#METABOLOMICS WORKBENCH cendalfr_20240411_073626 DATATRACK_ID:4770 STUDY_ID:ST003167 ANALYSIS_ID:AN005197 PROJECT_ID:PR001970
VERSION             	1
CREATED_ON             	April 11, 2024, 8:12 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               	Homo sapiens
#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           	-	D16 19BBz_2	Factor:low 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=2
SUBJECT_SAMPLE_FACTORS           	-	D16 19BBz_3	Factor:low 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=3
SUBJECT_SAMPLE_FACTORS           	-	D16 19BBz_4	Factor:low 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=4
SUBJECT_SAMPLE_FACTORS           	-	D26 19BBz_2	Factor:high 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=11
SUBJECT_SAMPLE_FACTORS           	-	D26 19BBz_3	Factor:high 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=12
SUBJECT_SAMPLE_FACTORS           	-	D26 19BBz_4	Factor:high 19BBz | Sample source:Cells	RAW_FILE_NAME(Raw File)=13
#COLLECTION
CO:COLLECTION_SUMMARY            	Human CD8 cells were isolated with positive selection as described. The CD8
CO:COLLECTION_SUMMARY            	cells were activated with anti-CD3/anti-CD28 beads (Dynabeads™ Human
CO:COLLECTION_SUMMARY            	T-Expander CD3/CD28, Gibco), as previously described107. After 48h activation,
CO:COLLECTION_SUMMARY            	the CD8 cells were spun (1000xg for 2h at 32C) with lentiviral supernatant
CO:COLLECTION_SUMMARY            	containing CD19-BBz/shMCJ-1 CAR, CD19-BBz/shMCJ-2 CAR or CD19-BBz/shMCJ-2 CAR
CO:COLLECTION_SUMMARY            	construct-packing virus with rhIL-2 (40 IU/ml) and protamine sulfate. After
CO:COLLECTION_SUMMARY            	transduction, the anti-CD3/anti-CD28 beads were removed and CD8 cells were
CO:COLLECTION_SUMMARY            	expanded with rhIL-2 (100 IU/ml) for the specified number of days.
CO:SAMPLE_TYPE                   	T-cells
#TREATMENT
TR:TREATMENT_SUMMARY             	The human CD19-BBz shRNA CAR lentiviral constructs were based on a previously
TR:TREATMENT_SUMMARY             	described CD19-BBz CAR containing the human CD19-binding scFV FMC63, CD8 hinge
TR:TREATMENT_SUMMARY             	domain, 4-1BB costimulatory domain and CD3 chain105. Using CD19-BBz CAR
TR:TREATMENT_SUMMARY             	plasmid as a cloning vector, we generated multiple vectors where we incorporated
TR:TREATMENT_SUMMARY             	the RNA polymerase III U6 promotor (on the 3' of the CD3 chain domain)
TR:TREATMENT_SUMMARY             	followed by an shRNA: 1) a CD19-BBz/shMCJ-1 CAR construct containing the shMCJ-1
TR:TREATMENT_SUMMARY             	5’-GAAGATTTCAACTCCTAGC-3’ sequence106, 2) a CD19-BBz/shMCJ-2 CAR construct
TR:TREATMENT_SUMMARY             	containing the shMCJ-2; 5’-AACCTCTAGAACAAGTTATC-3’, and 3) a
TR:TREATMENT_SUMMARY             	CD19-BBz/c-shRNA CAR vectors expressing the shRNA encoding scramble sequences.
TR:TREATMENT_SUMMARY             	Lentiviral supernatant was produced in the LentiX-293T packaging cell line
TR:TREATMENT_SUMMARY             	(Clonetech) as previously described. Lentiviral supernatants were collected
TR:TREATMENT_SUMMARY             	after 48 hours post-transfection.
