Summary of Study ST001023

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 IDST001023
Study TitleH3K27M cells and glutamine metabolomics 1 million cell test (part-I)
Study SummaryTesting TCA concentrations of Diffuse Intrinsic Pontine Gliomas (DIPG) cellines with H3K27M mutations. One million cells are tested with a TCA concentrations panel. We are a high volume center for treating malignant gliomas, which gives us an advantage in obtaining tissue for these relatively rare tumors. We have developed several DIPG patient derived cell lines and xenografts that bear all the key molecular features of this disease including the H3K27M mutation and global H3K27 hypomethylation. These cells are low in passage and we think these lines more closely resemble the patients tumor pathology then established cell lines that have been in culture/mice for numerous years.
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:SU001062
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 Group cell line
SA064316Sample # 1Cells DIPG XIII
SA064317Sample # 3Cells DIPG XVII
SA064318Sample # 2Media DIPG XIII
SA064319Sample # 4Media DIPG XVII
Showing results 1 to 4 of 4


Collection ID:CO001056
Collection Summary:DIPG XIII 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:TR001076
Treatment Summary:Metabolic profiling will be conducted similarly to published methods and standard methods from our metabolic core. Glucose, glutamine and lactate levels in culture medium will be measured using gas chromatography/mass spectroscopy (GC/MS). Briefly, cells will be seeded in 6 well plates in triplicate using our standard neurosphere media (MH+++) and cultured for 24 to 48h before experiments. Changes in metabolite concentrations relative to fresh media will be normalized to protein content of each well. Cellular metabolite levels will be measured using standard protocols using deuterated 2-hydroxyglutarate (d5-5HG) as an internal standard and analyzed using GC/MS. To determine the fraction of metabolites from Glu and Gln, 13C versions of each metabolite [U-13C]glucose or [U-13C]glutamine (Cambridge Isotope Lab) will be used. All of these experiements will be performed by the Mayo Clinic Metabolomics Research Core.

Sample Preparation:

Sampleprep ID:SP001069
Sampleprep Summary:TCA Concentrations in glioma cell lines

Combined analysis:

Analysis ID AN001680
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:CH001181
Instrument Name:Agilent 7890B
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Chromatography Type:GC


MS ID:MS001555
Analysis ID:AN001680
Instrument Name:Agilent 5977A
Instrument Type:Single quadrupole
MS Type:EI