#METABOLOMICS WORKBENCH hormel101_20180718_160139_mwtab.txt DATATRACK_ID:1461 STUDY_ID:ST001024 ANALYSIS_ID:AN001681 PROJECT_ID:PR000684
VERSION             	1
CREATED_ON             	July 19, 2018, 1:35 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                   	TCA cycle metabolomics of H3K27M cells grown in regular glutamine media,
ST:STUDY_TITLE                   	glutamine free media, and glutamine free media with alpha-ketoglutarate
ST:STUDY_TITLE                   	(Part-II)
ST:STUDY_SUMMARY                 	Testing TCA concentrations of Diffuse Intrinsic Pontine Gliomas (DIPG) cellines
ST:STUDY_SUMMARY                 	with H3K27M mutations. Preliminary studies show H3K27M tumor cells are addicted
ST:STUDY_SUMMARY                 	to Gln for survival. Removal of Gln from media resulted in tumor cell death
ST:STUDY_SUMMARY                 	which was rescued by the addition of α-KG. These data show that Gln is taken up
ST:STUDY_SUMMARY                 	and metabolized by H3K27M tumor cells and that Gln derived α-KG is critical for
ST:STUDY_SUMMARY                 	the survival of these tumors. Interestingly, tumor cell death with Gln
ST:STUDY_SUMMARY                 	deprivation was similar to the effect of the JMJD3 inhibitor GSKJ4. Therefore,
ST:STUDY_SUMMARY                 	Gln derived α-KG may be required for both anaplerosis and to drive JMJD3
ST:STUDY_SUMMARY                 	demethylation. We hypothesize that H3K27M tumors are reliant on α-KG that is
ST:STUDY_SUMMARY                 	derived from Gln to drive the TCA cycle and further decrease H3K27 methylation
ST:STUDY_SUMMARY                 	levels. Furthermore, inhibition of Gln metabolism may represent a novel
ST:STUDY_SUMMARY                 	therapeutic approach for tumors with this mutation. In this study, TCA cycle
ST:STUDY_SUMMARY                 	metabolomics are analyzed of H3K27M cells grown in regular glutamine media,
ST:STUDY_SUMMARY                 	glutamine free media, and glutamine free media with alpha-ketoglutarate.
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	Sample type:Cells | Grouping:IV Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 2	Sample type:Cells | Grouping:IV Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 3	Sample type:Cells | Grouping:IV Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 4	Sample type:Media | Grouping:IV Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 5	Sample type:Media | Grouping:IV Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 6	Sample type:Media | Grouping:IV Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 7	Sample type:Cells | Grouping:IV Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 8	Sample type:Cells | Grouping:IV Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 9	Sample type:Cells | Grouping:IV Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 10	Sample type:Media | Grouping:IV Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 11	Sample type:Media | Grouping:IV Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 12	Sample type:Media | Grouping:IV Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 13	Sample type:Cells | Grouping:IV Treatment 2	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 14	Sample type:Cells | Grouping:IV Treatment 2	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 15	Sample type:Cells | Grouping:IV Treatment 2	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 16	Sample type:Media | Grouping:IV Treatment 2	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 17	Sample type:Media | Grouping:IV Treatment 2	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 