#METABOLOMICS WORKBENCH cendalfr_20240411_081443 DATATRACK_ID:4771 STUDY_ID:ST003169 ANALYSIS_ID:AN005201 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        	Positive 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                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min
CH:FLOW_GRADIENT                 	95-5% B, 3-5 min hold at 5% 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                      	POSITIVE
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
L-alanine	1197023.88	1327983.88	1022429.81	1568968.62	1427793.12	1062461
L-arginine	6863230.5	7222230.5	6243684.5	7980496	7930764	6132052.5
L-asparagine	535468.31	476826.5	450014.75	631193.06	505875	375585
L-aspartate	1372252.62	1483275.12	1116382.38	1451198.62	1424129	1146679.62
L-glutamate	3973126.75	3948598.75	3736170	4412249.5	4241764.5	3648352
L-glutamine	8488004	8887393	7065745.5	10065109	9675385	7004350
L-histidine	453154.34	577390.25	269786.47	415080.84	422430	382865.53
L-leucine/L-isoleucine	24789086	27747430	24184186	28680718	26054418	20049822
L-lysine	775457.31	877422.56	377104.09	806658.81	727064.5	675014.25
L-methionine	7392514.5	8478231	7065848	8697247	8380496	6430578.5
L-phenylalanine	6978496.5	8475595	6852887.5	7940340	7643455.5	6032851.5
L-proline	6982688	6567523.5	7047762	7652542.5	7620930.5	6172780
L-serine	427444.22	485046.16	345151.25	514835.25	394962.66	229377.73
L-threonine	485082.16	524086.28	511597.22	528426.88	403367.66	419341.84
L-tryptophan	3562216.75	4013098.75	3605524.75	3124009.75	3073252.75	2051719.88
L-tyrosine	10081315	10097824	9413260	9469005	9446284	8265036
L-valine	5042666.5	5201343.5	4247850.5	5328159.5	5261880	4673793.5
AMP	456886.5	351903.41	297473.53	348723.72	443815.5	500947.84
Adenosine	707023.75	664739.56	720749.81	745050.44	850719.25	1729506
Guanine	323993.69	358170.78	292883.28	359655.97	364053.09	299765.22
CMP	71679.34	66506.85	50993.55	79041.96	74343.79	85925.59
Hypoxanthine	358667.34	377833.62	363256.12	335150.09	354565.38	366094.12
Urate	759133.5	815769.31	771022.94	702023.81	665949.5	692983.06
Nicotinamide	1961781.12	1801942.62	1629010.38	1850226.88	1589012	1885181.5
5-Oxoproline	10309443	12489417	8682290	10292717	11624371	8675300
S-Glutathionyl-L-cysteine	224014.3	296579.34	169744.86	320564.03	339876.75	191665.62
gamma-L-Glutamyl-L-cysteine	184416.39	189766.45	110685.09	78813.71	104129.03	163364.39
Ornithine	180562.64	259328.88	27569.28	77610.2	115839.06	36205.36
L-Citrulline	180369.12	164223.02	203855.31	113525.44	134043.44	139997.25
Creatine	4770057	8793808	4054201	4360741.5	3005996.75	10805841
Creatinine	2846940	3534683.25	2355705	2754858	2667432.25	3342950.75
N-Acetyl-L-citrulline	12353771	13820144	11396885	15381347	12215839	10168484
Guanidinoacetate	117404.18	72310.2	98977.09	56083.73	84498.34	67815.17
Pantothenol	440249.59	518334.91	404414.91	529658.81	479921.66	395565.91
Taurine	1408399.12	1351138.62	821823.5	1320948.75	1256985.25	1125754.62
Indole-3-acetaldehyde	285553.47	277493.47	263842.72	8207.73	5372.33	25682.28
Indole-3-acetate	1448145.5	1526587.38	1911843.12	1680266.88	1729845.5	1299315.88
6-Hydroxykynurenic acid	1486375.88	1398272.62	1111398	737258.19	744064.31	398366.47
kynurenine	23525.04	34734.91	31615.96	68574	65982.12	42198.25
N-formyl kynurenine	7078.12	5688.14	8759.02	36287.55	37959.28	22354.58
Anthranilate	230236.42	208531.12	234244.56	180784.58	203353.31	150428.47
g-Oxalo-crotonate	124694.13	104803.19	104126.43	135527.92	135116.95	78721.22
2-Oxoadipate	123582.88	135552.73	192910.25	185970.05	162221.08	104098.6
L-Adrenaline	215566	391056.41	542457.81	226354.56	263924.34	458769.59
