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• W2890911268 abstract "•(R)-2-hydroxyglutarate, produced by IDH1/2 mutants, inhibits the BCAT transaminases•IDH mutant gliomas display a transamination-dependent glutamate biosynthesis defect•BCAT loss increases reliance on glutaminase for glutamate and glutathione synthesis•Mutant IDH and glutaminase inhibition are synthetic lethal under oxidative stress IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas. IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas. IDH1 and IDH2 mutations occur in various cancers, including gliomas and leukemias (Losman and Kaelin, 2013Losman J.A. Kaelin Jr., W.G. What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer.Genes Dev. 2013; 27: 836-852Crossref PubMed Scopus (432) Google Scholar). Tumor-associated IDH mutants convert 2-oxoglutarate (2OG) to the (R) enantiomer of 2-hydroxyglutarate, (R)-2HG, which accumulates to millimolar levels in tumors (Losman and Kaelin, 2013Losman J.A. Kaelin Jr., W.G. What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer.Genes Dev. 2013; 27: 836-852Crossref PubMed Scopus (432) Google Scholar). (R)-2HG structurally resembles 2OG and competitively inhibits many 2OG-dependent dioxygenases, including JmjC histone demethylases and TET DNA hydroxylases (Losman and Kaelin, 2013Losman J.A. Kaelin Jr., W.G. What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer.Genes Dev. 2013; 27: 836-852Crossref PubMed Scopus (432) Google Scholar). The challenge has been to determine which dioxygenases play causal roles in transformation by (R)-2HG in a given tumor type and which are bystanders. In this regard, inhibition of TET2 promotes leukemic transformation and is also suspected of playing a role in IDH mutant gliomas (Flavahan et al., 2016Flavahan W.A. Drier Y. Liau B.B. Gillespie S.M. Venteicher A.S. Stemmer-Rachamimov A.O. Suvà M.L. Bernstein B.E. Insulator dysfunction and oncogene activation in IDH mutant gliomas.Nature. 2016; 529: 110-114Crossref PubMed Scopus (748) Google Scholar, Losman and Kaelin, 2013Losman J.A. Kaelin Jr., W.G. What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer.Genes Dev. 2013; 27: 836-852Crossref PubMed Scopus (432) Google Scholar). Although studies of (R)-2HG targets have focused on 2OG-dependent dioxygenases, cells have other 2OG-dependent enzymes, including various transaminases that reversibly couple nitrogen transfer from their specific amino acid substrates to the interconversion of 2OG and glutamate. In this regard, Reitman et al., 2011Reitman Z.J. Jin G. Karoly E.D. Spasojevic I. Yang J. Kinzler K.W. He Y. Bigner D.D. Vogelstein B. Yan H. Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome.Proc. Natl. Acad. Sci. USA. 2011; 108: 3270-3275Crossref PubMed Scopus (349) Google Scholar reported that the conditioned media of IDH mutant cells had decreased levels of branched chain α-ketoacids (BCKAs) compared to control cells. BCKAs are made from the branched chain amino acids (BCAAs) leucine, isoleucine, and valine by the BCAA transaminases BCAT1 and BCAT2 (Figure 1A). Based on these findings and the 2OG dependence of BCAT1 and BCAT2, we hypothesized that the BCKA depletion observed by Reitman et al., 2011Reitman Z.J. Jin G. Karoly E.D. Spasojevic I. Yang J. Kinzler K.W. He Y. Bigner D.D. Vogelstein B. Yan H. Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome.Proc. Natl. Acad. Sci. USA. 2011; 108: 3270-3275Crossref PubMed Scopus (349) Google Scholar was due to direct, competitive inhibition of BCAT activity by (R)-2HG and that this would create metabolic vulnerabilities that could be exploited therapeutically. We infected IDH1/2 wild-type HOG human glioma cells (Reitman et al., 2011Reitman Z.J. Jin G. Karoly E.D. Spasojevic I. Yang J. Kinzler K.W. He Y. Bigner D.D. Vogelstein B. Yan H. Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome.Proc. Natl. Acad. Sci. USA. 2011; 108: 3270-3275Crossref PubMed Scopus (349) Google Scholar) with a lentivirus encoding the canonical R132H IDH1 mutant, wild-type IDH1, or with the empty vector (EV) (Figure 1B). We then performed gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling of the resultant stable cell lines and of parental HOG cells that were treated with BCAT inhibitors (Compound 2 or gabapentin) (Hu et al., 2006Hu L.Y. Boxer P.A. Kesten S.R. Lei H.J. Wustrow D.J. Moreland D.W. Zhang L. Ahn K. Ryder T.R. Liu X. et al.The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases.Bioorg. Med. Chem. Lett. 2006; 16: 2337-2340Crossref PubMed Scopus (21) Google Scholar) or were infected to produce one of two BCAT1 small hairpin RNAs (shRNAs) or a control shRNA (Figures 1C and 1D). IDH1 R132H increased the abundance of the BCAAs and depleted glutamate and the BCKAs α-keto-β-methylvalerate (KMV) and α-ketoisocaproate (KIC), as did pharmacologically or genetically inhibiting BCAT1 (Figure 1D). Similarly, glioma stem-like cell (GSC) lines derived from IDH1 mutant gliomas had elevated BCAAs and lower levels of KIC and glutamate compared to IDH1 wild-type GSC lines (Figures S1A–S1C). Therefore, both engineered and patient-derived IDH1 mutant cells exhibit metabolic changes consistent with impaired BCAT activity. We next developed and validated a recombinant BCAT1 activity assay (Figures S1D–S1G). (R)-2HG competitively inhibited BCAT1, which is a cytosolic enzyme, with respect to 2OG over a physiologically relevant range of 2OG concentrations (Figures 1E–1H) (Koivunen et al., 2012Koivunen P. Lee S. Duncan C.G. Lopez G. Lu G. Ramkissoon S. Losman J.A. Joensuu P. Bergmann U. Gross S. et al.Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation.Nature. 2012; 483: 484-488Crossref PubMed Scopus (557) Google Scholar). (R)-2HG also inhibited the mitochondrial BCAT1 paralog, BCAT2 (Figure 1I), but minimally affected the GOT1 and GOT2 aspartate transaminases (Figures 1J and 1K). The alternative enantiomer, (S)-2HG, was a less effective BCAT inhibitor (Figure 1G), consistent with molecular docking studies that predict more extensive hydrogen bonding between (R)-2HG and BCAT2 (Figures 1L–1N, S1H, and S1I) relative to (S)-2HG. To ask if mutant IDH inhibits BCAT in cells, we assayed the conversion of 15N-leucine to 15N-glutamate in immortalized human astrocytes (NHA) and HOG cells infected to produce the IDH1 R132H mutant, wild-type IDH1, or with the EV (Figures 2A and 2C ). BCAT1 protein levels were lowered in late passage IDH1 mutant NHA cells (Figure S2C), consistent with a prior study (Tönjes et al., 2013Tönjes M. Barbus S. Park Y.J. Wang W. Schlotter M. Lindroth A.M. Pleier S.V. Bai A.H.C. Karra D. Piro R.M. et al.BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1.Nat. Med. 2013; 19: 901-908Crossref PubMed Scopus (308) Google Scholar) and with reduced BCAT1 mRNA levels in IDH1 mutant gliomas (Figure S2D), but were not downregulated in late passage HOG cells (Figures S2A and S2B). We therefore studied early passage NHA and HOG cells to avoid potential confounding effects in late passage NHA cells caused by decreased BCAT1 protein levels. Both the IDH1 mutant NHA and HOG cells had tumor-relevant (R)-2HG concentrations (Figure 2B) (Dang et al., 2009Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2009; 462: 739-744Crossref PubMed Scopus (2618) Google Scholar). Leucine transamination was impaired in IDH1 mutant NHA and HOG cells (Figures 2D, 2E, and S2F), mirroring the effects of Compound 2 (Figure S2E).Figure S2(R)-2HG Inhibits BCAA Catabolism in Multiple Cellular Contexts, Related to Figure 2Show full caption(A and B) Immunoblot analysis (n = 3) (A) and fraction of sites methylated (Beta) as determined by whole-genome bisulfite sequencing (n = 2) (B) of early and late passage HOG stable cell lines. Methylation of the