar
27. 1. MacLeod G
    2. Bozek DA
    3. Rajakulendran N
    4. Monteiro V
    5. Ahmadi M
    6. Steinhart Z
    7. Kushida MM
    8. Yu H
    9. Coutinho FJ
    10. Cavalli FMG
    11. Restall I
    12. Hao X
    13. Hart T
    14. Luchman HA
    15. Weiss S
    16. Dirks PB
    17. Angers S

    (2019) Genome-Wide CRISPR-Cas9 screens expose genetic vulnerabilities and mechanisms of temozolomide sensitivity in glioblastoma stem cells

    Cell Reports 27:971–986.

    https://doi.org/10.1016/j.celrep.2019.03.047

    • PubMed
    • Google Scholar
28. 1. Meyer M
    2. Reimand J
    3. Lan X
    4. Head R
    5. Zhu X
    6. Kushida M
    7. Bayani J
    8. Pressey JC
    9. Lionel AC
    10. Clarke ID
    11. Cusimano M
    12. Squire JA
    13. Scherer SW
    14. Bernstein M
    15. Woodin MA
    16. Bader GD
    17. Dirks PB

    (2015) Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

    PNAS 112:851–856.

    https://doi.org/10.1073/pnas.1320611111

    • PubMed
    • Google Scholar
29. 1. Michaelson JJ
    2. Shin MK
    3. Koh JY
    4. Brueggeman L
    5. Zhang A
    6. Katzman A
    7. McDaniel L
    8. Fang M
    9. Pufall M
    10. Pieper AA

    (2017) Neuronal PAS domain proteins 1 and 3 are master regulators of neuropsychiatric risk genes

    Biological Psychiatry 82:213–223.

    https://doi.org/10.1016/j.biopsych.2017.03.021

    • PubMed
    • Google Scholar
30. 1. Miller TE
    2. Liau BB
    3. Wallace LC
    4. Morton AR
    5. Xie Q
    6. Dixit D
    7. Factor DC
    8. Kim LJY
    9. Morrow JJ
    10. Wu Q
    11. Mack SC
    12. Hubert CG
    13. Gillespie SM
    14. Flavahan WA
    15. Hoffmann T
    16. Thummalapalli R
    17. Hemann MT
    18. Paddison PJ
    19. Horbinski CM
    20. Zuber J
    21. Scacheri PC
    22. Bernstein BE
    23. Tesar PJ
    24. Rich JN

    (2017) Transcription elongation factors represent in vivo Cancer dependencies in glioblastoma

    Nature 547:355–359.

    https://doi.org/10.1038/nature23000

    • Google Scholar
31. 1. Morris TJ
    2. Butcher LM
    3. Feber A
    4. Teschendorff AE
    5. Chakravarthy AR
    6. Wojdacz TK
    7. Beck S

    (2014) ChAMP: 450k chip analysis methylation pipeline

    Bioinformatics 30:428–430.

    https://doi.org/10.1093/bioinformatics/btt684

    • PubMed
    • Google Scholar
32. 1. Müller S
    2. Cho A
    3. Liu SJ
    4. Lim DA
    5. Diaz A

    (2018) CONICS integrates scRNA-seq with DNA sequencing to map gene expression to tumor sub-clones

    Bioinformatics 34:3217–3219.