Ethanolamine phosphate	337839.84	338737.34	214072.86	291651.41	360397.53	314050.91
Sphingosine	631332.44	368950.78	351429.72	546680.56	492058.91	650668.81
Acetylcholine	139253.92	115964.66	184844.95	113749.4	213390.48	370313.41
Choline	10643782	11372032	12102527	10968815	11590809	10520196
L-Carnitine	4897933.5	5952352.5	3980325.25	4253042.5	3873179.25	6830796
acyl-C2 (acetyl-carnitine)	4928999.5	5844600	3997628	4427018.5	5025709.5	5826114.5
acyl-C3 (propionyl-carnitine)	404599.66	445941.59	396862.59	369899.28	389751.72	449780.78
acyl-C4 (butanoyl-l-carnitine)	144729.33	152380.14	132910.02	127478.52	166861.56	101832.57
acyl-C4-OH (Hydroxybutyrylcarnitine)	194528.62	234728.06	143606.22	179798.36	199170.86	222721.3
acyl-C4-DC	22472.05	40887.15	17242.7	27167.21	27819.52	50310.48
acyl-C6 (hexanoyl-l-carnitine)	78703.78	68030.15	64582.07	74810.56	57538.34	75826.67
acyl-C14 (O-tetradecanoyl-L-carnitine)	474356.84	60020.62	18004.3	152064.39	235384.45	44302.54
acyl-C18 (Octadecanoyl-L-carnitine)	4398604.5	539934.06	98986.34	1182649.38	1317746.25	566479.94
acyl-C20	947354.69	1089732.12	851443.69	692495.62	740909.75	695332.75
acyl-C20:4	288275.88	595105.12	273191.84	126742.95	218085.8	130637.21
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
L-alanine	C00041	90.055092	0.656
L-arginine	C00062	175.118912	0.637
L-asparagine	C00152	133.060074	0.661
L-aspartate	C00049	134.044876	0.671
L-glutamate	C00025	148.060455	0.667
L-glutamine	C00064	147.076462	0.664
L-histidine	C00135	156.076797	0.63
L-leucine/L-isoleucine	C00123	132.102005	1.005
L-lysine	C00047	147.112869	0.636
L-methionine	C00073	150.058319	0.895
L-phenylalanine	C00079	166.086288	1.416
L-proline	C00148	116.070793	0.689
L-serine	C00065	106.050072	0.659
L-threonine	C00188	120.065681	0.662
L-tryptophan	C00078	205.097153	1.761
L-tyrosine	C00082	182.081131	0.899
L-valine	C00183	118.086433	0.668
AMP	C00020	348.070404	0.741
Adenosine	C00212	268.104034	0.895
Guanine	C00242	152.05452	0.897
CMP	C00055	324.059174	0.685
Hypoxanthine	C00262	137.045883	0.883
Urate	C00366	169.035522	0.894
Nicotinamide	C00153	123.055458	0.904
5-Oxoproline	C01879	130.049942	0.943
S-Glutathionyl-L-cysteine	C05526	427.095123	0.675
gamma-L-Glutamyl-L-cysteine	C00669	251.069855	0.895
Ornithine	C01602	133.09726	0.627
L-Citrulline	C00327	176.102966	0.669
Creatine	C00300	132.076859	0.674
Creatinine	C00791	114.066383	0.734
N-Acetyl-L-citrulline	C15532	218.113541	0.661
Guanidinoacetate	C00581	118.061272	0.674
Pantothenol	C00864	220.117981	1.711
Taurine	C00245	126.022072	0.666
Indole-3-acetaldehyde	C00637	160.075653	1.133
Indole-3-acetate	C00954	176.07074	1.969
6-Hydroxykynurenic acid	C08480	206.048141	0.904
kynurenine	C00328	209.091934	1.421
N-formyl kynurenine	C02700	237.087021	1.051
Anthranilate	C00108	138.055038	1.8
g-Oxalo-crotonate	C03453	159.027679	0.888
2-Oxoadipate	C00322	161.046036	1.97
L-Adrenaline	C00788	184.095642	0.662
Ethanolamine phosphate	C00346	142.026413	0.662
Sphingosine	C00319	300.289764	2.149
Acetylcholine	C01996	146.117569	0.739
Choline	C00114	104.107201	0.652
L-Carnitine	C00318	162.112473	0.666
acyl-C2 (acetyl-carnitine)	C02571	204.123032	0.886
acyl-C3 (propionyl-carnitine)	C03017	218.138626	1.061
acyl-C4 (butanoyl-l-carnitine)	C02862	232.154358	1.75
acyl-C4-OH (Hydroxybutyrylcarnitine)	HMDB13127	248.149384	0.897
acyl-C4-DC	HMDB13133	262.128693	0.893
acyl-C6 (hexanoyl-l-carnitine)	HMDB00756	260.185669	1.824
acyl-C14 (O-tetradecanoyl-L-carnitine)	HMDB05066	372.311218	2.05
acyl-C18 (Octadecanoyl-L-carnitine)	HMDB00848	428.373535	2.624
acyl-C20	ac124	458.417389	0.6
acyl-C20:4	ac125	448.341461	2.642
METABOLITES_END
#END