Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

May combine two claims.

(R)-2HG also inhibits BCAT2 but minimally affects GOT1 and GOT2 aspartate transaminases.

“(R)-2HG also inhibited the mitochondrial BCAT1 paralog, BCAT2 (Figure 1I), but minimally affected the GOT1 and GOT2 aspartate transaminases”

May combine two claims.

Lowering (R)-2HG with AGI-5198 in MGG152 and BT054 cell lines stimulated BCAT activity and restored glutamate levels without affecting BCAT1 or BCAT2 protein.

“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”

May combine two claims.

Suppressed BCAT activity was observed in HCT116 cells treated with (R)-2HG or with heterozygous IDH1 or IDH2 mutations, and BCAT activity was restored in HT1080 IDH1 +/R132C cells by AGI-5198.

“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”

May combine two claims.

(R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect.

“we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect”

• Also stated in: § (R)-2HG Inhibits BCAT1 and BCAT2 · plain

  “(R)-2HG competitively inhibited BCAT1, which is a cytosolic enzyme, with respect to 2OG over a physiologically-relevant range of 2OG concentrations”

• Also stated in: § Discussion · boosted

  “We show that (R)-2HG directly inhibits BCAT1 and BCAT2.”

May combine two claims.

GLS is highly expressed in brain compared to most normal tissues, and low-grade gliomas upregulate a more active GLS splice isoform.

“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., 2012) (Figures S3C and S3D).”

• cites Cassago et al., 2012Not checked yet

  Not checked in the free run.

May combine two claims.

Leucine transamination was impaired in IDH1 mutant NHA and HOG cells, mirroring the effects of Compound 2.

“Leucine transamination was impaired in IDH1 mutant NHA and HOG cells (Figures 2D, 2E, and S2F), mirroring the effects of Compound 2”

May combine two claims.

GSC lines from IDH1 mutant gliomas had elevated BCAAs and lower KIC and glutamate than IDH1 wild-type GSC lines.

“glioma stem-like cell (GSC) lines derived from IDH1 mutant gliomas (Figure S1B) had elevated BCAAs and lower levels of KIC and glutamate compared to IDH1 wild-type GSC lines”

May combine two claims.

IDH1 R132H expression in HOG cells increased BCAAs and depleted glutamate and the BCKAs KMV and KIC, similar to pharmacological or genetic BCAT1 inhibition.

“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).”

May combine two claims.

AGI-5198 lowered (R)-2HG and normalized intracellular glutamate and KIC levels in IDH1 mutant cells, indicating restored BCAT activity.

“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”

May combine two claims.

Cell-permeable (R)-2HG-TFMB, but not (S)-2HG-TFMB, inhibited leucine transamination in parental HOG cells.

“A cell-permeable version of (R)-2HG, (R)-2HG-TFMB (Losman et al., 2013), but not (S)-2HG-TFMB, inhibited leucine transamination in parental HOG cells”

• cites Losman et al., 2013Not checked yet

  Not checked in the free run.

May combine two claims.

In BCAT null cells, glutamate levels normalized over time and 2OG was dramatically upregulated.

“In contrast to IDH1 mutant HOG cells, however, glutamate levels normalized over time, associated with dramatic 2OG upregulation, in BCAT null cells”

May combine two claims.

CB-839 enhanced the effectiveness of radiation against IDH1 mutant HOG cells, but not IDH1 wild-type HOG cells, in vitro, unless the cells were cotreated with NAC.

“CB-839 also enhanced the effectiveness of radiation against IDH1 mutant HOG cells, but not IDH1 wild-type HOG cells, in vitro, unless the cells were cotreated with NAC”

May combine two claims.

BCAT null cells potently increased alanine transamination, an effect observed in IDH1 mutant cells only when supplemented with 2OG.

“BCAT null cells potently increased alanine transamination, an effect that was only observed in IDH1 mutant cells when supplemented with 2OG”

May combine two claims.

Compound 2 increased conversion of glutamine to glutamate, while CB-839 increased flux through BCATs.

“Compound 2 increased the conversion of glutamine to glutamate, while CB-839 increased flux through the BCATs”

May combine two claims.

The reduced/oxidized glutathione ratio was not impacted by mutant IDH in vitro or in vivo, and NADPH levels were not suppressed in IDH1 mutant HOG tumors.