CpG islands surrounding the transcriptional start sites for BCAT1 Promoters 1 and 2 (chr12:24,949,459 and chr12:24,903,075, respectively, in human genome assembly hg38) is shown. These transcriptional start sites are identical to those studied by Tönjes et al., 2013Tönjes M. Barbus S. Park Y.J. Wang W. Schlotter M. Lindroth A.M. Pleier S.V. Bai A.H.C. Karra D. Piro R.M. et al.BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1.Nat. Med. 2013; 19: 901-908Crossref PubMed Scopus (308) Google Scholar. Coordinates for the CpG islands associated with BCAT1 Promoters 1 and 2 are chr12:24,948,674-24,949,139 and chr12:24,902,666-24,903,312, respectively. We observed a pattern of BCAT1 promoter CpG island methylation in HOG cells that mirrors that seen in low-grade gliomas and secondary GBM patient samples analyzed by Tonjes and colleagues (i.e., low methylation of BCAT1 Promoter 1 and high methylation of BCAT1 Promoter 2). Expressing mutant IDH1 in HOG cells does not significantly impact methylation of either BCAT1 promoter at early or late passage.(C) Immunoblot analysis of early and late passage NHA stable lines (n = 3). 3DN signifies an enzymatically inactive IDH1 R132H variant in which three conserved aspartic acid residues within the IDH1 catalytic domain were replaced with asparagines.(D) BCAT1 mRNA levels in IDH1 mutant and wild-type glioma patient samples. Data are derived from analysis of 283 samples in the Brain Lower Grade Glioma TCGA dataset (n = 218 IDH1 mutant samples, n = 65 IDH1 wild-type samples).(E) 15N-leucine tracing assay in HOG cells pretreated with the indicated concentrations of Compound 2 for 17 hours (n = 3).(F) Labeling of intracellular leucine 10 minutes after 15N-leucine tracer administration in NHA (n = 3) and HOG (n = 2) stable cell lines confirming that the results in Figures 2D and 2E are not due to differential leucine tracer accumulation.(G) Ratios of the indicated metabolites in parental HCT116 cells treated with 10 mM (R)-2HG for 48 hours or isogenic HCT116 cells in which an IDH1 R132H or IDH2 R172K mutation was introduced into the endogenous IDH1 or IDH2 locus, respectively, by homologous recombination. n = 3.(H) Ratios of the indicated metabolites in HT1080 IDH1 R132C/+ fibrosarcoma cells treated with 1.5 μM AGI-5198 for 72 hours (n = 3).(I and J) Immunoblot analysis (I) and 15N-leucine tracing assay (J) of NHA and HOG stable cell lines infected to produce HA-IDH1 R132H, FLAG-IDH2 R172K, or with the empty vector (EV) (n = 3).(K) Steady-state 2HG and glutamate levels in HOG stable cell lines as in (I) (n = 3). TIC = total ion counts. Compared to mutant IDH1, mutant IDH2 more potently depletes glutamate and more potently inhibits BCAA catabolism. This effect correlates with higher (R)-2HG levels in IDH2 mutant cells relative to IDH1 mutant cells.(L) Levels of the indicated metabolites in conditioned media of HOG cells over time (n = 3). TIC are expressed relative to t = 0. Positive values indicate net secretion; negative values indicate net consumption.(M) U-13C-leucine, U-13C-isoleucine, and U-13C-glutamine tracing assays in HOG cells (n = 3). Fractional labeling of the citrate pool is shown.(N) Ratios of extracellular to intracellular TIC for the BCKAs KMV and KIC at the indicated time points (n = 3). Ratios are normalized to the 0 hour samples.(O) 1-13C-glutamate tracing assays in HOG cells (n = 3). Labeled and unlabeled glutamate isotopologues were quantified in both cellular and conditioned media extracts.(P) Immunoblot analysis of the indicated GSC lines treated with 3 μM AGI-5198 or DMSO for 3 days (n = 3).