Summary of Study ST001026

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR000684. The data can be accessed directly via it's Project DOI: 10.21228/M8868G This work is supported by NIH grant, U2C- DK119886.


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Study IDST001026
Study TitleTCA cycle metabolomics of H3K27M Cell Nucleus Fraction and Cell Mitonchonrdial Fraction (Part-IV)
Study SummaryTesting TCA concentrations of Diffuse Intrinsic Pontine Gliomas (DIPG) cellines with H3K27M mutations. Preliminary studies show H3K27M tumor cells are addicted to Gln for survival. Removal of Gln from media resulted in tumor cell death which was rescued by the addition of α-KG. These data show that Gln is taken up and metabolized by H3K27M tumor cells and that Gln derived α-KG is critical for the survival of these tumors. Interestingly, tumor cell death with Gln deprivation was similar to the effect of the JMJD3 inhibitor GSKJ4. Therefore, Gln derived α-KG may be required for both anaplerosis and to drive JMJD3 demethylation. We hypothesize that H3K27M tumors are reliant on α-KG that is derived from Gln to drive the TCA cycle and further decrease H3K27 methylation levels. Furthermore, inhibition of Gln metabolism may represent a novel therapeutic approach for tumors with this mutation. In this study, TCA cycle metabolomics are analyzed of H3K27M cells grown in regular glutamine media, glutamine free media, and glutamine free media with alpha-ketoglutarate. Additionally, cell nucleus and cell mitochrondial fractions are run separately.
Mayo Clinic
Last NameDaniels
First NameDavid
Address200 First Street SW Rochester, MN 55905
Submit Date2018-07-18
Analysis Type DetailGC-MS
Release Date2020-07-15
Release Version1
David Daniels David Daniels application/zip

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Project ID:PR000684
Project DOI:doi: 10.21228/M8868G
Project Title:Mayo Pilot and Feasibility: H3K27M cells and glutamine metabolomics quatitation studies
Project Summary:In children, tumors affecting the brain and nervous system result in more cancer-related deaths than any other type of tumor. It is thus critical to identify new approaches for therapy. Among pediatric patients, one of the most devastating brain tumor types is Diffuse Intrinsic Pontine Gliomas (DIPG). Our understanding of this deadly disease has recently been advanced by important discoveries, including the discovery that the majority of DIPG tumors harbor the histone H3K27M mutation. This mutation results in global hypomethylation of H3K27 residues and is the pathological hallmark for this disease. Glutamine (Gln) addiction has been reported in many cancers including malignant adult gliomas. Glutamine likely promotes cancer cell proliferation and survival likely through generation of the TCA cycle intermediate alpha-ketoglutarate (α-KG). Importantly, α-KG is a critical co-factor for histone lysine demethylases including JMJD3, the enzyme responsible for removing the methyl groups from H3K27me3. Our preliminary data shows H3K27M tumor cells require Gln for survival, and if Gln is removed from the culture media, cells can be rescued by the addition of α-KG. Furthermore, Gln deprivation leads to an increase in H3K27 trimethylation similar to direct inhibition of JMJD3. It is for these reasons we hypothesize that H3K27M tumors are dependent on Gln derived α-KG both for feeding the TCA cycle and for further decreasing H3K27 trimethylation. Inhibition of Gln metabolism will likely uncover novel therapeutic targets for this deadly disease. In Aim 1 we will study Gln and glucose metabolism in H3K27M tumor cells and compare this to Wild Type (WT) tumors and Embryonic Stem Cells (ESCs). In Aim 2 we will validate the therapeutic validity of inhibiting Gln metabolism in H3K27M tumors.
Institute:Mayo Clinic
Last Name:Daniels
First Name:David
Address:200 First Street SW Rochester, MN 55905


