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• W3048798848 abstract "•H3.3K27M mutations enhance glucose, glutamine, and TCA cycle metabolism•TCA cycle intermediate α-KG enables maintenance of H3K27 hypomethylation•Targeting enzymes related to α-KG synthesis including WT-IDH1 and/or GDH is therapeutic•H3.3K27M and mutant-IDH1 in gliomas are mutually exclusive and are synthetic lethal H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments. H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments. H3K27M-mutant gliomas, including diffuse intrinsic pontine gliomas (DIPGs), are lethal childhood brain tumors. DIPGs are inoperable due to their location within the pons/brainstem. Available treatment options, including chemo-/radiotherapy, are ineffective, and over 90% of patients die within 1.5 years of diagnosis (Morales La Madrid et al., 2015Morales La Madrid A. Hashizume R. Kieran M.W. Future clinical trials in DIPG: bringing epigenetics to the clinic.Front. Oncol. 2015; 5: 148Crossref PubMed Scopus (34) Google Scholar). Over 80% of DIPGs harbor recurrent mutations in histone H3-H3F3A and HIST1H3B/C (∼25%) where the lysine at position 27 is replaced by methionine (collectively H3K27M) (Fontebasso et al., 2013Fontebasso A.M. Liu X.Y. Sturm D. Jabado N. Chromatin remodeling defects in pediatric and young adult glioblastoma: a tale of a variant histone 3 tail.Brain Pathol. 2013; 23: 210-216Crossref PubMed Scopus (59) Google Scholar; Wu et al., 2012Wu G. Broniscer A. McEachron T.A. Lu C. Paugh B.S. Becksfort J. Qu C. Ding L. Huether R. Parker M. et al.Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas.Nat. Genet. 2012; 44: 251-253Crossref PubMed Scopus (923) Google Scholar). H3K27M mutations result in a global H3K27me3 reduction via multiple mechanisms, including aberrant PRC2 interactions and hampered H3K27me3 spreading (Bender et al., 2013Bender S. Tang Y. Lindroth A.M. Hovestadt V. Jones D.T. Kool M. Zapatka M. Northcott P.A. Sturm D. Wang W. et al.Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas.Cancer Cell. 2013; 24: 660-672Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar; Chan et al., 2013Chan K.M. Fang D. Gan H. Hashizume R. Yu C. Schroeder M. Gupta N. Mueller S. James C.D. Jenkins R. et al.The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression.Genes Dev. 2013; 27: 985-990Crossref PubMed Scopus (392) Google Scholar; Harutyunyan et al., 2019Harutyunyan A.S. Krug B. Chen H. Papillon-Cavanagh S. Zeinieh M. De Jay N. Deshmukh S. Chen C.C.L. Belle J. Mikael L.G. et al.H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.Nat. Commun. 2019; 10: 1262Crossref PubMed Scopus (64) Google Scholar; Lewis et al., 2013Lewis P.W. Muller M.M. Koletsky M.S. Cordero F. Lin S. Banaszynski L.A. Garcia B.A. Muir T.W. Becher O.J. Allis C.D. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma.Science. 2013; 340: 857-861Crossref PubMed Scopus (709) Google Scholar; Stafford et al., 2018Stafford J.M. Lee C.H. Voigt P. Descostes N. Saldana-Meyer R. Yu J.R. Leroy G. Oksuz O. Chapman J.R. Suarez F. et al.Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma.Sci. Adv. 2018; 4: eaau5935Crossref PubMed Scopus (60) Google Scholar). Epigenetic approaches, including increasing global H3K27me3 in H3K27M cells, are a key therapeutic strategy leading to cell death of H3K27M cells in vitro and in vivo (Anastas et al., 2019Anastas J.N. Zee B.M. Kalin J.H. Kim M. Guo R. Alexandrescu S. Blanco M.A. Giera S. Gillespie S.M. Das J. et al.Re-programing chromatin with a bifunctional LSD1/HDAC inhibitor induces therapeutic differentiation in DIPG.Cancer Cell. 2019; 36: 528-544.e10Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar; Grasso et al., 2015Grasso C.S. Tang Y. Truffaux N. Berlow N.E. Liu L. Debily M.A. Quist M.J. Davis L.E. Huang E.C. Woo P.J. et al.Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.Nat. Med. 2015; 21: 555-559Crossref PubMed Scopus (283) Google Scholar; Hashizume et al., 2014Hashizume R. Andor N. Ihara Y. Lerner R. Gan H. Chen X. Fang D. Huang X. Tom M.W. Ngo V. et al.Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma.Nat. Med. 2014; 20: 1394-1396Crossref PubMed Scopus (285) Google Scholar; Krug et al., 2019Krug B. De Jay N. Harutyunyan A.S. Deshmukh S. Marchione D.M. Guilhamon P. Bertrand K.C. Mikael L.G. McConechy M.K. Chen C.C.L. et al.Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas.Cancer Cell. 2019; 35: 782-797.e8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; Mohammad et al., 2017Mohammad F. Weissmann S. Leblanc B. Pandey D.P. Hojfeldt J.W. Comet I. Zheng C. Johansen J.V. Rapin N. Porse B.T. et al.EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas.Nat. Med. 2017; 23: 483-492Crossref PubMed Scopus (217) Google Scholar; Nagaraja et al., 2017Nagaraja S. Vitanza N.A. Woo P.J. Taylor K.R. Liu F. Zhang L. Li M. Meng W. Ponnuswami A. Sun W. et al.TraNSCriptional dependencies in diffuse intrinsic pontine glioma.Cancer Cell. 2017; 31: 635-652.e6Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar; Piunti et al., 2017Piunti A. Hashizume R. Morgan M.A. Bartom E.T. Horbinski C.M. Marshall S.A. Rendleman E.J. Ma Q. Takahashi Y.H. Woodfin A.R. et al.Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas.Nat. Med. 2017; 23: 493-500Crossref PubMed Scopus (177) Google Scholar). While H3K27M mutations are thought to suppress neuroglial differentiation through deregulation of epigenetic modifications, it is not known what additional mechanisms, if any, drive pathogenesis. Altered metabolism is a universal hallmark of cancer. Tumor cells take up and metabolize nutrients such as glucose and glutamine to support key anabolic processes and are actively driven by oncogenes and inactivated tumor suppressors (Venneti and Thompson, 2017Venneti S. Thompson C.B. Metabolic reprogramming in brain tumors.Annu. Rev. Pathol. 2017; 12: 515-545Crossref PubMed Scopus (43) Google Scholar). We hypothesized that H3K27M mutations rewire both glucose and glutamine metabolism. We also set out to determine if metabolic and epigenetic pathways are integrated in H3K27M tumors. Metabolic regulation of epigenetic modifications has been demonstrated in mIDH1/2 gliomas. Wild-type (WT) IDH1/2 converts isocitrate to α-KG, which serves as a co-factor for α-KG-dependent dioxygenases, including DNA demethylases and the Jumanji family of histone lysine demethylases (KDM) (Loenarz and Schofield, 2008Loenarz C. Schofield C.J. Expanding chemical biology of 2-oxoglutarate oxygenases.Nat. Chem. Biol. 2008; 4: 152-156Crossref PubMed Scopus (366) Google Scholar). Gain-of-function mIDH1/2 metabolizes α-KG to D-2HG. D-2HG is structurally similar to α-KG and blocks its function, resulting in global hypermethylation of CpG islands (CpGi) and histone residues, including H3K27me3 (Duncan et al., 2012Duncan C.G. Barwick B.G. Jin G. Rago C. Kapoor-Vazirani P. Powell D.R. Chi J.T. Bigner D.D. Vertino P.M. Yan H. A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation.Genome Res. 2012; 22: 2339-2355Crossref PubMed Scopus (131) Google Scholar; Losman and Kaelin, 2013Losman J.-A. Kaelin W.G. What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer.Genes Dev. 2013; 27: 836-852Crossref PubMed Scopus (356) Google Scholar; Lu et al., 2012Lu C. Ward P.S. Kapoor G.S. Rohle D. Turcan S. Abdel-Wahab O. Edwards C.R. Khanin R. Figueroa M.E. Melnick A. et al.IDH mutation impairs histone demethylation and results in a block to cell differentiation.Nature. 2012; 483: 474-478Crossref PubMed Scopus (1228) Google Scholar; Sasaki et al., 2012Sasaki M. Knobbe C.B. Munger J.C. Lind E.F. Brenner D. Brustle A. Harris I.S. Holmes R. Wakeham A. Haight J. et al.IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.Nature. 