18	Sample type:Media | Grouping:IV Treatment 2	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 19	Sample type:Cells | Grouping:XVII Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 20	Sample type:Cells | Grouping:XVII Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 21	Sample type:Cells | Grouping:XVII Control	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 22	Sample type:Media | Grouping:XVII Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 23	Sample type:Media | Grouping:XVII Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 24	Sample type:Media | Grouping:XVII Control	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 25	Sample type:Cells | Grouping:XVII Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 26	Sample type:Cells | Grouping:XVII Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 27	Sample type:Cells | Grouping:XVII Treatment 1	Species=Human; Volume=3-5 millions
SUBJECT_SAMPLE_FACTORS           	-	Sample # 28	Sample type:Media | Grouping:XVII Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 29	Sample type:Media | Grouping:XVII Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 30	Sample type:Media | Grouping:XVII Treatment 1	Species=Human; Volume=1 mL
SUBJECT_SAMPLE_FACTORS           	-	Sample # 31	Sample type:Cells | Grouping:XVII Treatment 2	Species=Human; Volume=3-5 millions x2 vials
SUBJECT_SAMPLE_FACTORS           	-	Sample # 32	Sample type:Cells | Grouping:XVII Treatment 2	Species=Human; Volume=3-5 millions x2 vials
SUBJECT_SAMPLE_FACTORS           	-	Sample # 33	Sample type:Cells | Grouping:XVII Treatment 2	Species=Human; Volume=3-5 millions x2 vials
SUBJECT_SAMPLE_FACTORS           	-	Sample # 34	Sample type:Media | Grouping:XVII Treatment 2	Species=Human; Volume=1 mL x2 vials
SUBJECT_SAMPLE_FACTORS           	-	Sample # 35	Sample type:Media | Grouping:XVII Treatment 2	Species=Human; Volume=1 mL x2 vials
SUBJECT_SAMPLE_FACTORS           	-	Sample # 36	Sample type:Media | Grouping:XVII Treatment 2	Species=Human; Volume=1 mL x2 vials
#COLLECTION
CO:COLLECTION_SUMMARY            	DIPG IV 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             	"Glutamine is required for survival in H3K27M cell lines. DIPG cell lines with
TR:TREATMENT_SUMMARY             	the H3K27M mutation were grown in regular media (45mM glucose + 4.5 mM
TR:TREATMENT_SUMMARY             	glutamine), glutamine (Gln) free media, and Gln free media with
TR:TREATMENT_SUMMARY             	alpha-ketoglutarate (α-KG). Study Design Factors IV Control / Treatment 1 /
TR:TREATMENT_SUMMARY             	Treatment2 = DIPG IV celline grown in regular media / glutamine free media /
TR:TREATMENT_SUMMARY             	glutamine free meida with alpha-ketoglutarate XVII Control / Treatment 1 /
TR:TREATMENT_SUMMARY             	Treatment2 = DIPG XVII celline grown in regular media / glutamine free media /
TR:TREATMENT_SUMMARY             	glutamine free meida with alpha-ketoglutarate"
#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 # 2	Sample # 3	Sample # 7	Sample # 8	Sample # 9	Sample # 13	Sample # 14	Sample # 15	Sample # 19	Sample # 20	Sample # 21	Sample # 25	Sample # 26	Sample # 27	Sample # 31	Sample # 32	Sample # 33	Sample # 4	Sample # 5	Sample # 6	Sample # 10	Sample # 11	Sample # 12	Sample # 16	Sample # 17	Sample # 18	Sample # 22	Sample # 23	Sample # 24	Sample # 28	Sample # 29	Sample # 30	Sample # 34	Sample # 35	Sample # 36
