#METABOLOMICS WORKBENCH cendalfr_20240411_081443 DATATRACK_ID:4771 STUDY_ID:ST003169 ANALYSIS_ID:AN005200 PROJECT_ID:PR001970
VERSION             	1
CREATED_ON             	April 11, 2024, 8:27 am
#PROJECT
PR:PROJECT_TITLE                 	Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T
PR:PROJECT_TITLE                 	cell therapy efficacy
PR:PROJECT_SUMMARY               	Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an
PR:PROJECT_SUMMARY               	important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial
PR:PROJECT_SUMMARY               	respiration is essential for survival and function of CAR-T cells, but
PR:PROJECT_SUMMARY               	developing strategies to specifically enhance mitochondrial respiration has been
PR:PROJECT_SUMMARY               	challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of
PR:PROJECT_SUMMARY               	mitochondrial Complex I, as a metabolic target to enhance mitochondrial
PR:PROJECT_SUMMARY               	respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their
PR:PROJECT_SUMMARY               	in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in
PR:PROJECT_SUMMARY               	TCR- specific CD8 cells also increases their efficacy against solid tumors in
PR:PROJECT_SUMMARY               	vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing
PR:PROJECT_SUMMARY               	MCJ expression increases mitochondrial metabolism and anti-tumor activity of
PR:PROJECT_SUMMARY               	human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a
PR:PROJECT_SUMMARY               	promising therapeutic strategy to improve the efficacy of adoptive T cell
PR:PROJECT_SUMMARY               	therapies.
PR:INSTITUTE                     	University of Colorado School of Medicine
PR:LABORATORY                    	Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon
PR:LAST_NAME                     	Cendali
PR:FIRST_NAME                    	Francesca
PR:ADDRESS                       	13199 East Montview Boulevard, Aurora, CO, 80045, USA
PR:EMAIL                         	francesca.cendali@cuanschutz.edu
PR:PHONE                         	3037246131
#STUDY
ST:STUDY_TITLE                   	Releasing the mitochondrial respiration brake MCJ/DnaJC15 enhances CD8 CAR-T
ST:STUDY_TITLE                   	cell therapy efficacy
ST:STUDY_SUMMARY                 	Metabolism of chimeric antigen receptor (CAR) T cells is emerging as an
ST:STUDY_SUMMARY                 	important area to improve CAR-T cell therapy in cancer treatment. Mitochondrial
ST:STUDY_SUMMARY                 	respiration is essential for survival and function of CAR-T cells, but
ST:STUDY_SUMMARY                 	developing strategies to specifically enhance mitochondrial respiration has been
ST:STUDY_SUMMARY                 	challenging. Here we identify MCJ/DnaJC15, an endogenous negative regulator of
ST:STUDY_SUMMARY                 	mitochondrial Complex I, as a metabolic target to enhance mitochondrial
ST:STUDY_SUMMARY                 	respiration in CD8 CAR-T cells. Loss of MCJ in CD8 CAR-T cells increases their
ST:STUDY_SUMMARY                 	in vitro and in vivo efficacy against mouse B cell leukemias. MCJ deficiency in
ST:STUDY_SUMMARY                 	TCR- specific CD8 cells also increases their efficacy against solid tumors in
ST:STUDY_SUMMARY                 	vivo. Furthermore, we reveal that human CD8 cells express MCJ and that silencing
ST:STUDY_SUMMARY                 	MCJ expression increases mitochondrial metabolism and anti-tumor activity of
ST:STUDY_SUMMARY                 	human CAR-T cells. Thus, targeting MCJ to enhance mitochondrial metabolism is a
ST:STUDY_SUMMARY                 	promising therapeutic strategy to improve the efficacy of adoptive T cell
ST:STUDY_SUMMARY                 	therapies.
ST:INSTITUTE                     	University of Colorado School of Medicine
ST:LABORATORY                    	Laboratory of Angelo D'Alessandro in collaboration with Mercedes Rincon
ST:LAST_NAME                     	Cendali
ST:FIRST_NAME                    	Francesca
ST:ADDRESS                       	13199 East Montview Boulevard, Aurora, CO, 80045, USA
ST:EMAIL                         	francesca.cendali@cuanschutz.edu
ST:PHONE                         	3037246131
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	1	Factor:WT CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=1
SUBJECT_SAMPLE_FACTORS           	-	2	Factor:WT CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=2
SUBJECT_SAMPLE_FACTORS           	-	3	Factor:WT CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=3
SUBJECT_SAMPLE_FACTORS           	-	4	Factor:MCJ KO CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=4
SUBJECT_SAMPLE_FACTORS           	-	5	Factor:MCJ KO CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=5
SUBJECT_SAMPLE_FACTORS           	-	6	Factor:MCJ KO CART in vivo | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=6
#COLLECTION
CO:COLLECTION_SUMMARY            	The E2a cells (106) were cultured in CMM for no more than 2 passages before
CO:COLLECTION_SUMMARY            	intravenous tail vail injection to B6 mice 3 days before CAR-T cell injection.