“the reduced/oxidized glutathione ratio was not impacted by mutant IDH in vitro or in vivo (Figures 5A and S5A) and, consistent with findings in IDH1 mutant macrophages (Sasaki et al., 2012), NADPH levels were not suppressed in IDH1 mutant HOG tumors”

• cites Sasaki et al., 2012Not checked yet

  Not checked in the free run.

May combine two claims.

TS516 cells had the greatest sensitivity to glutathione depletion by CB-839 among IDH1 wild-type GSC lines tested and had the highest basal expression of GLS and c-Myc.

“TS516 cells displayed the greatest sensitivity to glutathione depletion by CB-839 amongst the IDH1 wild-type GSC lines tested and had the highest basal expression of GLS (Figure S5I) and c-Myc”

May combine two claims.

In IDH mutant gliomas, but not wild-type gliomas, radiation and CB-839 strongly inhibited tumor growth only when given in combination, extending median survival from 13 days to 30 days.

“In IDH mutant gliomas, but not wild-type gliomas, radiation and CB-839 strongly inhibited tumor growth only when given in combination (Figures 7C to 7F, S7B, and S7C), extending median survival from 13 days in untreated mice to 30 days.”

May combine two claims.

IDH mutant GSC lines did not undergo apoptosis upon CB-839- or BSO-induced glutathione depletion, nor did they display hypersensitivity to tBH alone.

“IDH mutant GSC lines did not undergo apoptosis upon CB-839- or BSO-induced glutathione depletion (Figure S6H), nor did they display hypersensitivity to tBH treatment alone”

May combine two claims.

IDH1 mutant NHA and HOG cells were highly sensitive to the combination of CB-839 and tBH compared to IDH wild-type counterparts, and effects were reversed by cell-permeable GSH.

“IDH1 mutant NHA and HOG cells were highly sensitive to the combination of CB-839 and the oxidant tert-butyl hydroperoxide (tBH) compared to their IDH wild-type counterparts and their effects were reversed by a cell-permeable form of GSH”

May combine two claims.

Treatment of tumor-bearing mice with CB-839 depleted glutathione in IDH mutant HOG gliomas but not in isogenic wild-type tumors, mirroring its effects on glutamate.

“Treatment of tumor-bearing mice with CB-839 depleted glutathione in IDH mutant HOG gliomas, but not in isogenic wild-type tumors (Figure 5O), mirroring its effects on glutamate”

May combine two claims.

IDH mutant GSC lines display lower glutathione levels relative to IDH wild-type lines and a trend toward greater susceptibility to CB-839-induced glutathione depletion.

“IDH mutant GSC lines display lower glutathione levels relative to IDH wild-type lines and a trend toward greater susceptibility to CB-839-induced glutathione depletion”

May combine two claims.

U-13C-glutamine tracing in mice bearing orthotopic IDH1 mutant HOG tumors showed the tumor 2HG pool was not significantly labeled despite substantial plasma glutamine labeling and robust labeling of tumor glutamate and 2OG pools.

“U-13C-glutamine tracing in mice bearing orthotopic IDH1 mutant HOG tumors showed that the tumor 2HG pool was not labeled significantly (Figure S4L) despite substantial enrichment of labeled glutamine in plasma (Figure S4K) and robust labeling of tumor glutamate and 2OG pools.”

May combine two claims.

Glutathione levels are decreased in IDH1 mutant orthotopic xenograft gliomas and increased by AGI-5198 in engineered and endogenous IDH1 mutant glioma cell lines.

“We confirmed that glutathione levels are decreased in IDH1 mutant orthotopic xenograft gliomas (Figure 5A) and increased by AGI-5198 in both engineered (HOG) and endogenous (MGG152) IDH1 mutant glioma cell lines”

May combine two claims.

Only 1 of 3 IDH wild-type GSC lines was susceptible to synergistic killing by tBH and CB-839 (TS516), while all 3 IDH1 mutant lines were sensitized to tBH by CB-839.

“Whereas only 1 of 3 IDH wild-type GSC lines was susceptible to synergistic cell killing by tBH and CB-839 treatments (the c-Mychigh/GLShigh TS516 line), all 3 of the IDH1 mutant lines were sensitized to tBH by CB-839.”

