Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile, and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress due to downregulation of the E3 ligase RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.

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  1. 1.

    & Malignant gliomas in adults. N. Engl. J. Med. 359, 492–507 (2008).

  2. 2.

    et al. Identification of human brain tumour initiating cells. Nature 432, 396–401 (2004).

  3. 3.

    et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 64, 7011–7021 (2004).

  4. 4.

    et al. Blockade of EGFR signaling promotes glioma stem-like cell invasiveness by abolishing ID3-mediated inhibition of p27KIP1 and MMP3 expression. Cancer Lett. 328, 235–242 (2013).

  5. 5.

    et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760 (2006).

  6. 6.

    et al. Stem cell–like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res. 66, 7843–7848 (2006).

  7. 7.

    et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 15, 501–513 (2009).

  8. 8.

    et al. A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69–82 (2007).

  9. 9.

    et al. Acidic stress promotes a glioma stem cell phenotype. Cell Death Differ. 18, 829–840 (2011).

  10. 10.

    et al. Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake. Nat. Neurosci. 16, 1373–1382 (2013).

  11. 11.

    et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).

  12. 12.

    et al. Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma. PLoS One 9, e103327 (2014).

  13. 13.

    et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014).

  14. 14.

    et al. Single cell–derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Proc. Natl. Acad. Sci. USA 112, 851–856 (2015).

  15. 15.

    et al. In vivo radiation response of proneural glioma characterized by protective p53 transcriptional program and proneural–mesenchymal shift. Proc. Natl. Acad. Sci. USA 111, 5248–5253 (2014).

  16. 16.

    et al. Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma. Cancer Cell 24, 331–346 (2013).

  17. 17.

    et al. Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3. Proc. Natl. Acad. Sci. USA 110, 8644–8649 (2013).

  18. 18.

    et al. Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ. Cell Rep. 3, 260–273 (2013).

  19. 19.

    et al. Identification of a SOX2-dependent subset of tumor- and sphere-forming glioblastoma cells with a distinct tyrosine kinase inhibitor sensitivity profile. Neuro Oncol. 13, 1178–1191 (2011).

  20. 20.

    et al. Vascular progenitors from cord blood–derived iPSC possess augmented capacity for regenerating ischemic retinal vasculature. Circulation 129, 359–372 (2014).

  21. 21.

    et al. In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection. Cancer Res. 71, 779–789 (2011).

  22. 22.

    et al. Large neutral amino acid transporter enables brain drug delivery via prodrugs. J. Med. Chem. 51, 932–936 (2008).

  23. 23.

    et al. Endothelial cells of the human microvasculature express epidermal fatty acid–binding protein. Circ. Res. 81, 297–303 (1997).

  24. 24.

    et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379, 88–91 (1996).

  25. 25.

    , & Strategies to improve radiotherapy with targeted drugs. Nat. Rev. Cancer 11, 239–253 (2011).

  26. 26.

    et al. RYBP–PRC1 complexes mediate H2A ubiquitylation at Polycomb target sites independently of PRC2 and H3K27me3. Cell 148, 664–678 (2012).

  27. 27.

    , , , & SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4, 440–452 (2009).

  28. 28.

    et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004).

  29. 29.

    et al. The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood 107, 2170–2179 (2006).

  30. 30.

    et al. BMI-1 promotes Ewing sarcoma tumorigenicity independent of CDKN2A repression. Cancer Res. 68, 6507–6515 (2008).

  31. 31.

    et al. Regulation of tumor angiogenesis by EZH2. Cancer Cell 18, 185–197 (2010).

  32. 32.

    , , & RNF144A, an E3 ubiquitin ligase for DNA-PKcs, promotes apoptosis during DNA damage. Proc. Natl. Acad. Sci. USA 111, E2646–E2655 (2014).

  33. 33.

    et al. ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors. Int. J. Cancer 137, 2007–2018 (2015).

  34. 34.

    et al. Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution. Oncogene 28, 1807–1811 (2009).

  35. 35.

    et al. Clonal variation in drug and radiation response among glioma-initiating cells is linked to proneural–mesenchymal transition. Cell Rep. 17, 2994–3009 (2016).

  36. 36.

    et al. Nicotinamide metabolism regulates glioblastoma stem cell maintenance. JCI Insight 2, 90019 (2017).

  37. 37.

    & Making a tumour's bed: glioblastoma stem cells and the vascular niche. Nat. Rev. Cancer 7, 733–736 (2007).

  38. 38.

    et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013).

  39. 39.

    et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479–483 (2012).

  40. 40.

    et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340, 626–630 (2013).

  41. 41.

    The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev. 27, 985–990 (2013).

  42. 42.

    et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat. Genet. 44, 251–253 (2012).

  43. 43.

    et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482, 226–231 (2012).

  44. 44.

    et al. DNA methylation and somatic mutations converge on the cell cycle and define similar evolutionary histories in brain tumors. Cancer Cell 28, 307–317 (2015).

  45. 45.

    et al. Bmi1 controls tumor development in an Ink4a/Arf-independent manner in a mouse model for glioma. Cancer Cell 12, 328–341 (2007).

  46. 46.

    et al. BMI1 sustains human glioblastoma multiforme stem cell renewal. J. Neurosci. 29, 8884–8896 (2009).

  47. 47.

    et al. Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res. 68, 9125–9130 (2008).

  48. 48.

    et al. Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells. Stem Cell Res. 8, 141–153 (2012).