2012; 488: 656-659Crossref PubMed Scopus (361) Google Scholar; Turcan et al., 2012Turcan S. Rohle D. Goenka A. Walsh L.A. Fang F. Yilmaz E. Campos C. Fabius A.W. Lu C. Ward P.S. et al.IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype.Nature. 2012; 483: 479-483Crossref PubMed Scopus (1227) Google Scholar). Based on this premise, we also hypothesized that global H3K27me3 levels are metabolically regulated in H3K27M cells, and understanding these pathways could uncover therapeutic opportunities for treatment. We used an integrated approach to comprehensively determine metabolic alterations driven by H3.3K27M mutations using paired isogenic cell lines expressing H3.3K27M or H3.3WT, patient-derived tumor cell lines, and tumor samples. We expressed either H3.3K27M or H3.3WT in immortalized mouse neuronal stem cells (NSCs) (Johnson et al., 2010Johnson R.A. Wright K.D. Poppleton H. Mohankumar K.M. Finkelstein D. Pounds S.B. Rand V. Leary S.E. White E. Eden C. et al.Cross-species genomics matches driver mutations and cell compartments to model ependymoma.Nature. 2010; 466: 632-636Crossref PubMed Scopus (266) Google Scholar). H3.3K27M NSCs exhibited global decreased H3K27me3 and an increase in the opposing mark H3K27ac without changes in global H3K36me3, H3K4me3, or H3K4me1 levels (Figures 1A and S1A). H3.3K27M NSCs showed increased proliferation compared with H3.3WT cells (Figure S1B). H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) confirmed global genomic H3K27me3 reduction in H3.3K27M versus H3.3WT NSCs (Figure 1B). RNA sequencing (RNA-seq) revealed many differentially regulated genes in H3.3K27M versus H3.3WT NSCs, including downregulation of genes related to neuronal differentiation by gene set enrichment analysis (GSEA) (Figures 1C). Some of the most significantly upregulated pathways in H3.3K27M NSCs were related to carbohydrate metabolism and tricarboxylic acid (TCA) cycle regulation (Figure 1C). Similar results were observed in independent isogenic mouse cell lines expressing H3.3K27M or H3.3WT (Patel et al., 2019Patel S.K. Hartley R.M. Wei X. Furnish R. Escobar-Riquelme F. Bear H. Choi K. Fuller C. Phoenix T.N. Generation of diffuse intrinsic pontine glioma mouse models by brainstem targeted in utero electroporation.Neuro Oncol. 2019; 22: 381-392Google Scholar) (Figure S1C). We used an unbiased approach by determining overall changes in the proteome and metabolome in H3.3K27M versus H3.3WT NSCs. H3.3K27M showed differential regulation of 1,069 (503 upregulated, 566 downregulated) proteins compared with H3.3WT NSCs. Pathway analysis of the 503 upregulated proteins revealed glycolysis and the TCA cycle as the top upregulated pathways (Figures 1D). Metabolite analysis revealed 78 metabolites that were differentially regulated (48 upregulated, 30 downregulated) between H3.3K27M and H3.3WT NSCs (Figure 1E). Enrichment analysis of the 48 upregulated metabolites corroborated our proteomic findings to reveal upregulation of key glycolysis and TCA cycle-associated metabolites, including pyruvate, lactate, and α-KG in H3.3K27M versus H3.3WT NSCs (Figures 1E–1G). We confirmed upregulation of key proteins related to glucose, glutamine, and TCA cycle metabolism in a panel of low-passage, patient-derived cell lines. The glucose transporter SLC2A3 (GLUT3), hexokinase 2 (HK2), IDH1, and glutamate dehydrogenase (GDH) were expressed at higher levels in H3.3K27M compared with H3WT human cell lines (Figures 1G and 1H). As controls, GAPDH, GOT1, and the cysteine/glutamate antiporter SLC7A11 were relatively unchanged in H3.3K27M versus H3WT cells (Figures 1H and S1D). Because glucose and glutamine are major carbon sources in glycolysis and the TCA cycle, we used isotope tracing with 13C-uniformly labeled glucose and glutamine in vitro in both isogenic NSCs and patient-derived cell lines. Isotope tracing revealed