(Q) Glutamate levels in IDH1 wild-type TS516, TS676, and BT260 lines and in the positive control IDH1 mutant BT054 line treated with 3 μM AGI-5198 or DMSO for 3 days (n = 3).For all panels, data presented are means ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-tailed p values were determined by unpaired t test. n.s. = nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Immunoblot analysis (n = 3) (A) and fraction of sites methylated (Beta) as determined by whole-genome bisulfite sequencing (n = 2) (B) of early and late passage HOG stable cell lines. Methylation of the CpG islands surrounding the transcriptional start sites for BCAT1 Promoters 1 and 2 (chr12:24,949,459 and chr12:24,903,075, respectively, in human genome assembly hg38) is shown. These transcriptional start sites are identical to those studied by Tönjes et al., 2013Tönjes M. Barbus S. Park Y.J. Wang W. Schlotter M. Lindroth A.M. Pleier S.V. Bai A.H.C. Karra D. Piro R.M. et al.BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1.Nat. Med. 2013; 19: 901-908Crossref PubMed Scopus (308) Google Scholar. Coordinates for the CpG islands associated with BCAT1 Promoters 1 and 2 are chr12:24,948,674-24,949,139 and chr12:24,902,666-24,903,312, respectively. We observed a pattern of BCAT1 promoter CpG island methylation in HOG cells that mirrors that seen in low-grade gliomas and secondary GBM patient samples analyzed by Tonjes and colleagues (i.e., low methylation of BCAT1 Promoter 1 and high methylation of BCAT1 Promoter 2). Expressing mutant IDH1 in HOG cells does not significantly impact methylation of either BCAT1 promoter at early or late passage. (C) Immunoblot analysis of early and late passage NHA stable lines (n = 3). 3DN signifies an enzymatically inactive IDH1 R132H variant in which three conserved aspartic acid residues within the IDH1 catalytic domain were replaced with asparagines. (D) BCAT1 mRNA levels in IDH1 mutant and wild-type glioma patient samples. Data are derived from analysis of 283 samples in the Brain Lower Grade Glioma TCGA dataset (n = 218 IDH1 mutant samples, n = 65 IDH1 wild-type samples). (E) 15N-leucine tracing assay in HOG cells pretreated with the indicated concentrations of Compound 2 for 17 hours (n = 3). (F) Labeling of intracellular leucine 10 minutes after 15N-leucine tracer administration in NHA (n = 3) and HOG (n = 2) stable cell lines confirming that the results in Figures 2D and 2E are not due to differential leucine tracer accumulation. (G) Ratios of the indicated metabolites in parental HCT116 cells treated with 10 mM (R)-2HG for 48 hours or isogenic HCT116 cells in which an IDH1 R132H or IDH2 R172K mutation was introduced into the endogenous IDH1 or IDH2 locus, respectively, by homologous recombination. n = 3. (H) Ratios of the indicated metabolites in HT1080 IDH1 R132C/+ fibrosarcoma cells treated with 1.5 μM AGI-5198 for 72 hours (n = 3). (I and J) Immunoblot analysis (I) and 15N-leucine tracing assay (J) of NHA and HOG stable cell lines infected to produce HA-IDH1 R132H, FLAG-IDH2 R172K, or with the empty vector (EV) (n = 3). (K) Steady-state 2HG and glutamate levels in HOG stable cell lines as in (I) (n = 3). TIC = total ion counts. Compared to mutant IDH1, mutant IDH2 more potently depletes glutamate and more potently inhibits BCAA catabolism. This effect correlates with higher (R)-2HG levels in IDH2 mutant cells relative to IDH1 mutant cells. (L) Levels of the indicated metabolites in conditioned media of HOG cells over time (n = 3). TIC are expressed relative to t = 0. Positive values indicate net secretion; negative values indicate net consumption. (M) U-13C-leucine, U-13C-isoleucine, and U-13C-glutamine tracing assays in HOG cells (n = 3). Fractional labeling of the citrate pool is shown. (N) Ratios of extracellular to intracellular TIC for the BCKAs KMV and KIC at the indicated time points (n = 3). Ratios are normalized to the 0 hour samples. (O) 1-13C-glutamate tracing assays in HOG cells (n = 3). Labeled and unlabeled glutamate isotopologues were quantified in both cellular and conditioned media extracts. (P) Immunoblot analysis of the indicated GSC lines treated