May combine two claims.

(R)-2HG and BCAT1 shRNA decreased glutamate and glutathione levels without significantly reducing serine and cysteine levels.

“Both (R)-2HG and the BCAT1 shRNA decreased both glutamate and glutathione levels (Figures 5D to 5F) without significantly reducing the levels of the glutathione precursors serine, which gives rise to glycine, and cysteine”

May combine two claims.

Glutamate levels in IDH wild-type gliomas were impervious to GLS ablation, consistent with sustained BCAT activity.

“In contrast, glutamate levels in the IDH wild-type gliomas were impervious to GLS ablation (Figure 4K), consistent with sustained BCAT activity in these tumors”

May combine two claims.

Genetic ablation of BCAT1 and BCAT2 causes cells to engage GLS to make glutamate, recapitulating the effects of mutant IDH.

“We show that genetic ablation of BCAT1 and BCAT2 causes cells to engage GLS to make glutamate, recapitulating the effects of mutant IDH.”

May combine two claims.

Similar results were observed in subcutaneous TS603 IDH1 mutant GSC tumors treated with radiation and CB-839, associated with reduced proliferation, reduced angiogenesis, and modestly increased apoptosis.

“Similar results were observed when subcutaneous tumors formed by the TS603 IDH1 mutant GSC line were treated with radiation and CB-839 (Figures 7G, S7D, S7E, and S7K to S7M), associated with reduced proliferation, reduced angiogenesis and modestly increased apoptosis”

May combine two claims.

In isogenic IDH1 mutant HOG tumors, lower BCAT activity, decreased basal glutamate levels, and hyperdependence on GLS for glutamate and glutathione were observed.

“In our isogenic, IDH1 mutant HOG tumors, however, we observed lower BCAT activity, as evidenced by reduced flux of leucinederived nitrogen into glutamate, decreased basal glutamate levels, and hyperdependence on GLS for the production of glutamate and its downstream product, glutathione.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The TCGA LGG analysis included 283 samples with IDH1 mutation annotations: 218 IDH1-mutant and 65 IDH1-wild-type.

“IDH1 mutation annotations were found for 283 samples (n = 218 IDH1 mutant samples, n = 65 IDH1 wild-type samples).”

May combine two claims.

IDH1 mutations occur frequently in low-grade gliomas and secondary glioblastomas and lead to excess production of (R)-2HG.

“IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG.”

• cites Losman and Kaelin, 2013Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Inhibition of TET2 promotes leukemic transformation and is suspected to play a role in IDH mutant gliomas.

“Inhibition of TET2 promotes leukemic transformation and is also suspected of playing a role in IDH mutant gliomas (Flavahan et al., 2016; Losman and Kaelin, 2013).”

• cites Flavahan et al., 2016 sourceNot checked yet

  Not checked in the free run.

• cites Losman and Kaelin, 2013Not checked yet

  Not checked in the free run.

May combine two claims.

Tumor-associated IDH mutants convert 2OG to (R)-2HG, which accumulates to millimolar levels in tumors.

“Tumor-associated IDH mutants convert 2-oxoglutarate (2OG) to the (R) enantiomer of 2-hydroxyglutarate, (R)-2HG, which accumulates to mM levels in tumors (Losman and Kaelin, 2013).”

• cites Losman and Kaelin, 2013Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

(R)-2HG competitively inhibits many 2OG-dependent dioxygenases, including JmjC histone demethylases and TET DNA hydroxylases.

“(R)-2HG structurally resembles 2OG and competitively inhibits many 2OG-dependent dioxygenases, including JmjC histone demethylases and TET DNA hydroxylases (Losman and Kaelin, 2013).”

• cites Losman and Kaelin, 2013Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

BCKAs are produced from the branched chain amino acids leucine, isoleucine, and valine by BCAT1 and BCAT2.

“BCKAs are made from the branched chain amino acids (BCAAs) leucine, isoleucine, and valine by the BCAA transaminases BCAT1 and BCAT2 (Figure 1A).”

May combine two claims.

BCAT inhibition and resulting glutamate depletion decrease glutathione levels and induce reliance on GLS for glutathione synthesis in IDH mutant glioma cells.

“Therefore, BCAT inhibition and resulting glutamate depletion decrease glutathione levels and induce reliance on GLS for glutathione synthesis in IDH mutant glioma cells.”