both increased glycolysis (Figures S1E–S1G) and glutaminolysis (Figures S1H–S1J) in H3.3K27M versus H3WT cells. We evaluated the significance of our findings for patient tumors using multiple approaches. We queried data from a gene expression repository (PedcBioPortal) that included H3K27M (n = 83), H3WT (n = 101), and histone H3G34R/V hemispheric (n = 19) pediatric high-grade gliomas (Mackay et al., 2017Mackay A. Burford A. Carvalho D. Izquierdo E. Fazal-Salom J. Taylor K.R. Bjerke L. Clarke M. Vinci M. Nandhabalan M. et al.Integrated molecular meta-analysis of 1,000 pediatric high-grade and diffuse intrinsic pontine glioma.Cancer Cell. 2017; 32: 520-537.e5Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Our analysis revealed increased expression of SLC2A3, several glycolytic enzymes, including HK2, and GLUD1/2 (encoding GDH) in H3K27M versus either H3WT or H3G34R/V tumors (Figure 1I). We grouped high-grade pediatric gliomas, independent of H3K27M status, into high and low expression subgroups based on median expression value for SLC2A3/GLUT3, HK2, GLUD1, and PFKFB2. High expression tumors mainly localized to the pons/brainstem versus hemispheric regions, consistent with the primary location of H3K27M gliomas (Figure S2A). High expression of glycolysis-KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway genes, or HK2 or PFKFB2, was associated with poor overall prognosis independent of H3K27M status (Figures S2B–S2D). Expression levels of SLC2A3 and enzymes including HK2, IDH1, and GLUD1 were significantly higher in malignant compared with non-malignant cells in single-cell (sc) RNA-seq analyses from H3K27M tumors (Filbin et al., 2018Filbin M.G. Tirosh I. Hovestadt V. Shaw M.L. Escalante L.E. Mathewson N.D. Neftel C. Frank N. Pelton K. Hebert C.M. et al.Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq.Science. 2018; 360: 331-335Crossref PubMed Scopus (174) Google Scholar) (Figure 1J). As observed in patient-derived cell lines, GAPDH, SCL7A11, and GOT1 levels were not significantly different between malignant and non-malignant cells (Figure S2E). Malignant cells in H3K27M tumors are heterogeneous and include highly proliferative oligodendrocyte precursor (OPC)-like cells along with oligodendrocyte (OC)-like and astrocyte (AC)-like cells (Filbin et al., 2018Filbin M.G. Tirosh I. Hovestadt V. Shaw M.L. Escalante L.E. Mathewson N.D. Neftel C. Frank N. Pelton K. Hebert C.M. et al.Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq.Science. 2018; 360: 331-335Crossref PubMed Scopus (174) Google Scholar). All three subtypes of malignant cells showed high expression of IDH1 and GLUD1 compared with HK2 and SLC2A3 (Figure 1K). To assess potential mechanisms of enhanced metabolism, we interrogated master regulators of glucose/glutamine metabolism, including activation of mTor, N-Myc, C-Myc, and Hif-1α. Western blotting did not reveal elevation of these factors, including Hif-1α in H3.3K27M versus H3.3WT NSCs (Figures S2F–S2G). H3K27M tumor cells exhibit various epigenetic alterations at several gene promoters that can regulate gene expression. These include global reduction in H3K27me3, elevated H3K27ac levels, and enrichment of the activating mark H3K4me3 (including bivalent H3K4me3/H3K27me3 at neurodevelopment-related gene promoters; Figure S2H) (Bender et al., 2013Bender S. Tang Y. Lindroth A.M. Hovestadt V. Jones D.T. Kool M. Zapatka M. Northcott P.A. Sturm D. Wang W. et al.Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas.Cancer Cell. 2013; 24: 660-672Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar; Harutyunyan et al., 2019Harutyunyan A.S. Krug B. Chen H. Papillon-Cavanagh S. Zeinieh M. De Jay N. Deshmukh S. Chen C.C.L. Belle J. Mikael L.G. et al.H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.Nat. Commun. 