with 3 μM AGI-5198 or DMSO for 3 days (n = 3). (Q) Glutamate levels in IDH1 wild-type TS516, TS676, and BT260 lines and in the positive control IDH1 mutant BT054 line treated with 3 μM AGI-5198 or DMSO for 3 days (n = 3). For all panels, data presented are means ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-tailed p values were determined by unpaired t test. n.s. = nonsignificant. A cell-permeable version of (R)-2HG, (R)-2HG-TFMB (Losman et al., 2013Losman J.A. Looper R.E. Koivunen P. Lee S. Schneider R.K. McMahon C. Cowley G.S. Root D.E. Ebert B.L. Kaelin Jr., W.G. (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible.Science. 2013; 339: 1621-1625Crossref PubMed Scopus (549) Google Scholar), but not (S)-2HG-TFMB, inhibited leucine transamination in parental HOG cells (Figure 2F). Conversely, leucine transamination in IDH1 mutant cells was rescued by AGI-5198, which blocks (R)-2HG production (Losman et al., 2013Losman J.A. Looper R.E. Koivunen P. Lee S. Schneider R.K. McMahon C. Cowley G.S. Root D.E. Ebert B.L. Kaelin Jr., W.G. (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible.Science. 2013; 339: 1621-1625Crossref PubMed Scopus (549) Google Scholar) (Figure 2G). Metabolic evidence of suppressed BCAT activity was also observed in HCT116 colorectal cancer cells treated with (R)-2HG or engineered to have heterozygous IDH1 or IDH2 mutations (Figure S2G), while BCAT activity was restored in HT1080 IDH1 +/R132C fibrosarcoma cells treated with AGI-5198 (Figure S2H). Therefore, loss of BCAT activity in IDH mutant cells is caused by (R)-2HG. Mutant IDH2, like mutant IDH1, also potently repressed BCAT activity when introduced into NHA and HOG cells (Figures S2I–S2K). BCAA catabolism yields glutamate and BCKAs. We found that HOG cells avidly consume BCAAs from culture media (Figure S2L) and that the resultant BCKAs KMV and KIC are secreted by these cells rather than being further metabolized (Figures S2M and S2N). We therefore focused on glutamate, which is used to make many nitrogenous metabolites and acts as a neurotransmitter. Glutamate does not efficiently cross the blood-brain barrier (BBB) and brain interstitial glutamate concentrations are ∼100-fold lower than in serum (Espey et al., 1998Espey M.G. Kustova Y. Sei Y. Basile A.S. Extracellular glutamate levels are chronically elevated in the brains of LP-BM5-infected mice: a mechanism of retrovirus-induced encephalopathy.J. Neurochem. 1998; 71: 2079-2087Crossref PubMed Scopus (58) Google Scholar, Smith, 2000Smith Q.R. Transport of glutamate and other amino acids at the blood-brain barrier.J. Nutr. 2000; 130: 1016S-1022SCrossref PubMed Google Scholar). Neural cells heavily rely on transaminases, particularly BCATs, to sustain intracellular glutamate levels (Cooper and Jeitner, 2016Cooper A.J. Jeitner T.M. Central role of glutamate metabolism in the maintenance of nitrogen homeostasis in normal and hyperammonemic brain.Biomolecules. 2016; 6 (pii: E16)Crossref PubMed Scopus (118) Google Scholar). Moreover, we found that imported glutamate makes a negligible contribution to intracellular glutamate levels in HOG cells grown ex vivo, reaffirming the importance of glutamate synthesis to maintain the relatively large intracellular glutamate pool (Figure S2O). To ask whether glutamate suppression by mutant IDH (Figures 1D and S1C) can be linked to loss of BCAT activity, we quantified steady-state 2HG, glutamate, and KIC levels in IDH1 mutant and wild-type HOG cell lines that were treated with AGI-5198 or DMSO (Figure 2H), while grown in cell culture media that contained glucose, glutamate, pyruvate, and amino acid levels based on physiological concentrations in the brain (Espey et al., 1998Espey M.G. Kustova Y. Sei Y. Basile A.S. Extracellular glutamate levels are chronically elevated in the brains of LP-BM5-infected mice: a mechanism of retrovirus-induced encephalopathy.J. Neurochem. 