    https://doi.org/10.1093/bioinformatics/bty316

    • PubMed
    • Google Scholar
33. 1. Neftel C
    2. Laffy J
    3. Filbin MG
    4. Hara T
    5. Shore ME
    6. Rahme GJ
    7. Richman AR
    8. Silverbush D
    9. Shaw ML
    10. Hebert CM
    11. Dewitt J
    12. Gritsch S
    13. Perez EM
    14. Gonzalez Castro LN
    15. Lan X
    16. Druck N
    17. Rodman C
    18. Dionne D
    19. Kaplan A
    20. Bertalan MS
    21. Small J
    22. Pelton K
    23. Becker S
    24. Bonal D
    25. Nguyen QD
    26. Servis RL
    27. Fung JM
    28. Mylvaganam R
    29. Mayr L
    30. Gojo J
    31. Haberler C
    32. Geyeregger R
    33. Czech T
    34. Slavc I
    35. Nahed BV
    36. Curry WT
    37. Carter BS
    38. Wakimoto H
    39. Brastianos PK
    40. Batchelor TT
    41. Stemmer-Rachamimov A
    42. Martinez-Lage M
    43. Frosch MP
    44. Stamenkovic I
    45. Riggi N
    46. Rheinbay E
    47. Monje M
    48. Rozenblatt-Rosen O
    49. Cahill DP
    50. Patel AP
    51. Hunter T
    52. Verma IM
    53. Ligon KL
    54. Louis DN
    55. Regev A
    56. Bernstein BE
    57. Tirosh I
    58. Suvà ML

    (2019) An integrative model of cellular states, plasticity, and genetics for glioblastoma

    Cell 178:835–849.

    https://doi.org/10.1016/j.cell.2019.06.024

    • PubMed
    • Google Scholar
34. 1. O'Connor L
    2. Gilmour J
    3. Bonifer C

    (2016)

    The role of the ubiquitously expressed transcription factor Sp1 in Tissue-specific transcriptional regulation and in disease

    The Yale Journal of Biology and Medicine 89:513–525.

    • PubMed
    • Google Scholar
35. 1. Orzan F
    2. De Bacco F
    3. Crisafulli G
    4. Pellegatta S
    5. Mussolin B
    6. Siravegna G
    7. D'Ambrosio A
    8. Comoglio PM
    9. Finocchiaro G
    10. Boccaccio C

    (2017) Genetic evolution of glioblastoma Stem-Like cells from primary to recurrent tumor

    Stem Cells 35:2218–2228.

    https://doi.org/10.1002/stem.2703

    • PubMed
    • Google Scholar
36. 1. Park NI
    2. Guilhamon P
    3. Desai K
    4. McAdam RF
    5. Langille E
    6. O'Connor M
    7. Lan X
    8. Whetstone H
    9. Coutinho FJ
    10. Vanner RJ
    11. Ling E
    12. Prinos P
    13. Lee L
    14. Selvadurai H
    15. Atwal G
    16. Kushida M
    17. Clarke ID
    18. Voisin V
    19. Cusimano MD
    20. Bernstein M
    21. Das S
    22. Bader G
    23. Arrowsmith CH
    24. Angers S
    25. Huang X
    26. Lupien M
    27. Dirks PB

    (2017) ASCL1 reorganizes chromatin to direct neuronal fate and suppress tumorigenicity of glioblastoma stem cells

    Cell Stem Cell 21:209–224.

    https://doi.org/10.1016/j.stem.2017.06.004

    • PubMed
    • Google Scholar
37. 1. Patel AP
    2. Tirosh I
    3. Trombetta JJ
    4. Shalek AK
    5. Gillespie SM
    6. Wakimoto H
    7. Cahill DP
    8. Nahed BV
    9. Curry WT
    10. Martuza RL
    11. Louis DN
    12. Rozenblatt-Rosen O
    13. Suvà ML
    14. Regev A
    15. Bernstein BE

    (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma

    Science 344:1396–1401.

    https://doi.org/10.1126/science.1254257

    • PubMed
    • Google Scholar
38. 1. Pollard SM
    2. Yoshikawa K
    3. Clarke ID
    4. Danovi D
    5. Stricker S
    6. Russell R
    7. Bayani J
    8. Head R
    9. Lee M
    10. Bernstein M
    11. Squire JA
    12. Smith A
    13. Dirks P

    (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens

    Cell Stem Cell 4:568–580.

    https://doi.org/10.1016/j.stem.2009.03.014

    • PubMed
    • Google Scholar
39. Software

    1. R package survminer

    (2020) Drawing Survival Curves Using “Ggplot2”, version 0.4.6

    Drawing Survival Curves Using “Ggplot2”.