2019; 10: 1262Crossref PubMed Scopus (64) Google Scholar; Krug et al., 2019Krug B. De Jay N. Harutyunyan A.S. Deshmukh S. Marchione D.M. Guilhamon P. Bertrand K.C. Mikael L.G. McConechy M.K. Chen C.C.L. et al.Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas.Cancer Cell. 2019; 35: 782-797.e8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; Larson et al., 2019Larson J.D. Kasper L.H. Paugh B.S. Jin H. Wu G. Kwon C.H. Fan Y. Shaw T.I. Silveira A.B. Qu C. et al.Histone H3.3 K27M accelerates spontaneous brainstem glioma and drives restricted changes in bivalent gene expression.Cancer Cell. 2019; 35: 140-155.e7Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar; Piunti et al., 2017Piunti A. Hashizume R. Morgan M.A. Bartom E.T. Horbinski C.M. Marshall S.A. Rendleman E.J. Ma Q. Takahashi Y.H. Woodfin A.R. et al.Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas.Nat. Med. 2017; 23: 493-500Crossref PubMed Scopus (177) Google Scholar). ChIP-seq analyses showed that promoters for Slc2a3, Hk2, Glud1, and Idh1 were enriched for the activation mark H3K4me3 and excluded H3K27me3 in H3.3K27M versus H3.3WT NSCs (Figure 1L). Slc2a3 was also enriched for H3K27ac in H3.3K27M compared with H3.3WT NSCs (Figure 1L). H3K4me1-enriched sites (enhancers) did not show changes near these gene loci (Figure 1L). Consistent with our control western blot data (Figures 1H and S1C), Gapdh, Got1, and Slc7a11 did not show differences in enrichment between H3.3K27M and H3.3WT NSCs for any of these marks (Figure S2I). The histone H3 mark enrichments at Slc2a3 in our NSC model system also aligned with earlier ChIP-seq studies in three human H3K27M DIPG cell lines (Piunti et al., 2017Piunti A. Hashizume R. Morgan M.A. Bartom E.T. Horbinski C.M. Marshall S.A. Rendleman E.J. Ma Q. Takahashi Y.H. Woodfin A.R. et al.Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas.Nat. Med. 2017; 23: 493-500Crossref PubMed Scopus (177) Google Scholar) (Figure S2J). Our data from multiple models, including H3.3K27M versus H3.3WT isogenic cells, patient tumor cell lines, and bulk and single-cell RNA-seq analyses demonstrate that H3K27M cells show enhanced glycolysis and TCA cycle metabolism. We used non-invasive, in vivo magnetic resonance spectroscopy (MRS) imaging to assess metabolite levels in patients with high-grade midline gliomas in a retrospective and blinded manner in 15 patients. As observed in midline gliomas, 73% (11/15) were histopathologically confirmed to be H3K27M (n = 11), and 27% (4/15) were H3WT midline gliomas (Figure 2A). Metabolites, including glutamine (Gln), glutamate (Glu), myoinositol (mI), glycine (Gly), glucose (Glc), choline (Cho), citrate (Cit), alanine (Ala), and lactate (Lac), were assessed in all tumors. H3K27M midline gliomas contained significantly higher levels of citrate (p = 0.0029) and glutamine (p = 0.0227) compared with H3WT tumors (Figures 2B and 2C). Overall, in vivo MRS imaging data paralleled our gene expression, protein, and metabolite data derived from cell lines and tumor tissues. From our metabolomic studies, we noted that α-KG levels were higher in H3.3K27M compared with H3.3WT cells (Figure 1F). Histone KDMs, including the H3K27 demethylases KDM6A/6B, use α-KG as a crucial co-factor, metabolizing it to succinate (Suc), while demethylating H3K27me3 (Figure 3A) (Loenarz and Schofield, 2008Loenarz C. Schofield C.J. Expanding chemical biology of 2-oxoglutarate oxygenases.Nat. Chem. Biol. 2008; 4: 152-156Crossref PubMed Scopus (366) Google Scholar). A high α-KG/Suc ratio favors H3K27me3 demethylation (Carey et al., 2015Carey B.W. Finley L.W. Cross J.R. Allis C.D. Thompson C.B. Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (455) Google Scholar), and H3.3K27M cells showed high α-KG/Suc ratios (Figure S3A). We therefore hypothesized that α-KG could regulate H3K27me3 levels in H3.3K27M cells. α-KG can be derived from both glutamine and glucose metabolism. Glucose carbons that enter the TCA cycle to generate citrate/isocitrate can give rise to α-KG via enzymatic activity of IDH1/2, while GDH metabolizes glutamine-derived glutamate to α-KG (Figure 3A). To test if glutamine metabolism regulates H3K27me3 levels, we used a panel of cells, including H3.3K27M NSC and patient-derived H3.3K27M and H3.1K27M cells. Glutamine deprivation from cell culture media resulted in increased H3K27me3 levels in H3.3K27M NSCs, DIPG-007, and DIPG-IV cells but not SF7761 and DIPG-XIII∗P cells (Figure 3B and S3A–S3N). This effect in H3K27M NSCs was rescued by addition of cell-permeable α-KG (Figure 3C). Glutamine withdrawal did not significantly affect proliferation of H3.3WT NSCs, but it inhibited that of H3.3K27M NSCs, an effect reversed by downstream metabolites α-KG or glutamate (Figures 3D, S3B, and S3C). In human cell lines, DIPG-007 cells were more sensitive to glutamine withdrawal than SF7761 cells and showed a partial decrease in proliferation that was reversed by α-KG (Figure 3E). We next tested whether glucose metabolism regulated H3K27me3. Glucose withdrawal did not alter H3K27me3 levels in H3.3K27M and H3.3WT NSCs, but it increased H3K27me3 levels in DIPG-007, SF7761, DIPG-IV, and DIPG-XIII∗P cells (Figures 3F and S3D–S3L). This effect was observed as early as 24 h after glucose withdrawal and was rescued by α-KG in DIPG-007 cells (Figures 3G, S3E, and S3F). Moreover, partial glucose withdrawal was sufficient to increase H3K27me3 levels in SF7661 but not in DIPG-007 cells (Figure S3G). This observation was borne out in cell viability studies, where maximal reduction in DIPG-007 cell numbers required both glucose and glutamine withdrawal but was achieved in SF7761 cells on glucose withdrawal alone (Figure 3H). Moreover, α-KG partially rescued viability on glucose withdrawal in both DIPG-007 and SF7761 cells but not in H3.3K27M NSCs (Figures 3H and S3H). Glucose and glutamine withdrawal did not alter H3K27me3 levels in H3WT SJGBM2 cells (Figure S3I). Overall, the impact of glutamine and glucose withdrawal on H3K27me3 levels showed both heterogeneity and redundancy among the tested cell lines (Figure 3I). Our data suggest that irrespective of the metabolic pathway used, α-KG is critical for maintaining low H3K27me3 in H3.3K27M cells. We verified this observation by adding cell-permeable α-KG to increase the α-KG/Suc ratio or cell-permeable Suc to lower the α-KG/Suc ratio in H3.3K27M NSCs. Global H3K27me3 levels were further lowered on α-KG addition but were increased on Suc treatment (Figures 3J and S3M). As anticipated, addition of Suc to H3.3K27M NSCs lowered proliferation, while α-KG or the upstream metabolite glutamate increased proliferation that was abrogated by the H3K27 demethylase KDM6A/6B inhibitor GSK-J4 (Figure S3N). To determine the effects of α-KG and Suc on gene expression, we performed RNA-seq on H3.3K27M NSCs treated with vehicle or with α-KG or Suc. Compared with vehicle-treated controls, Suc induced pathways related to neuronal differentiation (consistent with increased H3K27me3), whereas α-KG induced gene expression pathways related to proliferation (Figures 3K–3L). Having established that some H3K27M cells are reliant on glutamine to maintain low H3K27me3, we next sought to determine whether suppressing glutamine metabolism in these cells could have an impact on global H3K27me3 levels and cell proliferation. We targeted GDH (Figure 4A) with two independent shRNAs, which increased H3K27me3 and slowed proliferation (Figures 4B and 4C). The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) (Lemberg et al., 2018Lemberg K.M. Vornov J.J. Rais R. Slusher B.S. We're not DON yet: optimal dosing and prodrug delivery of 6-Diazo-5-oxo-L-norleucine.Mol. Cancer Ther. 2018; 17: 1824-1832Crossref PubMed Scopus (45) Google Scholar) increased H3K27me3 levels in