1998; 71: 2079-2087Crossref PubMed Scopus (58) Google Scholar, Schug et al., 2015Schug Z.T. Peck B. Jones D.T. Zhang Q. Grosskurth S. Alam I.S. Goodwin L.M. Smethurst E. Mason S. Blyth K. et al.Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.Cancer Cell. 2015; 27: 57-71Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, Tisdall and Smith, 2006Tisdall M.M. Smith M. Cerebral microdialysis: research technique or clinical tool.Br. J. Anaesth. 2006; 97: 18-25Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). AGI-5198 lowered (R)-2HG and normalized intracellular glutamate and KIC levels in IDH1 mutant cells, with the latter change indicative of restored BCAT activity (Figure 2H). Overexpressing an (R)-2HG-insensitive transaminase, ornithine aminotransferase (OAT), and supplementing media with the OAT substrates 2OG (in a cell-permeable form) and L-ornithine partially reversed mutant IDH-induced glutamate depletion (Figures 2I–2K). IDH1 mutant GSC lines, like our engineered cell lines, also displayed reduced glutamate levels and BCAA catabolism relative to IDH1 wild-type lines despite comparable BCAT1 and BCAT2 protein levels (Figures 2L–2N). Lowering (R)-2HG in MGG152 and BT054 cell lines with AGI-5198 potently stimulated BCAT activity and restored glutamate levels without affecting BCAT1 or BCAT2 protein levels (Figures 2O, 2P, S2P, and S2Q). Cells can also make glutamate from glutamine using glutaminase (GLS). Interestingly, GLS is highly expressed in the brain compared to most normal tissues (Figures S3A and S3B) and low-grade gliomas upregulate a more active GLS splice isoform (Cassago et al., 2012Cassago A. Ferreira A.P. Ferreira I.M. Fornezari C. Gomes E.R. Greene K.S. Pereira H.M. Garratt R.C. Dias S.M. Ambrosio A.L. Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.Proc. Natl. Acad. Sci. USA. 2012; 109: 1092-1097Crossref PubMed Scopus (182) Google Scholar) (Figures S3C and S3D). Furthermore, human brain tumors are highly glutamine avid (Venneti et al., 2015Venneti S. Dunphy M.P. Zhang H. Pitter K.L. Zanzonico P. Campos C. Carlin S.D. La Rocca G. Lyashchenko S. Ploessl K. et al.Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo.Sci. Transl. Med. 2015; 7: 274ra17Crossref PubMed Scopus (216) Google Scholar, Tardito et al., 2015Tardito S. Oudin A. Ahmed S.U. Fack F. Keunen O. Zheng L. Miletic H. Sakariassen P.O. Weinstock A. Wagner A. et al.Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.Nat. Cell Biol. 2015; 17: 1556-1568Crossref PubMed Scopus (330) Google Scholar). We hypothesized that decreased BCAT function in IDH mutant cells would increase their reliance on GLS activity for glutamate production. Indeed, the conversion of glutamine to glutamate was increased in IDH1 mutant NHA and HOG cells compared to their wild-type counterparts (Figures 3A–3C and 3E ). This was not due to increased protein levels of GLS or c-Myc, which regulates GLS expression (Figures 3D and S3E) (Gao et al., 2009Gao P. Tchernyshyov I. Chang T.C. Lee Y.S. Kita K. Ochi T. Zeller K.I. De Marzo A.M. Van Eyk J.E. Mendell J.T. Dang C.V. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism.Nature. 2009; 458: 762-765Crossref PubMed Scopus (1575) Google Scholar). Moreover, expression of mutant IDH1 in NHA and HOG cells cooperated with the GLS inhibitor CB-839 (Gross et al., 2014Gross M.I. Demo S.D. Dennison J.B. Chen L. Chernov-Rogan T. Goyal B. Janes J.R. Laidig G.J. Lewis E.R. Li J. et al.Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer.Mol. Cancer Ther. 2014; 13: 890-901Crossref PubMed Scopus (607) Google Scholar) to deplete intracellular glutamate levels >75% in vitro (Figure 3F) and the glutamate pools in IDH1 mutant GSC lines were hypersensitive to CB-839 (Figure 3G).Figure 3BCAT Repression Promotes Glutamine-Dependent Glutamate SynthesisShow full caption(A) Schema of GLS-dependent nitrogen transfer in α-15N-glutamine tracing assays.