    http://finzi.psych.upenn.edu/library/survminer/html/ggsurvplot.html
40. 1. Rajakulendran N
    2. Rowland KJ
    3. Selvadurai HJ
    4. Ahmadi M
    5. Park NI
    6. Naumenko S
    7. Dolma S
    8. Ward RJ
    9. So M
    10. Lee L
    11. MacLeod G
    12. Pasiliao C
    13. Brandon C
    14. Clarke ID
    15. Cusimano MD
    16. Bernstein M
    17. Batada N
    18. Angers S
    19. Dirks PB

    (2019) Wnt and notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells

    Genes & Development 33:498–510.

    https://doi.org/10.1101/gad.321968.118

    • PubMed
    • Google Scholar
41. 1. Reimand J
    2. Arak T
    3. Adler P
    4. Kolberg L
    5. Reisberg S
    6. Peterson H
    7. Vilo J

    (2016) G:profiler-a web server for functional interpretation of gene lists (2016 update)

    Nucleic Acids Research 44:W83–W89.

    https://doi.org/10.1093/nar/gkw199

    • PubMed
    • Google Scholar
42. 1. Restall I
    2. Bozek DA
    3. Chesnelong C
    4. Weiss S
    5. Luchman HA

    (2018) Live-Cell imaging assays to study glioblastoma brain tumor stem cell migration and invasion

    Journal of Visualized Experiments 1:e58152.

    https://doi.org/10.3791/58152

    • Google Scholar
43. 1. Rheinbay E
    2. Suvà ML
    3. Gillespie SM
    4. Wakimoto H
    5. Patel AP
    6. Shahid M
    7. Oksuz O
    8. Rabkin SD
    9. Martuza RL
    10. Rivera MN
    11. Louis DN
    12. Kasif S
    13. Chi AS
    14. Bernstein BE

    (2013) An aberrant transcription factor network essential for wnt signaling and stem cell maintenance in glioblastoma

    Cell Reports 3:1567–1579.

    https://doi.org/10.1016/j.celrep.2013.04.021

    • PubMed
    • Google Scholar
44. Preprint

    1. Sallari RC
    2. Sinnott-Armstrong NA
    3. French JD
    4. Kron KJ
    5. Ho J
    6. Moore JH
    7. Stambolic V
    8. Edwards SL
    9. Lupien M
    10. Kellis M

    (2017) Convergence of dispersed regulatory mutations predicts driver genes in prostate Cancer

    bioRxiv.

    https://doi.org/10.1101/097451

    • Google Scholar
45. 1. Schep AN
    2. Wu B
    3. Buenrostro JD
    4. Greenleaf WJ

    (2017) chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data

    Nature Methods 14:975–978.

    https://doi.org/10.1038/nmeth.4401

    • PubMed
    • Google Scholar
46. 1. Schmitt AD
    2. Hu M
    3. Ren B

    (2016) Genome-wide mapping and analysis of chromosome architecture

    Nature Reviews Molecular Cell Biology 17:743–755.

    https://doi.org/10.1038/nrm.2016.104

    • PubMed
    • Google Scholar
47. 1. Shin H
    2. Liu T
    3. Manrai AK
    4. Liu XS

    (2009) CEAS: cis-regulatory element annotation system

    Bioinformatics 25:2605–2606.

    https://doi.org/10.1093/bioinformatics/btp479

    • PubMed
    • Google Scholar
48. 1. Stergachis AB
    2. Neph S
    3. Reynolds A
    4. Humbert R
    5. Miller B
    6. Paige SL
    7. Vernot B
    8. Cheng JB
    9. Thurman RE
    10. Sandstrom R
    11. Haugen E
    12. Heimfeld S
    13. Murry CE
    14. Akey JM
    15. Stamatoyannopoulos JA

    (2013) Developmental fate and cellular maturity encoded in human regulatory DNA landscapes

    Cell 154:888–903.