glutamine-dependent H3.3K27M NSCs and DIPG-007 but not in glucose-independent SF7761 cells (Figure 4D). Moreover, glutaminase (GLS) inhibitors CB-839 and BPTES increased H3K27me3 levels in DIPG-007 cells (Figures S4A and S4B). DON treatment in vivo, significantly suppressed growth compared with vehicle-treated animals in H3.3K27M NSC xenografts (Figures 4E and S4C). Cancer cells can take up glutamine via the transporter SLC1A5 and metabolize it to glutamate by GLS. Both SLC1A5 and GLS were elevated in glutamine-dependent DIPG-007 compared with non-dependent SF7761 cells (Figure S4D). GSK3α/β phosphorylation (pGSK3α/β) drives glutamine dependency in cancer cells (Momcilovic et al., 2018Momcilovic M. Bailey S.T. Lee J.T. Fishbein M.C. Braas D. Go J. Graeber T.G. Parlati F. Demo S. Li R. et al.The GSK3 signaling axis regulates adaptive glutamine metabolism in lung squamous cell carcinoma.Cancer Cell. 2018; 33: 905-921.e5Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Accordingly, glutamine-sensitive DIPG-007, but not glutamine-resistant SF7761, showed elevated pGSK3α/β (Figure S4D). To determine if this heterogeneity was mirrored in patient H3K27M DIPGs, we assessed SLC1A5 expression by immunohistochemistry (IHC) in tumor samples (n = 6). Similar to cell lines, SLC1A5 expression was heterogeneous across H3.3K27M DIPGs (Figures 4F, 4G, S4E, and S4F). Within tumors with low overall SLC1A5 levels, individual tumor cells still expressed high SLC1A5 levels (Figures 4G, arrows). Moreover, higher expression of SLC1A5 was associated with a poor prognosis in pediatric high-grade gliomas (Figure S4G). Patient-derived samples mirrored our cell culture results, suggesting that there is marked heterogeneity in glutamine metabolism in H3K27M DIPGs. We next targeted HK2, which was elevated in H3.3K27M cells and tumor samples (Figures 1H–1J). Two independent HK2 shRNAs increased H3K27me3 levels and suppressed proliferation in DIPG-007 cells (Figures 4H–4I). Similarly, pharmacological inhibition with 2-deoxy-D-glucose (2-DG), an HK2 inhibitor, raised H3K27me3 levels in both DIPG-007 and SF7761 cells (Figure 4J). Other azole HK2 inhibitors (Agnihotri et al., 2019Agnihotri S. Mansouri S. Burrell K. Li M. Mamatjan Y. Liu J. Nejad R. Kumar S. Jalali S. Singh S.K. et al.Ketoconazole and posaconazole selectively target HK2-expressing glioblastoma cells.Clin. Cancer Res. 2019; 25: 844-855Crossref PubMed Scopus (16) Google Scholar) produced a similar effect (Figures S4H–S4I). We took advantage of the blood-brain barrier (BBB) penetrability of 2-DG (Pardridge et al., 1982Pardridge W.M. Crane P.D. Mietus L.J. Oldendorf W.H. Kinetics of regional blood-brain barrier transport and brain phosphorylation of glucose and 2-deoxyglucose the barbiturate-anesthetized rat.J. Neurochem. 1982; 38: 560-568Crossref PubMed Scopus (68) Google Scholar) and found it suppressed tumor growth in DIPG-007 orthotopic xenografts compared with vehicle treatment (Figures 4K–4L). Furthermore, GLUD1 and HK2 knockdown significantly lowered α-KG/Suc ratios (Figure S4J). We wanted to assess if inhibiting glutamine or glucose metabolism increased H3K27me3 levels in H3.3K27M tumors in vivo. To specifically examine changes in global H3K27me3 in H3K27M mutant cells, we combined IHC of mutant-specific H3K27M and H3K27me3 and compared results in vehicle versus DON- or 2-DG-treated animals. Both DON- and 2-" @default.
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• W3048798848 date "2020-09-01" @default.
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• W3048798848 title "Integrated Metabolic and Epigenomic Reprograming by H3K27M Mutations in Diffuse Intrinsic Pontine Gliomas" @default.
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• W3048798848 doi "https://doi.org/10.1016/j.ccell.2020.07.008" @default.
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