  49. 49.

    et al. In vivo RNAi screen for BMI1 targets identifies TGF-β/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis. Cancer Cell 23, 660–676 (2013).

  50. 50.

    et al. Self-renewal as a therapeutic target in human colorectal cancer. Nat. Med. 20, 29–36 (2014).

  51. 51.

    et al. Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cell 13, 69–80 (2008).

  52. 52.

    et al. EZH2 is essential for glioblastoma cancer stem cell maintenance. Cancer Res. 69, 9211–9218 (2009).

  53. 53.

    et al. Phosphorylation of EZH2 activates STAT3 signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma stem-like cells. Cancer Cell 23, 839–852 (2013).

  54. 54.

    et al. Inhibition of EZH2 reverses chemotherapeutic drug TMZ chemosensitivity in glioblastoma. Int. J. Clin. Exp. Pathol. 7, 6662–6670 (2014).

  55. 55.

    et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat. Med. 23, 483–492 (2017).

  56. 56.

    et al. Therapeutic targeting of Polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. Nat. Med. 23, 493–500 (2017).

  57. 57.

    , , & BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J. Neurosci. 30, 10096–10111 (2010).

  58. 58.

    et al. Combined aberrant expression of Bmi1 and EZH2 is predictive of poor prognosis in glioma patients. J. Neurol. Sci. 335, 191–196 (2013).

  59. 59.

    et al. Tumor evolution of glioma intrinsic gene expression subtype associates with immunological changes in the microenvironment. Preprint at bioRxiv (2016).

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We thank PTC Therapeutics for providing PTC209 and PTC596, as well as performing measurement of drug levels. We thank E.P. Sulman (MD Anderson Cancer Center) and A.W. Boyd (Queensland Institute of Medical Research) for subtype-characterized GSCs (PN11, PN23, PN-JK2, PN-MMK1, MES20, MES28 and MES-MN1). We would like to thank N. DeWitt for editorial assistance, as well as the Cleveland Clinic Lerner Research Institute imaging core and proteomics core service teams. We also thank members of J.N.R.'s lab for input about the manuscript. Finally, we would like to thank our funding sources: the National Institutes of Health grants CA203101 (L.K.); CA183510 (T.E.M.); CA217065 (R.C.G.); CA217066 (B.C.P.); CA043703 (J.S.B.-S.); CA169117, CA184090, NS091080 and NS099175 (S.B.); CA197718, CA154130, CA169117, CA171652, NS087913 and NS089272 (J.N.R.); the Peter D. Cristal Chair, the Kimble Family Foundation, the Ferry Foundation, the Jerry Kaufman GBM Research Fund, and CA217956 (A.E.S.); the General Program of the National Natural Science Foundation of China (81572891) (X.J.); Canadian Institutes of Health Research Banting Fellowship (S.C.M.).

Author information


  1. Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

    • Xun Jin
    • , Leo J Y Kim
    • , Qiulian Wu
    • , Lisa C Wallace
    • , Briana C Prager
    • , Tanwarat Sanvoranart
    • , Ryan C Gimple
    • , Xiuxing Wang
    • , Stephen C Mack
    • , Tyler E Miller
    • , Ping Huang
    • , Claudia L Valentim
    • , Qi-gang Zhou
    • , Shideng Bao
    •  & Jeremy N Rich
  2. Tianjin Medical University Cancer Institute and Hospital, Tianjin, P.R. China.

    • Xun Jin
  3. First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.

    • Xun Jin
  4. Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.

    • Leo J Y Kim
    • , Briana C Prager
    • , Ryan C Gimple
    •  & Tyler E Miller
  5. Medical Scientist Training Program, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.

    • Leo J Y Kim
    • , Briana C Prager
    • , Ryan C Gimple
    •  & Tyler E Miller
  6. Division of Regenerative Medicine, Department of Medicine, University of San Diego, San Diego, California, USA.

    • Leo J Y Kim
    • , Qiulian Wu
    • , Briana C Prager
    • , Ryan C Gimple
    • , Xiuxing Wang
    •  & Jeremy N Rich
  7. Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, USA.

    • Briana C Prager
    • , Shideng Bao
    •  & Jeremy N Rich
  8. Department of Pediatrics, Division of Pediatric Hematology and Oncology, Baylor College of Medicine, Houston, Texas, USA.

    • Stephen C Mack
  9. Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

    • Jill S Barnholtz-Sloan
    • , Shideng Bao
    • , Andrew E Sloan
    •  & Jeremy N Rich
  10. Department of Neurological Surgery, University Hospitals–Cleveland Medical Center, Cleveland, Ohio, USA.

    • Andrew E Sloan


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X.J. and J.N.R. designed the overall experiments, analyzed data, and wrote the manuscript. X.J., L.J.Y.K., L.C.W., T.S., S.C.M., T.E.M., Q.W., P.H., X.W., C.L.V., and Q.Z. performed cell culture and/or animal experiments. X.J., L.J.Y.K., B.C.P., R.C.G., and S.C.M. performed bioinformatics analysis of published expression data sets. J.S.B.-S., S.B., and A.E.S. provided intellectual input and patient tissues. All authors provided scientific input, edited and approved the final manuscript.

Competing interests

J.N.R. received an honorarium from PTC Therapeutics as an advisory board member.

Corresponding authors

Correspondence to Andrew E Sloan or Jeremy N Rich.

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