Subject ID:SU001065
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606


Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id sample type Grouping
SA064476ms6808 - 18Mito Fraction IV aKG
SA064477ms6808 - 17Mito Fraction IV aKG
SA064478ms6808 - 16Mito Fraction IV aKG
SA064470ms6808 - 10Mito Fraction IV GF
SA064471ms6808 - 12Mito Fraction IV GF
SA064472ms6808 - 11Mito Fraction IV GF
SA064473ms6808 - 6Mito Fraction IV REG
SA064474ms6808 - 5Mito Fraction IV REG
SA064475ms6808 - 4Mito Fraction IV REG
SA064485ms6808 - 35Mito Fraction XVII aKG
SA064486ms6808 - 36Mito Fraction XVII aKG
SA064487ms6808 - 34Mito Fraction XVII aKG
SA064479ms6808 - 28Mito Fraction XVII GF
SA064480ms6808 - 30Mito Fraction XVII GF
SA064481ms6808 - 29Mito Fraction XVII GF
SA064482ms6808 - 24Mito Fraction XVII REG
SA064483ms6808 - 22Mito Fraction XVII REG
SA064484ms6808 - 23Mito Fraction XVII REG
SA064494ms6808 - 15Nucleus fraction IV aKG
SA064495ms6808 - 13Nucleus fraction IV aKG
SA064496ms6808 - 14Nucleus fraction IV aKG
SA064488ms6808 - 8Nucleus fraction IV GF
SA064489ms6808 - 7Nucleus fraction IV GF
SA064490ms6808 - 9Nucleus fraction IV GF
SA064491ms6808 - 2Nucleus fraction IV REG
SA064492ms6808 - 3Nucleus fraction IV REG
SA064493ms6808 - 1Nucleus fraction IV REG
SA064503ms6808 - 31Nucleus fraction XVII aKG
SA064504ms6808 - 32Nucleus fraction XVII aKG
SA064505ms6808 - 33Nucleus fraction XVII aKG
SA064497ms6808 - 25Nucleus fraction XVII GF
SA064498ms6808 - 26Nucleus fraction XVII GF
SA064499ms6808 - 27Nucleus fraction XVII GF
SA064500ms6808 - 19Nucleus fraction XVII REG
SA064501ms6808 - 20Nucleus fraction XVII REG
SA064502ms6808 - 21Nucleus fraction XVII REG
Showing results 1 to 36 of 36


Collection ID:CO001059
Collection Summary:DIPG IV and DIPG XVII cell lines are collected in this experiment. Susupension cells are harvested using centrifugation at 1200 rpm for 5 min. 1 mL of the supernatant media werr collected in an eppendorf tube and snapped frozen. The cell pellets were broken up into single cell suspension and counted. 1 million cells were taken from the stock and washed 1 x with PBS using table top centrifuge with 10 sec quick spin. The resulting cell pellet was snap frozen. Both the frozen media and cell pellet are stored in -80 C prior transfer.
Sample Type:Glioma cells


Treatment ID:TR001079
Treatment Summary:Glutamine is required for survival in H3K27M cell lines. DIPG cell lines with the H3K27M mutation were grown in regular media (45mM glucose + 4.5 mM glutamine), glutamine (Gln) free media, and Gln free media with alpha-ketoglutarate (α-KG). Study Design Factors IV REG / GF / aKG = DIPG IV celline grown in regular media / glutamine free media / glutamine free meida with alpha-ketoglutarate XVII REG / GF / aKG = DIPG XVII celline grown in regular media / glutamine free media / glutamine free meida with alpha-ketoglutarate

Sample Preparation:

Sampleprep ID:SP001072
Sampleprep Summary:TCA Concentrations in glioma cell lines separated into nucleus and mitochondrial fractions.

Combined analysis:

Analysis ID AN001683
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890B
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5977A
Units nmol/vial


Chromatography ID:CH001184
Instrument Name:Agilent 7890B
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Chromatography Type:GC


MS ID:MS001558
Analysis ID:AN001683
Instrument Name:Agilent 5977A
Instrument Type:Single quadrupole
MS Type:EI