    https://doi.org/10.1016/j.cell.2013.07.020

    • PubMed
    • Google Scholar
49. 1. Stuart T
    2. Butler A
    3. Hoffman P
    4. Hafemeister C
    5. Papalexi E
    6. Mauck WM
    7. Hao Y
    8. Stoeckius M
    9. Smibert P
    10. Satija R

    (2019) Comprehensive Integration of Single-Cell Data

    Cell 177:1888–1902.

    https://doi.org/10.1016/j.cell.2019.05.031

    • Google Scholar
50. 1. Stupp R
    2. Mason WP
    3. van den Bent MJ
    4. Weller M
    5. Fisher B
    6. Taphoorn MJB
    7. Belanger K
    8. Brandes AA
    9. Marosi C
    10. Bogdahn U
    11. Curschmann J
    12. Janzer RC
    13. Ludwin SK
    14. Gorlia T
    15. Allgeier A
    16. Lacombe D
    17. Cairncross JG
    18. Eisenhauer E
    19. Mirimanoff RO

    (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma

    New England Journal of Medicine 352:987–996.

    https://doi.org/10.1056/NEJMoa043330

    • Google Scholar
51. 1. Subramanian A
    2. Tamayo P
    3. Mootha VK
    4. Mukherjee S
    5. Ebert BL
    6. Gillette MA
    7. Paulovich A
    8. Pomeroy SL
    9. Golub TR
    10. Lander ES
    11. Mesirov JP

    (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

    PNAS 102:15545–15550.

    https://doi.org/10.1073/pnas.0506580102

    • PubMed
    • Google Scholar
52. 1. Suvà ML
    2. Rheinbay E
    3. Gillespie SM
    4. Patel AP
    5. Wakimoto H
    6. Rabkin SD
    7. Riggi N
    8. Chi AS
    9. Cahill DP
    10. Nahed BV
    11. Curry WT
    12. Martuza RL
    13. Rivera MN
    14. Rossetti N
    15. Kasif S
    16. Beik S
    17. Kadri S
    18. Tirosh I
    19. Wortman I
    20. Shalek AK
    21. Rozenblatt-Rosen O
    22. Regev A
    23. Louis DN
    24. Bernstein BE

    (2014) Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells

    Cell 157:580–594.

    https://doi.org/10.1016/j.cell.2014.02.030

    • Google Scholar
53. 1. Tang Z
    2. Li C
    3. Kang B
    4. Gao G
    5. Li C
    6. Zhang Z

    (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses

    Nucleic Acids Research 45:W98–W102.

    https://doi.org/10.1093/nar/gkx247

    • Google Scholar
54. Software

    1. Therneau T

    (2015)

    A Package for Survival Analysis in S, version 2.38

    A Package for Survival Analysis in S.
55. 1. Trépant A-L
    2. Bouchart C
    3. Rorive S
    4. Sauvage S
    5. Decaestecker C
    6. Demetter P
    7. Salmon I

    (2015) Identification of OLIG2 as the most specific glioblastoma stem cell marker starting from comparative analysis of data from similar DNA chip microarray platforms

    Tumor Biology 36:1943–1953.

    https://doi.org/10.1007/s13277-014-2800-5

    • Google Scholar
56. 1. Venere M
    2. Fine HA
    3. Dirks PB
    4. Rich JN

    (2011) Cancer stem cells in gliomas: identifying and understanding the apex cell in Cancer's hierarchy

    Glia 59:1148–1154.

    https://doi.org/10.1002/glia.21185

    • PubMed
    • Google Scholar
57. 1. von Neubeck C
    2. Seidlitz A
    3. Kitzler HH
    4. Beuthien-Baumann B
    5. Krause M

    (2015) Glioblastoma multiforme: emerging treatments and stratification markers beyond new drugs

    The British Journal of Radiology 88:20150354.

    https://doi.org/10.1259/bjr.20150354

    • PubMed
    • Google Scholar
58. 1. Wang B
    2. Mezlini AM
    3. Demir F
    4. Fiume M
    5. Tu Z
    6. Brudno M
    7. Haibe-Kains B
    8. Goldenberg A