May combine two claims.

Both loss of BCAT activity and increased 2OG consumption contribute to decreased transamination-dependent glutamate synthesis in IDH mutant cells, leading to increased reliance on GLS.

“Therefore, both loss of BCAT activity and increased 2OG consumption contribute to decreased transamination-dependent glutamate synthesis in IDH mutant cells (Figure S3O), leading to increased reliance on GLS”

May combine two claims.

CB-839-induced growth inhibition was due to redox stress because it was rescued by AGI-5198 and by media supplementation with glutamate or NAC.

“This was due to redox stress because CB-839-induced growth inhibition was rescued by AGI-5198 and by media supplementation with glutamate or the antioxidant N-acetylcysteine (NAC)”

Classified as probably not a substantial claim. May combine two claims.

GLS inhibitors selectively inhibit proliferation of IDH mutant cells ex vivo, but the mechanism remains unknown.

“GLS inhibitors selectively inhibit the proliferation of IDH mutant cells ex vivo, but the mechanism underlying this selectivity remains unknown (Emadi et al., 2014; Seltzer et al., 2010).”

• cites Emadi et al., 2014Not checked yet

  Not checked in the free run.

• cites Seltzer et al., 2010Not checked yet

  Not checked in the free run.

May combine two claims.

The effects of the single GLS sgRNA were on-target because they were rescued by an sgRNA-insensitive GLS cDNA and corroborated with CB-839.

“The effects of the single GLS sgRNA used in these experiments were on-target because they could be rescued by an sgRNA-insensitive GLS cDNA (Figures S4E and S4F) and were corroborated with CB-839”

May combine two claims.

Glutamate is limiting for glutathione synthesis because glutathione levels were decreased in HOG and NHA cells treated with Compound 2 or GLS inhibitors CB-839 and BPTES, all of which lower glutamate.

“Glutamate is limiting for glutathione synthesis because glutathione levels were decreased in HOG and NHA cells treated with Compound 2 (Figure 5H) or with the GLS inhibitors CB-839 and BPTES (Figures 5I and S5G), all of which lower glutamate levels”

May combine two claims.

15N-glutamate levels in tumors vastly exceeded those in plasma, consistent with intracellular BCAT-dependent 15N-glutamate synthesis.

“The 15N-glutamate levels in tumors vastly exceeded those in plasma, consistent with intracellular BCAT-dependent 15Nglutamate synthesis”

May combine two claims.

BCATs and GLS reciprocally support glutamate synthesis, and GLS can be engaged to overcome loss of BCAT activity caused by mutant IDH.

“Therefore, BCATs and GLS reciprocally support glutamate synthesis and GLS can be engaged to overcome the loss of BCAT activity caused by mutant IDH.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Prior reports indicate that the interstitial glutamate concentration in human brain tissue is about 1 μM.

“cells were cultured in low glutamate Serum-like Modified Eagles Medium (SMEM) (1 μM glutamate, consistent with reported glutamate concentrations in the interstitial space of human brain tissue) (Espey et al., 1998; Smith, 2000)”

• cites Espey et al., 1998Not checked yet

  Not checked in the free run.

• cites Smith, 2000Not checked yet

  Not checked in the free run.

May combine two claims.

Early indications suggest this strategy will be less effective against IDH mutant gliomas, possibly because mutant IDH acts through a hit-and-run mechanism.

“There are early indications, however, that this strategy will be less effective against IDH mutant gliomas, possibly because mutant IDH acts through a ‘hit-and-run’ mechanism in this setting (Waitkus et al., 2018).”

• cites Waitkus et al., 2018Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Tumor size and survival data were analyzed by investigators who knew the treatment assignments.

“Tumor size measurements and survival analyses were performed by researchers who were unblinded to treatment assignments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cell segmentation was improved by adjusting inForm parameters to anticipate counterstain-based cell features and then visually checking performance.

“Cell segmentation was optimized by tuning inForm parameters to anticipate cell features from the counterstain and then visually assessing performance.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The LC-MS/MS method used selected reaction monitoring and was able to detect 263 water-soluble metabolites, including amino acids, central carbon metabolism intermediates, nucleotides, redox cofactors, and α-ketoacids.