#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 (30 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                 	0-0.3 min 5% B, 0.3-.8 min hold at 95% B, .8-.81 min 95-5% B, .81-1 min hold at
CH:FLOW_GRADIENT                 	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               	Exploris120
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	D16 19BBz_2	D16 19BBz_3	D16 19BBz_4	D26 19BBz_2	D26 19BBz_3	D26 19BBz_4
Factors	Factor:low 19BBz | Sample source:Cells	Factor:low 19BBz | Sample source:Cells	Factor:low 19BBz | Sample source:Cells	Factor:high 19BBz | Sample source:Cells	Factor:high 19BBz | Sample source:Cells	Factor:high 19BBz | Sample source:Cells
L-arginine	4836214	6785196	6755478	10302230	9424327	7626944
L-asparagine	37503.3	42690.48	67833.41	60725.81	71021.97	65893.31
L-aspartate	28981.37	41678.82	14709.47	16476.69	14864.61	12181.24
L-glutamate	802946.3	845804.8	600798.2	183256.9	206508	356782.1
L-glutamine	3439261	3939573	3915508	4815704	4532506	4128390
L-histidine	369375.3	513238.9	481658.2	682251.3	629307.6	553394.5
L-lysine	357767.5	453960	490274.5	584052.2	651972.6	428345.4
L-methionine	3574919	4810836	7134604	14361280	19849850	8304524
L-phenylalanine	13252870	24823610	33770950	53692260	60064010	36744040
L-proline	3105265	3811522	2677829	2095116	2496928	2296737
L-serine	32324.36	33420.47	25410.65	65750.81	63232.71	29389.21
L-threonine	142195.6	153783.9	124187.9	212152.8	209963.6	125985.2
L-tryptophan	2942730	4261352	7024666	9710075	10706360	6115136
L-valine	2231444	3414796	3889653	6098210	6347984	3617895
L-cystine	64583	107327	98759.41	144405	131719.7	126396.3
ADP	59791.2	94461.95	58093.92	22428.92	30390.95	21674.16
AMP	1419016	1362332	4631006	409236.2	863352.3	3470705
Adenosine	79032.62	80378.93	107412.7	110002.9	480938.2	120737.6
GMP	58893.68	70706.32	96280.62	16548.04	24222.9	62658.27
Guanine	313441.5	386815.1	373353.7	516909.1	572704.1	300565.6
CDP	317518.8	398792	446556.6	571555.1	543438.1	443421.8
Cytosine	243299.5	312363.8	375206	279655.5	278067.8	294016.2
Thymine	41042.51	78981.81	102438.5	178650.7	196509.5	116619.8
IMP	202223.9	287439.4	207479.2	7695.023	12793.13	13010.5
Inosine	5338734	8588710	13515070	26243070	28806180	14170390
Hypoxanthine	5187894	7013674	8552895	12458630	14052880	11026250
Xanthine	128902.8	188495.3	306592.7	574331.6	585139.2	309872.5
5-Hydroxyisourate	105950.3	126693.8	138815.2	142087.2	105340.5	127299.7
Urate	180137	332912.8	401141.8	605900.7	855114.8	542341.6
3',5'-Cyclic AMP	372240.7	374187.1	279015.5	166721.4	189206	232901
Pyridoxal	1499482	2381946	2602829	5575752	4712484	3858727
4-Pyridoxate	92575.34	147170.7	135681.3	274874	221752.3	183305.3
Pyridoxamine 5'-phosphate	21560.37	43064.54	55883.83	174109.2	185150.3	91242.63
Nicotinamide	4105021	4327748	5960306	9815957	9175447	7905774
Glutathione	3386297	6186050	49255.19	306551.4	704639.2	2637615
Glutathione disulfide	799686.1	1212538	367034.6	15968.2	44457.63	346490
5-Oxoproline	5487150	9509547	12104630	19642490	22481430	13354610
S-Glutathionyl-L-cysteine	193332.7	73193.23	137240.8	11178.67	9449.616	53960.24
gamma-L-Glutamyl-D-alanine	98246.91	117512.2	129246.5	95466.15	127197.7	98780.81
Folate	64723.56	111532.2	154574.2	316011.2	403372.9	174976.3
Ornithine	32679.47	71855.24	89619.16	192331.6	195803	93312.7
Argininosuccinate	12842.21	15907.12	233074.6	60032.23	56678.19	66238.17
Cadaverine	30384.57	57404.28	29127.74	85229.2	80728.38	59815.26
Spermidine	133397.6	260838.5	511260.2	92443.25	161260.7	729062.2
Spermine	34560.02	100809.7	291594.1	193208.4	257733.1	346499.1
D-Glucosamine	149951.5	220186.3	222498.9	244619.9	262140.4	217882.6
Creatine	680199.1	781028.2	993825.8	869068.5	870444.2	907648.2
Guanidinoacetate	36957.72	49598.77	79591.41	121270.3	157379.3	93209.66
Pantothenol	6085636	10852810	12377360	16262290	24435500	12412770
L-Methionine S-oxide	65106.59	101449.9	130656.8	341548.4	469314.5	434722.1