    (2014) Similarity network fusion for aggregating data types on a genomic scale

    Nature Methods 11:333–337.

    https://doi.org/10.1038/nmeth.2810

    • PubMed
    • Google Scholar
59. 1. Wang Q
    2. Hu B
    3. Hu X
    4. Kim H
    5. Squatrito M
    6. Scarpace L
    7. deCarvalho AC
    8. Lyu S
    9. Li P
    10. Li Y
    11. Barthel F
    12. Cho HJ
    13. Lin YH
    14. Satani N
    15. Martinez-Ledesma E
    16. Zheng S
    17. Chang E
    18. Sauvé CG
    19. Olar A
    20. Lan ZD
    21. Finocchiaro G
    22. Phillips JJ
    23. Berger MS
    24. Gabrusiewicz KR
    25. Wang G
    26. Eskilsson E
    27. Hu J
    28. Mikkelsen T
    29. DePinho RA
    30. Muller F
    31. Heimberger AB
    32. Sulman EP
    33. Nam DH
    34. Verhaak RGW

    (2017) Tumor evolution of Glioma-Intrinsic gene expression subtypes associates with immunological changes in the microenvironment

    Cancer Cell 32:42–56.

    https://doi.org/10.1016/j.ccell.2017.06.003

    • PubMed
    • Google Scholar
60. 1. Wang L
    2. Babikir H
    3. Müller S
    4. Yagnik G
    5. Shamardani K
    6. Catalan F
    7. Kohanbash G
    8. Alvarado B
    9. Di Lullo E
    10. Kriegstein A
    11. Shah S
    12. Wadhwa H
    13. Chang SM
    14. Phillips JJ
    15. Aghi MK
    16. Diaz AA

    (2019) The phenotypes of proliferating glioblastoma cells reside on a single Axis of variation

    Cancer Discovery 9:1708–1719.

    https://doi.org/10.1158/2159-8290.CD-19-0329

    • PubMed
    • Google Scholar
61. 1. Weirauch MT
    2. Yang A
    3. Albu M
    4. Cote AG
    5. Montenegro-Montero A
    6. Drewe P
    7. Najafabadi HS
    8. Lambert SA
    9. Mann I
    10. Cook K
    11. Zheng H
    12. Goity A
    13. van Bakel H
    14. Lozano JC
    15. Galli M
    16. Lewsey MG
    17. Huang E
    18. Mukherjee T
    19. Chen X
    20. Reece-Hoyes JS
    21. Govindarajan S
    22. Shaulsky G
    23. Walhout AJM
    24. Bouget FY
    25. Ratsch G
    26. Larrondo LF
    27. Ecker JR
    28. Hughes TR

    (2014) Determination and inference of eukaryotic transcription factor sequence specificity

    Cell 158:1431–1443.

    https://doi.org/10.1016/j.cell.2014.08.009

    • PubMed
    • Google Scholar
62. 1. Yin W
    2. Tang G
    3. Zhou Q
    4. Cao Y
    5. Li H
    6. Fu X
    7. Wu Z
    8. Jiang X

    (2019) Expression profile analysis identifies a novel Five-Gene signature to improve prognosis prediction of glioblastoma

    Frontiers in Genetics 10:419.

    https://doi.org/10.3389/fgene.2019.00419

    • PubMed
    • Google Scholar
63. 1. Zheng H
    2. Xie W

    (2019) The role of 3D genome organization in development and cell differentiation

    Nature Reviews Molecular Cell Biology 20:535–550.

    https://doi.org/10.1038/s41580-019-0132-4

    • PubMed
    • Google Scholar
64. 1. Zuo S
    2. Zhang X
    3. Wang L

    (2019) A RNA sequencing-based six-gene signature for survival prediction in patients with glioblastoma

    Scientific Reports 9:2615.