CO:COLLECTION_SUMMARY            	The E2a-bearing mice were irradiated with 500cGy sublethal dosage the day before
CO:COLLECTION_SUMMARY            	CAR-T cell injection. The murine CD8 CAR-T cells were generated and expanded
CO:COLLECTION_SUMMARY            	with rhIL-2 (60 IU/ml) as described above. The expanded CD8 CAR-T cells were
CO:COLLECTION_SUMMARY            	washed with PBS, and 106 CAR+ cells were injected into the E2a-bearing mice via
CO:COLLECTION_SUMMARY            	intravenous tail vail injection. The mice were followed for survival with the
CO:COLLECTION_SUMMARY            	humane end point of development of hind limb paralysis, or weight loss of more
CO:COLLECTION_SUMMARY            	than 20% of the original weight, or signs of major discomfort as determined by
CO:COLLECTION_SUMMARY            	the institutional veterinarian.
CO:SAMPLE_TYPE                   	T-cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Metabolomics differences in this experiment are observed at baseline
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	CAR-T cells were isolated as described above either from in vitro culture or
SP:SAMPLEPREP_SUMMARY            	from bone marrow harvested in an in vivo study. The cells were washed in PBS and
SP:SAMPLEPREP_SUMMARY            	frozen at -80C until the assay is ready to run. Metabolites from cells were
SP:SAMPLEPREP_SUMMARY            	extracted at 2x106 cells/ml at 4°C (30 min) in the presence of 5:3:2
SP:SAMPLEPREP_SUMMARY            	MeOH:MeCN:water (v/v/v). The samples were spun down and the resulting
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to new tubes and dried under a vacuum. The resulting
SP:SAMPLEPREP_SUMMARY            	residue was reconstituted in 0.1% formic acid at a 3x concentration, then
SP:SAMPLEPREP_SUMMARY            	analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive MS as
SP:SAMPLEPREP_SUMMARY            	previously described in detail.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Negative ion Mode
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Exploris120
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
CH:SOLVENT_A                     	95% water/5% acetonitrile; 1 mM ammonium acetate
CH:SOLVENT_B                     	95% acetonitrile/5% water; 1 mM ammonium acetate
CH:FLOW_GRADIENT                 	0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min
CH:FLOW_GRADIENT                 	100-0% B, 3-5 min hold at 0% B
CH:FLOW_RATE                     	0.450ml/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms,
MS:MS_COMMENTS                   	microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV,
MS:MS_COMMENTS                   	capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas
MS:MS_COMMENTS                   	0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were
MS:MS_COMMENTS                   	annotated and integrated using Maven in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak Areas
MS_METABOLITE_DATA_START
Samples	1	2	3	4	5	6
Factors	Factor:WT CART in vivo | Sample source:Cells	Factor:WT CART in vivo | Sample source:Cells	Factor:WT CART in vivo | Sample source:Cells	Factor:MCJ KO CART in vivo | Sample source:Cells	Factor:MCJ KO CART in vivo | Sample source:Cells	Factor:MCJ KO CART in vivo | Sample source:Cells
3',5'-Cyclic AMP	300820.16	291389.66	283999.19	364222	413530.12	385803.25
UDP-glucose	78019.73	264155.34	188787.3	226078.61	118955.24	79944.06
ADP-D-ribose	30781.57	31617.88	20426.83	77489.49	29884.48	67544.46
Flavin mononucleotide	116998.47	63665.12	183561.33	16340.81	8665.42	82865.02
NAD+	455241.22	475134	376562.84	431355.25	322604.38	308721.53
NADH	16624.96	20875.69	21708.31	25141.91	17288.89	21527.54
Phosphate	4708291.5	6032085.5	3931543.75	5765092	5028842	5412158
Diphosphate	239318.67	360335.62	183459.67	342133.66	268239.47	305427.28
D-Glucose	6532598.5	6874687.5	4593389.5	8340493.5	4728896	6785037.5
D-Fructose 1-6-bisphosphate	86957.12	123120.79	105511.62	117419.12	85078.4	39957.36
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	11577917	12131144	10963801	10314201	10525097	9543872
2/3-Phospho-D-glycerate	128030.65	131101.64	16124.37	171737.69	66588.05	64000.11
