#METABOLOMICS WORKBENCH hormel101_20180718_154812 DATATRACK_ID:1460 STUDY_ID:ST001023 ANALYSIS_ID:AN001680 PROJECT_ID:PR000684 VERSION 1 CREATED_ON July 19, 2018, 1:34 pm #PROJECT PR:PROJECT_TITLE Mayo Pilot and Feasibility: H3K27M cells and glutamine metabolomics quatitation PR:PROJECT_TITLE studies PR:PROJECT_SUMMARY In children, tumors affecting the brain and nervous system result in more PR:PROJECT_SUMMARY cancer-related deaths than any other type of tumor. It is thus critical to PR:PROJECT_SUMMARY identify new approaches for therapy. Among pediatric patients, one of the most PR:PROJECT_SUMMARY devastating brain tumor types is Diffuse Intrinsic Pontine Gliomas (DIPG). Our PR:PROJECT_SUMMARY understanding of this deadly disease has recently been advanced by important PR:PROJECT_SUMMARY discoveries, including the discovery that the majority of DIPG tumors harbor the PR:PROJECT_SUMMARY histone H3K27M mutation. This mutation results in global hypomethylation of PR:PROJECT_SUMMARY H3K27 residues and is the pathological hallmark for this disease. Glutamine PR:PROJECT_SUMMARY (Gln) addiction has been reported in many cancers including malignant adult PR:PROJECT_SUMMARY gliomas. Glutamine likely promotes cancer cell proliferation and survival likely PR:PROJECT_SUMMARY through generation of the TCA cycle intermediate alpha-ketoglutarate (α-KG). PR:PROJECT_SUMMARY Importantly, α-KG is a critical co-factor for histone lysine demethylases PR:PROJECT_SUMMARY including JMJD3, the enzyme responsible for removing the methyl groups from PR:PROJECT_SUMMARY H3K27me3. Our preliminary data shows H3K27M tumor cells require Gln for PR:PROJECT_SUMMARY survival, and if Gln is removed from the culture media, cells can be rescued by PR:PROJECT_SUMMARY the addition of α-KG. Furthermore, Gln deprivation leads to an increase in PR:PROJECT_SUMMARY H3K27 trimethylation similar to direct inhibition of JMJD3. It is for these PR:PROJECT_SUMMARY reasons we hypothesize that H3K27M tumors are dependent on Gln derived α-KG PR:PROJECT_SUMMARY both for feeding the TCA cycle and for further decreasing H3K27 trimethylation. PR:PROJECT_SUMMARY Inhibition of Gln metabolism will likely uncover novel therapeutic targets for PR:PROJECT_SUMMARY this deadly disease. In Aim 1 we will study Gln and glucose metabolism in H3K27M PR:PROJECT_SUMMARY tumor cells and compare this to Wild Type (WT) tumors and Embryonic Stem Cells PR:PROJECT_SUMMARY (ESCs). In Aim 2 we will validate the therapeutic validity of inhibiting Gln PR:PROJECT_SUMMARY metabolism in H3K27M tumors. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Daniels PR:FIRST_NAME David PR:ADDRESS 200 First Street SW Rochester, MN 55905 PR:EMAIL daniels.david@mayo.edu PR:PHONE 507-284-2511 #STUDY ST:STUDY_TITLE H3K27M cells and glutamine metabolomics 1 million cell test (part-I) ST:STUDY_SUMMARY Testing TCA concentrations of Diffuse Intrinsic Pontine Gliomas (DIPG) cellines ST:STUDY_SUMMARY with H3K27M mutations. One million cells are tested with a TCA concentrations ST:STUDY_SUMMARY panel. We are a high volume center for treating malignant gliomas, which gives ST:STUDY_SUMMARY us an advantage in obtaining tissue for these relatively rare tumors. We have ST:STUDY_SUMMARY developed several DIPG patient derived cell lines and xenografts that bear all ST:STUDY_SUMMARY the key molecular features of this disease including the H3K27M mutation and ST:STUDY_SUMMARY global H3K27 hypomethylation. These cells are low in passage and we think these ST:STUDY_SUMMARY lines more closely resemble the patients tumor pathology then established cell ST:STUDY_SUMMARY lines that have been in culture/mice for numerous years. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Daniels ST:FIRST_NAME David ST:ADDRESS 200 First Street SW Rochester, MN 55905 ST:EMAIL daniels.david@mayo.edu ST:PHONE 507-284-2511 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Sample # 1 Group:Cells | cell line:DIPG XIII # cells=1000000 SUBJECT_SAMPLE_FACTORS - Sample # 3 Group:Cells | cell line:DIPG XVII # cells=1000000 SUBJECT_SAMPLE_FACTORS - Sample # 2 Group:Media | cell line:DIPG XIII # cells=5500000 SUBJECT_SAMPLE_FACTORS - Sample # 4 Group:Media | cell line:DIPG XVII # cells=6886000 #COLLECTION CO:COLLECTION_SUMMARY DIPG XIII and DIPG XVII cell lines are collected in this experiment. Susupension CO:COLLECTION_SUMMARY cells are harvested using centrifugation at 1200 rpm for 5 min. 1 mL of the CO:COLLECTION_SUMMARY supernatant media werr collected in an eppendorf tube and snapped frozen. The CO:COLLECTION_SUMMARY cell pellets were broken up into single cell suspension and counted. 1 million CO:COLLECTION_SUMMARY cells were taken from the stock and washed 1 x with PBS using table top CO:COLLECTION_SUMMARY centrifuge with 10 sec quick spin. The resulting cell pellet was snap frozen. CO:COLLECTION_SUMMARY Both the frozen media and cell pellet are stored in -80 C prior transfer. CO:SAMPLE_TYPE Glioma cells #TREATMENT TR:TREATMENT_SUMMARY Metabolic profiling will be conducted similarly to published methods and TR:TREATMENT_SUMMARY standard methods from our metabolic core. Glucose, glutamine and lactate levels TR:TREATMENT_SUMMARY in culture medium will be measured using gas chromatography/mass spectroscopy TR:TREATMENT_SUMMARY (GC/MS). Briefly, cells will be seeded in 6 well plates in triplicate using our TR:TREATMENT_SUMMARY standard neurosphere media (MH+++) and cultured for 24 to 48h before TR:TREATMENT_SUMMARY experiments. Changes in metabolite concentrations relative to fresh media will TR:TREATMENT_SUMMARY be normalized to protein content of each well. Cellular metabolite levels will TR:TREATMENT_SUMMARY be measured using standard protocols using deuterated 2-hydroxyglutarate TR:TREATMENT_SUMMARY (d5-5HG) as an internal standard and analyzed using GC/MS. To determine the TR:TREATMENT_SUMMARY fraction of metabolites from Glu and Gln, 13C versions of each metabolite TR:TREATMENT_SUMMARY [U-13C]glucose or [U-13C]glutamine (Cambridge Isotope Lab) will be used. All of TR:TREATMENT_SUMMARY these experiements will be performed by the Mayo Clinic Metabolomics Research TR:TREATMENT_SUMMARY Core. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY TCA Concentrations in glioma cell lines #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent HP5-MS (30m × 0.25mm, 0.25 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5977A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/vial MS_METABOLITE_DATA_START Samples Sample # 1 Sample # 3 Sample # 2 Sample # 4 Factors Group:Cells | cell line:DIPG XIII Group:Cells | cell line:DIPG XVII Group:Media | cell line:DIPG XIII Group:Media | cell line:DIPG XVII Lactate 19.7 26.3 8590 7275 Succinate 0.12 0.30 1.7 4.2 Fumarate 0.41 0.63 1.9 2.7 Oxaloacetate 0.006 0.007 0.22 0.30 Ketoglutarate 0.274 0.237 6.1 6.7 Malate 1.61 1.85 3.8 5.2 Aspartate 1.9 2.3 0.4 7.2 2-Hydroxyglutarate 0.203 0.042 0.8 0.3 Glutamate 27.0 18.4 5.2 37.9 cis-Aconitic Acid 0.598 0.394 65.4 47.2 Citrate 30.8 21.4 83.3 69.3 Isocitrate 12.5 7.8 20.0 11.2 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Lactate Succinate Fumarate Oxaloacetate Ketoglutarate Malate Aspartate 2-Hydroxyglutarate Glutamate cis-Aconitic Acid Citrate Isocitrate METABOLITES_END #END