Factors	Sample type:Cells | Grouping:IV Control	Sample type:Cells | Grouping:IV Control	Sample type:Cells | Grouping:IV Control	Sample type:Cells | Grouping:IV Treatment 1	Sample type:Cells | Grouping:IV Treatment 1	Sample type:Cells | Grouping:IV Treatment 1	Sample type:Cells | Grouping:IV Treatment 2	Sample type:Cells | Grouping:IV Treatment 2	Sample type:Cells | Grouping:IV Treatment 2	Sample type:Cells | Grouping:XVII Control	Sample type:Cells | Grouping:XVII Control	Sample type:Cells | Grouping:XVII Control	Sample type:Cells | Grouping:XVII Treatment 1	Sample type:Cells | Grouping:XVII Treatment 1	Sample type:Cells | Grouping:XVII Treatment 1	Sample type:Cells | Grouping:XVII Treatment 2	Sample type:Cells | Grouping:XVII Treatment 2	Sample type:Cells | Grouping:XVII Treatment 2	Sample type:Media | Grouping:IV Control	Sample type:Media | Grouping:IV Control	Sample type:Media | Grouping:IV Control	Sample type:Media | Grouping:IV Treatment 1	Sample type:Media | Grouping:IV Treatment 1	Sample type:Media | Grouping:IV Treatment 1	Sample type:Media | Grouping:IV Treatment 2	Sample type:Media | Grouping:IV Treatment 2	Sample type:Media | Grouping:IV Treatment 2	Sample type:Media | Grouping:XVII Control	Sample type:Media | Grouping:XVII Control	Sample type:Media | Grouping:XVII Control	Sample type:Media | Grouping:XVII Treatment 1	Sample type:Media | Grouping:XVII Treatment 1	Sample type:Media | Grouping:XVII Treatment 1	Sample type:Media | Grouping:XVII Treatment 2	Sample type:Media | Grouping:XVII Treatment 2	Sample type:Media | Grouping:XVII Treatment 2
Lactate	75.6	209.3	101.9	77.3	80.3	79.0	89.3	100.2	76.4	547.1	425.8	519.4	544.8	496.3	522.7	111.4	202.9	231.0	13485.1	13823.9	14040.5	12478.6	11388.3	11701.5	9015.0	8978.4	9310.8	16075.9	15706.2	15508.0	15912.3	16674.3	17126.3	2228.9	2540.7	3210.2
Succinate	1.21	3.30	1.52	0.42	0.46	0.36	0.81	0.93	0.79	1.61	1.60	1.56	1.26	1.09	1.12	4.13	8.38	5.29	1.99	2.07	2.12	1.07	1.00	0.98	49.41	47.05	49.38	12.30	11.73	10.95	4.41	4.32	4.34	68.30	67.81	78.26
Fumarate	2.14	4.17	2.87	0.74	0.88	1.02	1.50	2.12	1.70	1.65	2.37	1.85	0.72	0.62	0.76	0.51	0.98	1.77	4.11	3.45	3.64	1.40	1.30	1.18	4.57	4.09	4.47	4.68	4.55	4.82	2.12	2.00	2.07	1.81	1.68	1.98
Oxaloacetate	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Ketoglutarate	0.131	0.387	0.265	0.093	0.125	0.137	0.366	0.373	0.323	0.725	0.589	0.707	0.153	0.138	0.146	55.293	63.484	34.487	2.0	1.9	1.8	2.1	2.0	2.0	3068.4	3030.8	3037.4	11.6	11.4	11.2	4.1	3.8	3.8	2472.7	2473.1	2697.8
Malate	10.85	23.43	13.19	1.19	1.46	1.43	5.29	7.02	5.26	6.27	6.65	6.61	1.21	1.08	1.00	2.29	5.12	8.68	9.7	9.8	10.1	2.4	2.3	2.3	14.1	13.5	14.7	14.2	14.0	13.5	5.0	4.9	4.8	4.1	4.3	6.4
Aspartate	51.9	129.9	66.7	1.7	2.0	1.3	5.5	6.5	6.0	12.7	13.2	12.9	2.1	1.8	2.1	0.1	0.3	1.7	110.0	114.1	112.7	0.6	0.8	0.9	2.7	4.8	4.0	0.7	0.9	0.7	0.5	0.5	0.5	0.5	0.7	0.6
2-Hydroxyglutarate	0.089	0.176	0.106	0.234	0.314	0.292	0.259	0.295	0.252	0.119	0.116	0.120	0.101	0.111	0.105	2.363	5.233	2.575	0.20	0.13	0.12	0.15	0.11	0.19	1.85	1.65	1.93	0.42	0.38	0.42	0.27	0.22	0.24	18.81	20.30	16.08
Glutamate	103.7	224.2	128.6	7.9	11.3	10.3	35.4	40.1	35.1	114.0	120.8	114.8	9.7	8.4	7.8	38.5	79.0	74.0	260.1	269.8	263.6	1.3	1.8	2.6	39.6	61.4	67.1	4.8	6.8	5.8	0.7	0.8	0.8	368.5	376.3	356.6
cis-Aconitic Acid	0.052	0.123	0.078	0.033	0.043	0.052	0.083	0.102	0.083	0.094	0.075	0.080	0.042	0.040	0.041	0.048	0.134	0.129	11.17	4.28	2.62	1.83	1.32	1.48	2.83	2.11	2.55	6.82	6.20	5.85	5.84	5.40	5.95	1.11	1.37	2.07
Citrate	2.4	3.7	3.1	1.3	1.9	2.8	3.8	4.3	3.6	4.4	3.2	3.8	2.0	1.8	1.8	2.6	5.2	4.5	43.3	43.5	42.4	37.1	32.0	36.9	63.8	55.5	58.9	95.2	84.4	80.2	58.1	53.5	52.7	19.8	20.1	42.4
Isocitrate	0.1	0.1	0.1	0.0	0.1	0.1	0.1	0.2	0.1	0.1	0.1	0.1	0.1	0.0	0.0	0.1	0.2	0.1	0.52	0.24	0.00	0.11	0.10	0.11	0.27	0.18	0.22	0.70	0.55	0.47	0.44	0.32	0.43	0.26	0.25	0.36
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