Pyruvate	788084.44	764327.5	576449.06	730107.69	512558.25	573920.06
Lactate	24824090	22721558	14517621	18108718	19331342	11601363
Maltose	113289.34	122825.24	219278.3	17408.34	23193.05	66636.04
Mannitol	1752122.12	2208180.75	1009614.69	1603708.38	1605212.62	1215720.75
D-Ribose	188523.23	235587.89	118950.27	218383.62	133937.47	175212.58
Citrate	351997.97	395456.75	39168.57	318693.91	161904.8	187807.08
2-Oxoglutarate	82676.9	22868.48	15903.86	54510.99	87693.96	72807.1
Succinate	914513.5	1466651.62	766620.31	1040283.69	986377.44	969151.31
Fumarate	98240.48	194259.62	91462.41	160623.19	124196.01	102530.76
Malate	323225.22	254501.98	47477.34	256219.8	226898.81	193257.67
Glutathione	4450507.5	4735921	3779081.25	3976812	3853983.75	4707613.5
Glutathione disulfide	371886.5	571333.94	341167.12	340488.22	271317.78	193323.48
Dimethylglycine	271605.47	409256.66	232446.3	338687.66	325075.47	218092.42
3-Phosphonooxypyruvate	12572125	7406658.5	14424561	10828133	15684227	12670995
N-Acetylneuraminate	302473.34	296080.34	182300.88	302430.75	244089.98	179568.95
N-Glycoloyl-neuraminate	578931.44	757934.06	427743.03	709567.31	479860.53	317187.09
Thiocysteine	2737592.5	1500180.88	2448839.25	2005125.38	2784178.25	2608847.75
2-Aminomuconate	123057.24	159401.62	161439.28	170675.58	95521.85	121663.28
Hexanoic acid (caproate)	746891.31	1049169.38	961799.81	1987071.62	1722644	1411357.88
Heptanoic acid	465283.12	470386.75	531094.94	614395.44	569229.81	485931.97
Octanoic acid (caprylate)	1109976.38	1080138.62	1074314.62	1314804.25	1313111	1024136.31
Nonanoic acid (pelargonate)	10606719	11259147	13723145	14377468	15216276	11314125
Decanoic acid (caprate)	5107329.5	3797142.75	4907762.5	5416038.5	4476081.5	2461855.25
Dodecanoic acid	3431239	2237240.75	2911579.75	3453956	3732525.75	2788487.75
Tetradecanoic acid	4620953	4267733.5	3724754	6238420.5	5590310	5348239.5
Hexadecanoic acid	33964304	31077094	25996198	36264532	35238320	44074524
Octadecanoic acid	1723014.38	16486327	15357951	15804653	5443690.5	4279789.5
Hexadecenoic acid	2263770.5	1666643.62	1764144.38	2198247.25	2535006.75	1980509.62
Octadecenoic acid	1917746.5	1674860.12	1556654.88	1868171	1844765.12	1814959.62
Linoleate	517200.59	507455.84	508105	376877.12	493465.09	520202.09
Dodecanedioic acid	5190450.5	4577127.5	4802036	4328291.5	5174980	5433636
(8Z-11Z-14Z)-Icosatrienoic acid	63345.96	67534.84	79913.84	14210.91	24139.67	49462.86
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
3',5'-Cyclic AMP	C00575	328.04538	1.032
UDP-glucose	C00029	565.047791	0.795
ADP-D-ribose	C01882	558.064331	0.798
Flavin mononucleotide	C00061	455.101807	0.691
NAD+	C00003	662.102051	0.834
NADH	C00004	664.107178	0.838
Phosphate	C00009	96.969635	1.175
Diphosphate	C00013	176.935577	1.154
D-Glucose	C00031	179.056107	0.663
D-Fructose 1-6-bisphosphate	C00354	338.993134	0.661
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	C00118	168.989471	1.766
2/3-Phospho-D-glycerate	C00631	184.985397	0.61
Pyruvate	C00022	87.008774	0.662
Lactate	C01432	89.024406	0.793
Maltose	C00208	341.108063	0.698
Mannitol	C00392	181.071747	0.664
D-Ribose	C00121	149.045563	0.666
Citrate	C00158	191.019714	0.632
2-Oxoglutarate	C00026	145.014603	0.644
Succinate	C00042	117.019394	0.797
Fumarate	C00122	115.003708	0.811
Malate	C00149	133.014008	0.644
Glutathione	C00051	306.076599	0.795
Glutathione disulfide	C00127	611.144775	0.796
Dimethylglycine	C01026	102.056061	0.607
3-Phosphonooxypyruvate	C03232	182.967133	0.744
N-Acetylneuraminate	C00270	308.098907	0.639
N-Glycoloyl-neuraminate	C03410	324.093781	0.623
Thiocysteine	C01962	151.986206	0.743
2-Aminomuconate	C02220	156.028748	0.668
Hexanoic acid (caproate)	C01585	115.076462	1.871
Heptanoic acid	C17714	129.092117	1.962
Octanoic acid (caprylate)	C06423	143.107758	2.087
Nonanoic acid (pelargonate)	C01601	157.123428	2.191
Decanoic acid (caprate)	C01571	171.139053	2.268
Dodecanoic acid	C02679	199.17038	2.47
Tetradecanoic acid	C06424	227.20166	2.742
Hexadecanoic acid	C00249	255.232941	3.154
Octadecanoic acid	C01530	283.264282	3.776
Hexadecenoic acid	C08362	253.217285	2.766
Octadecenoic acid	C00712	281.249115	3.189
Linoleate	C01595	279.232971	2.843
Dodecanedioic acid	C02678	229.144531	2.121
(8Z-11Z-14Z)-Icosatrienoic acid	C03242	305.248688	2.937
METABOLITES_END
#END