#METABOLOMICS WORKBENCH cendalfr_20240411_082827 DATATRACK_ID:4772 STUDY_ID:ST003170 ANALYSIS_ID:AN005203 PROJECT_ID:PR001970
VERSION             	1
CREATED_ON             	April 11, 2024, 8:36 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           	-	A	Factor:WT CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=1
SUBJECT_SAMPLE_FACTORS           	-	B	Factor:WT CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=2
SUBJECT_SAMPLE_FACTORS           	-	C	Factor:WT CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=3
SUBJECT_SAMPLE_FACTORS           	-	D	Factor:MCJ KO CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=4
SUBJECT_SAMPLE_FACTORS           	-	E	Factor:MCJ KO CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=5
SUBJECT_SAMPLE_FACTORS           	-	F	Factor:MCJ KO CART med | Sample source:Cells	RAW_FILE_NAME(Raw File Name)=6
#COLLECTION
CO:COLLECTION_SUMMARY            	Murine CD8 cells were isolated from lymph nodes and spleen using negative
CO:COLLECTION_SUMMARY            	selection with CD8+ T cell isolation kit (for generation of CAR-T cells)
CO:COLLECTION_SUMMARY            	(Miltenyi, order# 130-104-075) or positive selection (for OT-I CD8 cells) with
CO:COLLECTION_SUMMARY            	CD8 (Ly-2) Microbeads (Miltenyi, order# 130-117-044). Mouse CD8 cells were
CO:COLLECTION_SUMMARY            	activated with plate-bound anti-CD3 (5 g/ml) and soluble anti-CD28 mAbs (1
CO:COLLECTION_SUMMARY            	g/ml) for 48h for WT and cMCJ KO CD8 studies and OT-I CD8 cells studies.
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               	Thermo Vanquish
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	A	B	C	D	E	F
Factors	Factor:WT CART med | Sample source:Cells	Factor:WT CART med | Sample source:Cells	Factor:WT CART med | Sample source:Cells	Factor:MCJ KO CART med | Sample source:Cells	Factor:MCJ KO CART med | Sample source:Cells	Factor:MCJ KO CART med | Sample source:Cells
L-alanine	2899601.25	1423708	2347212.25	2074696	1722981	1278456
L-arginine	4378797.5	2704065	4461140.5	4934806	4223950.5	6972328
L-asparagine	703490.31	544056.44	833265.69	567966.38	395946.41	376895.38
L-aspartate	5977391.5	5584386.5	7831730.5	4871708	3764491.75	2684774.25
L-cysteine	4048.84	0	8091.44	0	4655.7	5678.73
L-glutamate	6848666.5	10564893	12352985	7319765.5	6202989.5	5458377.5
L-glutamine	4403248.5	2513811.75	4662096	2399477.75	2220893.25	1995128
L-histidine	1053737.62	319320.62	526109.19	940233.31	1038589.25	901908
L-leucine/L-isoleucine	18551056	5101423.5	10462659	23616624	26848600	26839890
L-lysine	1556454.62	723165	1213436.62	1772048	1616108.5	1824667.5
L-methionine	2163834	655633.06	1371486.62	3615653.25	4054355	4977298.5
L-phenylalanine	9279409	2495139.75	5557782.5	12300485	12438457	14553895
L-proline	3687341	1669128.5	2985754.75	4483041.5	4816232.5	5357467.5
L-serine	3460968.25	1172575.5	2322425.75	2751616.75	2296694.75	1262677.88