Indole	947706.2	1416332	2078088	3352337	3634027	1988830
Indole-3-acetaldehyde	123822.1	273367.4	391438	491273.9	557152.7	461290.9
Indoxyl	20825.97	42783.77	79627.47	106773.8	107797.8	64166.84
kynurenine	22458.14	46536.79	50832.14	84404.9	87888.23	63908.09
N-formyl kynurenine	222115.3	274022.3	211305.7	381391.3	309354.5	314259
Anthranilate	103091.8	177953.5	124998.4	125605.8	108039.6	102153.1
L-Adrenaline	299009.5	515065.9	646757.6	858608.3	1224887	651910.8
Dopamine	78815.91	136858.3	206040	474414.2	520266.4	246524.2
L-Carnitine	285153.3	374549.8	425253.4	357406.2	393370.9	370966.4
acyl-C2 (acetyl-carnitine)	577962.3	983788.2	1111839	1023382	1253770	864629.7
acyl-C3 (propionyl-carnitine)	344720.3	530737.3	684161.8	1114068	1134817	998383.4
acyl-C4 (butanoyl-l-carnitine)	174964	347169.6	466629.3	642511.8	691257.1	513051.2
acyl-C4-OH (Hydroxybutyrylcarnitine)	29291.88	52056.3	68738.69	37887.25	29717.67	35012.85
acyl-C4-DC	122402.1	262860	550056.6	542175.7	544125.7	266261.8
acyl-C5 (isovalerylcarnitine)	126088	230650.2	196766.1	339702.1	500660.2	382690.2
acyl-C12:1 (O-dodecenoyl-carnitine)	1300313	1667830	1524680	1459152	1409776	1672712
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
L-arginine	C00062	175.1189	0.1624834
L-asparagine	C00152	133.0599	0.1869574
L-aspartate	C00049	134.0447	0.1816977
L-glutamate	C00025	148.0605	0.1781275
L-glutamine	C00064	147.0765	0.1701856
L-histidine	C00135	156.0769	0.1645671
L-lysine	C00047	147.1129	0.1566485
L-methionine	C00073	150.0581	0.2222577
L-phenylalanine	C00079	166.0864	0.3099783
L-proline	C00148	116.0706	0.1752843
L-serine	C00065	106.0495	0.1925863
L-threonine	C00188	120.0655	0.1625859
L-tryptophan	C00078	205.0972	0.564035
L-valine	C00183	118.0862	0.1802121
L-cystine	C00491	241.0303	0.1849434
ADP	C00008	428.0364	0.2346289
AMP	C00020	348.0703	0.2327737
Adenosine	C00212	268.1037	0.259257
GMP	C00144	364.065	0.2181699
Guanine	C00242	152.0565	0.2412697
CDP	C00112	404.0309	0.1871555
Cytosine	C00380	112.0505	0.2396484
Thymine	C00178	127.0502	0.298997
IMP	C00130	349.0544	0.2426962
Inosine	C00294	269.0895	0.5679792
Hypoxanthine	C00262	137.0459	0.2307134
Xanthine	C00385	153.0404	0.218978
5-Hydroxyisourate	C11821	185.032	0.1681975
Urate	C00366	169.0351	0.2325194
3',5'-Cyclic AMP	C00575	330.0647	0.1496044
Pyridoxal	C00250	168.0656	0.2134997
4-Pyridoxate	C00847	184.0598	0.1859921
Pyridoxamine 5'-phosphate	C00647	249.061	0.3945555
Nicotinamide	C00153	123.0552	0.2344992
Glutathione	C00051	308.0909	0.2235605
Glutathione disulfide	C00127	613.1601	0.2945337
5-Oxoproline	C01879	130.05	0.2328438
S-Glutathionyl-L-cysteine	C05526	427.0947	0.1980999
gamma-L-Glutamyl-D-alanine	C03738	219.0992	0.2123491
Folate	C00504	442.1479	0.4976575
Ornithine	C01602	133.098	0.2267196
Argininosuccinate	C03406	291.1266	0.2523319
Cadaverine	C01672	103.1247	0.2230587
Spermidine	C00315	146.1652	0.1456083
Spermine	C00750	203.2231	0.1301141
D-Glucosamine	C00329	180.0868	0.2034096
Creatine	C00300	132.0768	0.1719973
Guanidinoacetate	C00581	118.0607	0.5595359
Pantothenol	C00864	220.118	0.3161802
L-Methionine S-oxide	C02989	166.0518	0.2092958
Indole	C00463	118.0651	0.5703475
Indole-3-acetaldehyde	C00637	160.0757	0.285743
Indoxyl	C05658	134.06	0.2504088
kynurenine	C00328	209.0921	0.3017523
N-formyl kynurenine	C02700	237.0901	0.2051545
Anthranilate	C00108	138.0539	0.188619
L-Adrenaline	C00788	184.0969	0.3172795
Dopamine	C03758	154.0862	0.2825861
L-Carnitine	C00318	162.1125	0.1659907
acyl-C2 (acetyl-carnitine)	C02571	204.1232	0.2201084
acyl-C3 (propionyl-carnitine)	C03017	218.1387	0.2798177
acyl-C4 (butanoyl-l-carnitine)	C02862	232.1543	0.3462324
acyl-C4-OH (Hydroxybutyrylcarnitine)	HMDB13127	248.1492	0.2568511
acyl-C4-DC	HMDB13133	262.1284	0.2265172
acyl-C5 (isovalerylcarnitine)	C20826	246.17	0.5272858
acyl-C12:1 (O-dodecenoyl-carnitine)	HMDB13326	342.264	0.5726996
METABOLITES_END
#END