“with selected reaction monitoring (SRM) with positive/negative polarity switching to detect a total of 263 water-soluble metabolites including amino acids and amino acid biosynthetic pathway intermediates, central carbon metabolism intermediates, nucleotides and their precursors, redox cofactors, and α-ketoacids (Yuan et al., 2012).”

• cites Yuan et al., 2012Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

IHC analysis algorithms were built from representative samples covering variation in tissue morphology, stain distribution, tissue quality, and other factors.

“Analysis algorithms were developed using select representative samples that span variation in tissue morphology, stain distribution, tissue quality, and other factors.”

May combine two claims.

A prior in vivo study using 13C-labeled glucose and glutamine tracers showed patient-derived xenografts from primary GBMs consume glutamine but do not readily convert it to glutamate; instead they oxidize glucose to make glutamate.

“A prior in vivo study using 13C-labeled glucose and glutamine tracers showed that patient-derived xenografts made from primary GBMs, ~95% of which are IDH wild-type, consume glutamine, but do not readily convert it to glutamate (Marin-Valencia et al., 2012).”

• cites Marin-Valencia et al., 2012 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

H&E- and IHC-stained tumor sections were examined by a board-certified pathologist.

“Tumor sections stained with H&E or stained for immunohistochemistry (IHC) were reviewed by board-certified pathologist R.D.C.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The CD31 segmentation and classification batch output was reviewed by a pathologist for accuracy and then iteratively retrained.

“The batch output was pathologyreviewed for segmentation and classification accuracy, then optimized via iterative re-training.”

Classified as probably not a substantial claim.

The GC-MS platform was built to measure most proteinogenic amino acids and important central carbon metabolism intermediates.

“Our GC-MS platform is designed to detect the majority of proteinogenic amino acids as well as key intermediates in central carbon metabolism.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Bioluminescence values for Figures 7D, S7B, and S7C were background-corrected.

“For data in Figures 7D, S7B, and S7C, values were corrected for background luminescence.”

Classified as probably not a substantial claim.

2HG was measured by GC-MS using standards of known amounts of pure (R)-2HG spanning 20 ng to 6 μg.

“2HG was quantified in these experimental samples and in standards containing known quantities of pure (R)-2HG (20 ng – 6 μg range) by GC-MS as described above.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the in vivo 15N-leucine tracing study, 8-week-old ICR SCID mice were assigned randomly to a standard diet or a diet containing 50% 15N-leucine 96 hours before organs and plasma were collected.

“8 week old ICR SCID mice (Taconic) were randomized to standard diet or MouseExpress 15Nleucine-containing diet (50% labeled, 50% unlabeled, Cambridge Isotope Laboratories) 96 hours before organs and plasma were harvested.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Every plasmid listed in the study was verified by DNA sequencing.

“All of the plasmids described above were authenticated by DNA sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A recent study found that glutamine contributes to (R)-2HG synthesis in an IDH1 mutant chondrosarcoma model.

“In contrast, a recent study found that glutamine contributes to (R)-2HG synthesis in an IDH1 mutant chondrosarcoma model (SalamancaCardona et al., 2017).”

• cites SalamancaCardona et al., 2017Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Blocking (R)-2HG production causes regressions in IDH1 mutant AML.

“Blocking (R)-2HG production causes regressions in IDH1 mutant AML (Waitkus et al., 2018).”

• cites Waitkus et al., 2018Not checked yet

  Not checked in the free run.

May combine two claims.

Inhibiting GLS in IDH1 mutant cells, in which BCAT is suppressed by (R)-2HG, blocks this adaptation and impairs fitness by lowering glutamate, glutathione, and resistance to oxidative stress.

“Inhibiting GLS in IDH1 mutant cells, in which BCAT is suppressed by (R)-2HG, blocks this adaptation and dramatically impairs their fitness by lowering glutamate, glutathione, and resistance to oxidative stress.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The DAB scoring threshold was set manually by a pathologist to match how they would score brightfield slides microscopically.

“The threshold was manually set by a pathologist as they would score the brightfield slides under a microscope.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the primary xenograft model, once tumors measured 50-350 mm3, mice were assigned randomly to treatment groups 30-45 days after injection.

“Mice were monitored for tumor development and randomized to distinct treatment cohorts 30–45 days post-injection, once tumors had reached 50–350 mm3 in size.”