L-threonine	2266535	1505199.38	2384315.75	1677710.38	1276456	902260.44
L-tryptophan	4972201.5	1271265.5	2854987	6331887.5	5395861.5	6540048.5
L-tyrosine	9781055	2439037.75	5448181.5	12511029	12843873	14191539
L-valine	7786622.5	3211052.75	4734124	9403460	10358271	9624791
L-cystine	17054.77	40252.64	43958.91	26954.08	14206.53	15569.73
Adenosine	744224.25	90820.98	263896.41	972697.19	587653.5	675454.56
GMP	140813.75	26505.21	52639.27	519133.22	128707.75	200740.3
CMP	245513.5	179945.58	221565.33	347736.5	321683.28	330805.59
Cytidine	268586.66	104568.44	268844.16	258058.48	238843.33	219376.11
Cytosine	755998.81	471160.28	811429.31	691954.5	834295.56	873001.31
Thymine	61353.93	9672.73	17846.94	54683.97	65031.56	47290.68
Uracil	141063.98	43676.78	104610.27	101250.62	151095.73	192534.17
Hypoxanthine	616633.38	167498.89	420965.59	703927.44	751096.19	810906.75
Xanthine	279047.47	41528.81	158168.95	351033.34	367374.09	347689.12
Pyridoxal	26197.57	29369.78	26379.74	26285.33	28834.73	27215.24
Nicotinamide	50033.61	41158.32	31674.04	1599020	1582243.88	1941346
NAD+	169119.92	97853.34	159148.12	318612.53	266344.31	311710.59
Glutathione disulfide	308701.34	68639.66	239406.64	125322.65	183067.95	215601.55
5-Oxoproline	8317864	748918.06	2611579	12060173	12192916	8213134.5
Cys-Gly	259763.73	119837.75	195899.56	591315.12	574105.25	599195.06
gamma-L-Glutamyl-L-cysteine	43406.39	0	34799.02	87423.65	75943.65	80581.55
5-10-Methenyltetrahydrofolate	6786.34	17884.08	1984.07	2326.09	0	7652.7
Ornithine	1176229.38	199346.05	502341.03	1649606.12	1402677.38	1003263.5
L-Citrulline	918793.5	252538.55	650328.69	882434.25	745029.56	358113.59
Spermidine	160086.17	62881.47	168013.23	7178412	789808.81	430480.09
Creatine	4041169	3179826.75	4313000.5	5077945.5	5148315.5	5679788.5
Creatinine	251845.62	152382.91	189605.12	372297.34	322271.19	257966.98
Guanidinoacetate	37512.12	24503.65	34844.28	30042.47	40801.73	34055.34
Pantothenol	411571.5	172507.56	313711.78	488571.88	542859.81	635116.62
Taurine	5265075.5	5270539	6398993.5	6138257.5	5176429.5	4157402
Hypotaurine	256271.69	258927.19	317501.28	195892.12	196788.69	131817.86
Indole-3-acetate	356172.97	150644.39	288192.38	295362.75	436574.47	747935.5
kynurenine	10813.21	3551.59	4352.9	4409.37	6276.17	14509.63
Sphingosine	3161847.75	1051735	2036684.62	658386	1846358.62	2768849.25
Sphingosine 1-phosphate	147247.88	5067.22	32281.68	383647.84	275147.69	175304.27
Acetylcholine	341317.66	165527.55	300137.22	459234.47	533833.88	540494.5
Choline	16901946	9498761	13981761	25780640	26762016	25263662
L-Carnitine	1296800	312192.47	798093.06	1923007.38	1906532.38	1680421.38
acyl-C2 (acetyl-carnitine)	0	0	2395.08	5016.06	5124.45	3883.29
acyl-C3 (propionyl-carnitine)	88985.52	43960.79	59945.25	151073	169809.11	163341.39
acyl-C4 (butanoyl-l-carnitine)	31386.37	11047.66	21979.24	54573.37	59169.31	56681.35