    https://doi.org/10.1038/s41598-019-39273-4

    • PubMed
    • Google Scholar

Author details

1. Paul Guilhamon

   1. Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
   2. Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8276-5987

2. Charles Chesnelong

   Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Michelle M Kushida

   Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Ana Nikolic

   1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
   2. Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada
   3. Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Divya Singhal

   1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
   2. Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada
   3. Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Graham MacLeod

   Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada

   Contribution

   Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6401-9307

7. Seyed Ali Madani Tonekaboni

   1. Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
   2. Department of Medical Biophysics, University of Toronto, Toronto, Canada

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

8. Florence MG Cavalli

   Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

   Contribution

   Data curation

   Competing interests

   No competing interests declared

9. Christopher Arlidge

   Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Nishani Rajakulendran

    Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada

    Contribution

    Resources

    Competing interests

    No competing interests declared

11. Naghmeh Rastegar

    Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

    Contribution

    Investigation

    Competing interests

    No competing interests declared

12. Xiaoguang Hao

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
    3. Department of Cell Biology & Anatomy, University of Calgary, Calgary, Canada

    Contribution

    Investigation

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2695-0111

13. Rozina Hassam

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
    3. Department of Cell Biology & Anatomy, University of Calgary, Calgary, Canada

    Contribution

    Investigation

    Competing interests

    No competing interests declared

14. Laura J Smith

    Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada

    Contribution

    Investigation, Visualization

    Competing interests

    No competing interests declared

15. Heather Whetstone

    Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

    Contribution

    Investigation

    Competing interests

    No competing interests declared

16. Fiona J Coutinho

    Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada

    Contribution

    Project administration

    Competing interests

    No competing interests declared

17. Bettina Nadorp

    Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

18. Katrina I Ellestad

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada

    Contribution

    Investigation

    Competing interests

    No competing interests declared

19. H Artee Luchman

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
    3. Department of Cell Biology & Anatomy, University of Calgary, Calgary, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

20. Jennifer Ai-wen Chan

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada
    3. Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Canada

    Contribution

    Resources

    Competing interests

    No competing interests declared

21. Molly S Shoichet

    Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1830-3475

22. Michael D Taylor

    1. Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada
    2. Division of Neurosurgery, University of Toronto, Toronto, Canada
    3. Departments of Molecular Genetics and Surgery, University of Toronto, Toronto, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

23. Benjamin Haibe-Kains

    1. Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
    2. Department of Medical Biophysics, University of Toronto, Toronto, Canada
    3. Department of Computer Science, University of Toronto, Toronto, Canada
    4. Ontario Institute for Cancer Research, Toronto, Canada
    5. Vector Institute, Toronto, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

24. Samuel Weiss

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
    3. Department of Cell Biology & Anatomy, University of Calgary, Calgary, Canada
    4. Department of Physiology & Pharmacology, University of Calgary, Calgary, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

25. Stephane Angers

    1. Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada
    2. Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Canada

    Contribution

    Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7241-9044

26. Marco Gallo

    1. Clark Smith Brain Tumour Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada
    2. Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada
    3. Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
    4. Department of Physiology & Pharmacology, University of Calgary, Calgary, Canada

    Contribution

    Supervision, Writing - review and editing

    Competing interests

    No competing interests declared

27. Peter B Dirks

    1. Developmental and Stem Cell Biology Program and Arthur and Sonia Labatt Brain tumor Research Centre, The Hospital for Sick Children, Toronto, Canada
    2. Division of Neurosurgery, University of Toronto, Toronto, Canada
    3. Ontario Institute for Cancer Research, Toronto, Canada

    Contribution

    Conceptualization, Supervision, Writing - original draft, Writing - review and editing

    For correspondence

    peter.dirks@sickkids.ca

    Competing interests

    No competing interests declared

28. Mathieu Lupien

    1. Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
    2. Department of Medical Biophysics, University of Toronto, Toronto, Canada

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    mlupien@uhnres.utoronto.ca

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0929-9478

• 8,507

  views

• 999

  downloads

• 57

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.