Probably not a substantial claim: may not pass S3 (load-bearing).

No statistical method was used to predetermine sample size in the animal studies.

“No statistical method was used to predetermine sample size in animal studies.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Three mice with IDH wild-type HOG gliomas had small, diffuse tumors at dissection, so less than 3 mg of tissue was available for analysis.

“3 mice bearing IDH wild-type HOG gliomas displayed small, diffuse tumors upon dissection that precluded recovery of sufficient tissue (<3 mg) for analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In every metabolomics experiment, cells were collected within 6 hours of transfer to SMEM, a limit meant to prevent nutrient depletion and starvation.

“For all metabolomics experiments, cells were harvested no longer than 6 hours after switching to SMEM media in order to avoid nutrient depletion from the media and resulting cell starvation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The BCAT1 and BCAT2 sgRNA templates were aimed at exon-intron boundaries so that ectopically expressed BCAT1 and BCAT2 cDNAs would not be edited.

“BCAT1 and BCAT2 sgRNA templates were designed to target exon-intron boundaries to prevent editing of ectopically-expressed BCAT1 and BCAT2 cDNAs.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the TS603 subcutaneous xenograft CB-839 PD study, mice were randomly assigned to CB-839 or vehicle.

“7–9 weeks after tumor cell implantation, mice were randomized to receive CB839 (200 mg/kg BID via oral gavage) or vehicle control.”

May combine two claims.

Glutamate loss is key because restoring glutamate levels using a GLS hypermorph or OAT rescued the effects of mutant IDH1 on redox homeostasis.

“Glutamate loss is key in this context because we could rescue the effects of mutant IDH1 on redox homeostasis by restoring glutamate levels using a GLS hypermorph or OAT, which is (R)-2HG-insensitive.”

May combine two claims.

The findings were concordant using multiple cell lines, including engineered isogenic cells and patient-derived non-isogenic GSC lines, and were observed in both subcutaneous and orthotopic tumor models.

“Our findings were concordant using multiple cell lines, including engineered isogenic cells and patient-derived non-isogenic GSC lines, and were observed in both subcutaneous and orthotopic tumor models.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For glutathione assays using SMEM, cells were lysed within 8 hours of culture to prevent nutrient depletion and starvation.

“For all glutathione quantification experiments involving SMEM media, cells were cultured for no longer than 8 hours before lysis in order to avoid nutrient depletion from the media and resulting cell starvation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sequencing confirmed that the amplified BCAT1 cDNA matched reference sequence NM_005504.6.

“The amplified BCAT1 cDNA was confirmed by sequencing to match the reference sequence NM_005504.6.”

Classified as probably not a substantial claim.

Isotopologue measurements were corrected for the natural abundance of isotopes.

“Mass isotopologues were quantified and corrected for natural isotope abundance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the orthotopic glioma xenograft experiments, the technicians who measured bioluminescence were unaware of which treatment each mouse received.

“Bioluminescence imaging of tumor-bearing mice was performed by technicians who were blinded to treatment assignments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the HOG orthotopic xenograft CB-839 PK/PD studies, mice were randomly assigned to CB-839 or vehicle.

“3–4 weeks after tumor cell implantation, mice were randomized to receive CB-839 (200 mg/kg BID via oral gavage) or vehicle control.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In all proliferation assays, SMEM medium was replaced every 48 hours and volume increased by 50% to prevent nutrient depletion and starvation.

“In order to avoid nutrient depletion from the media and resulting cell starvation, SMEM media was changed every 48 hours and the volume increased by 50% in all proliferation assays.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For tumor 2HG measurements, a published brain tissue density value was used to convert sample mass into volume.

“For quantification of 2HG in tumors, sample mass was used to estimate sample volume based on a published value of brain tissue density (Bothe et al., 1984).”

• cites Bothe et al., 1984Not checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: Tumor tissue density is similar to the published brain tissue density used to convert tumor mass to volume.

May combine two claims.

The work was limited by the dearth of available tumorigenic IDH1 mutant glioma lines (and no low-grade glioma lines) and by CB839's poor BBB penetration.

“Nonetheless, our work was limited by the dearth of available tumorigenic IDH1 mutant glioma lines (and no low-grade glioma lines) and by CB839’s poor BBB penetration.”