acyl-C4-OH (Hydroxybutyrylcarnitine)	2786.45	0	2465.62	6528.4	6092.74	3931.44
acyl-C5 (isovalerylcarnitine)	115314.91	25418.54	46752.33	171694.3	211221.27	125328.62
acyl-C5-OH	2651.68	0	2306.64	3251.13	3092	9224.33
acyl-C6 (hexanoyl-l-carnitine)	5302.28	0	5888.43	12565.7	8557.76	0
acyl-C12:1 (O-dodecenoyl-carnitine)	233369.12	202824.5	229522.17	232325.06	322816.88	276151.25
acyl-C14 (O-tetradecanoyl-L-carnitine)	80482.43	15904.25	52073.17	24801.63	61691.72	96589.43
acyl-C14:1 (Tetradecenoyl Carnitine)	111206.41	21080.8	27550.81	18586.01	81515.98	56359.87
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
L-alanine	C00041	90.055	0.683
L-arginine	C00062	175.119	0.661
L-asparagine	C00152	133.061	0.674
L-aspartate	C00049	134.045	0.684
L-cysteine	C00097	122.028	0.598
L-glutamate	C00025	148.060	0.688
L-glutamine	C00064	147.076	0.679
L-histidine	C00135	156.077	0.638
L-leucine/L-isoleucine	C00123	132.102	0.991
L-lysine	C00047	147.113	0.642
L-methionine	C00073	150.058	0.904
L-phenylalanine	C00079	166.086	1.481
L-proline	C00148	116.071	0.710
L-serine	C00065	106.050	0.674
L-threonine	C00188	120.066	0.687
L-tryptophan	C00078	205.097	1.799
L-tyrosine	C00082	182.081	0.933
L-valine	C00183	118.086	0.745
L-cystine	C00491	241.031	0.674
Adenosine	C00212	268.104	0.930
GMP	C00144	364.065	0.805
CMP	C00055	324.059	0.712
Cytidine	C00475	244.093	0.729
Cytosine	C00380	112.051	0.697
Thymine	C00178	127.049	0.737
Uracil	C00106	113.035	0.950
Hypoxanthine	C00262	137.046	0.917
Xanthine	C00385	153.041	0.926
Pyridoxal	C00250	168.065	1.079
Nicotinamide	C00153	123.055	1.010
NAD+	C00003	664.117	0.789
Glutathione disulfide	C00127	613.160	0.912
5-Oxoproline	C01879	130.050	0.926
Cys-Gly	C01419	179.048	0.896
gamma-L-Glutamyl-L-cysteine	C00669	251.070	0.931
5-10-Methenyltetrahydrofolate	C00445	457.170	0.743
Ornithine	C01602	133.097	0.633
L-Citrulline	C00327	176.103	0.688
Spermidine	C00315	146.165	0.602
Creatine	C00300	132.077	0.696
Creatinine	C00791	114.066	0.702
Guanidinoacetate	C00581	118.061	0.689
Pantothenol	C00864	220.118	1.746
Taurine	C00245	126.022	0.690
Hypotaurine	C00519	110.027	0.685
Indole-3-acetate	C00954	176.071	1.981
kynurenine	C00328	209.092	1.475
Sphingosine	C00319	300.290	2.131
Sphingosine 1-phosphate	C06124	380.253	2.197
Acetylcholine	C01996	146.118	0.733
Choline	C00114	104.107	0.675
L-Carnitine	C00318	162.112	0.688
acyl-C2 (acetyl-carnitine)	C02571	204.123	0.930
acyl-C3 (propionyl-carnitine)	C03017	218.139	1.079
acyl-C4 (butanoyl-l-carnitine)	C02862	232.154	1.834
acyl-C4-OH (Hydroxybutyrylcarnitine)	HMDB13127	248.149	0.937
acyl-C5 (isovalerylcarnitine)	C20826	246.170	1.856
acyl-C5-OH	ac107	262.165	1.118
acyl-C6 (hexanoyl-l-carnitine)	HMDB00756	260.186	1.848
acyl-C12:1 (O-dodecenoyl-carnitine)	HMDB13326	342.269	2.738
acyl-C14 (O-tetradecanoyl-L-carnitine)	HMDB05066	372.311	2.172
acyl-C14:1 (Tetradecenoyl Carnitine)	HMDB13329	370.300	3.146
METABOLITES_END
#END