(B and C) α-15N-glutamine tracing assays in NHA (B) and HOG (C) stable cell lines (n = 3).(D) Immunoblot analysis (n = 2).(E) 15N-(amide)-glutamine tracing in HOG stable lines (n = 4). Fractional labeling of ammonia 8 hr after tracer addition is shown.(F) Glutamate levels in NHA and HOG stable cell lines treated with 100 nM CB-839 or DMSO for 2 hr (n = 4).(G) Glutamate levels in GSC lines treated with CB-839 or DMSO for 2 hr (n = 3).(H and I) Immunoblots (H) and 15N-leucine and α-15N-glutamine tracing assays (I) in HOG stable cells expressing the indicated sgRNAs (together with Cas9) and, where indicated, sgRNA-insensitive BCAT1 and BCAT2 cDNAs. n = 3.(J) Schema depicting the major routes of glutamate synthesis in glial cells.(K and L) 15N-BCAA and α-15N-glutamine tracing assays of NHA (K) and HOG (L) cells (n = 3). Fractional labeling of the glutamate pool at 6 hr is shown.(M) α-15N-glutamine (n = 6) and 15N-leucine (n = 3) tracing assays in HOG cells treated with 100 μM Compound 2 or 100 nM CB-839, respectively. Cells were given 15N-labeled amino acids for 20 min.(N) Immunoblot analysis of HOG stable lines infected to produce the GLS Y394L mutant or EV. KGA and GAC splice isoforms are indicated. n = 3.(O) Glutamate levels in HOG stable cell lines like in (N) (n = 3).For (N) and (O), see also Figures S3P–S3R. For all panels, data presented are means ± SD; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Two-tailed p values were determined by unpaired t test.See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Schema of GLS-dependent nitrogen transfer in α-15N-glutamine tracing assays. (B and C) α-15N-glutamine tracing assays in NHA (B) and HOG (C) stable cell lines (n = 3). (D) Immunoblot analysis (n = 2). (E) 15N-(amide)-glutamine tracing in HOG stable lines (n = 4). Fractional labeling of ammonia 8 hr after tracer addition is shown. (F) Glutamate levels in NHA and HOG stable cell lines treated with 100 nM CB-839 or DMSO for 2 hr (n = 4). (G) Glutamate levels in GSC lines treated with CB-839 or DMSO for 2 hr (n = 3). (H and I) Immunoblots (H) and 15N-leucine and α-15N-glutamine tracing assays (I) in HOG stable cells expressing the indicated sgRNAs (together with Cas9) and, where indicated, sgRNA-insensitive BCAT1 and BCAT2 cDNAs. n = 3. (J) Schema depicting the major routes of glutamate synthesis in glial cells. (K and L) 15N-BCAA and α-15N-glutamine tracing assays of NHA (K) and HOG (L) cells (n = 3). Fractional labeling of the glutamate pool at 6 hr is shown. (M) α-15N-glutamine (n = 6) and 15N-leucine (n = 3) tracing assays in HOG cells treated with 100 μM Compound 2 or 100 nM CB-839, respectively. Cells were given 15N-labeled amino acids for 20 min. (N) Immunoblot analysis of HOG stable lines infected to produce the GLS Y394L mutant or EV. KGA and GAC splice isoforms are indicated. n = 3. (O) Glutamate levels in HOG stable cell lines like in (N) (n = 3). For (N) and (O), see also Figures S3P–S3R. For all panels, data presented are means ± SD; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Two-tailed p values were determined by unpaired t test. See also Figure S3. To model this further we inactivated BCAT1 and BCAT2 in HOG cells with CRISPR/Cas9, leading to an on-target loss of leucine transamination (Figure 3H and 3I, left panel). 15N-glutamine tracing confirmed that, similar to IDH mutant cells, glutamine catabolism was induc" @default.
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• W2890911268 date "2018-09-01" @default.
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• W2890911268 title "Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma" @default.
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• W2890911268 doi "https://doi.org/10.1016/j.cell.2018.08.038" @default.
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