#METABOLOMICS WORKBENCH cendalfr_20240411_073626 DATATRACK_ID:4770 STUDY_ID:ST003167 ANALYSIS_ID:AN005196 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        	Negative 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; 10mM Ammonium acetate, .01% Formic Acid
CH:SOLVENT_B                     	50% Methanol/ 50% Acetonitrile; 10mM Ammonium acetate, .01% Formic Acid
CH:FLOW_GRADIENT                 	0-0.2 min 5% B, 0.2-.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                      	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	D16 19BBz_1	D16 19BBz_2	D16 19BBz_3	D26 19BBz_2	D26 19BBz_3	D26 19BBz_4
Factors	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
ATP	19014.66	26227.63	16447.19	2206.066	1390.958	2917.118
Phosphate	21833840	22965410	23621400	21054690	18596380	20222680
Diphosphate	3173455	3622059	3854649	3952140	3478727	2549453
D-Glucose	351309.9	433952.1	325824.6	369659.3	306164.4	312890.2
D-Fructose 1-6-bisphosphate	146827.2	259145.7	228674.7	12428.3	17114.98	24310.78
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	930077.3	690660.1	558928	976922.1	1457511	735174.5
2/3-Phospho-D-glycerate	60150.83	46892.61	80066.84	381.0097	502.2223	12902.58
Phosphoenolpyruvate	4157.771	8876.774	15065.4	0	0	2800.342
Pyruvate	84402.01	133531.6	90980.66	234081.5	210604.5	95224.38
Lactate	4611334	7797466	7531226	7079256	6972118	3402812
Mannitol	42364.34	82909.54	109113.8	153467.3	187324.9	48378.62
Citrate	141050.3	176027.1	157177	98102.71	146129.8	99637.65
2-Oxoglutarate	8487.82	12050.6	6578.336	1287.222	2813.854	3613.314
Succinate	49954.75	84106.76	74866.57	112383	107117.8	44798.08
Fumarate	72002.36	60629.3	46702.17	13292.89	17005.37	24117.62
Malate	452675.2	403095.8	306856.8	76249.38	109483.5	166152.3
4-Acetamidobutanoate	4583.899	7702.877	8920.666	17016.33	18558.4	3859.461
Octanoic acid (caprylate)	2993856	4182712	1153829	4866070	5023588	1946367
Nonanoic acid (pelargonate)	109969.9	117714.5	81422.14	112241.5	123672.1	74494.55
Decanoic acid (caprate)	45847.74	55780.47	42193.36	59552.89	65901.07	39853.85
Dodecanoic acid	12245.82	17391.16	14257.24	10060.08	13218.41	8528.238
Tetradecanoic acid	17870.01	18869.06	16064.83	20769.71	20445.1	14913.76
Hexadecanoic acid	600047.1	638391.4	517900.9	1131847	765317	512133.7
Hexadecenoic acid	31826.61	26635.73	33079.71	21976.22	25815.62	20764.3
Octadecenoic acid	151570.7	157885.7	134179.4	157201.1	132413.5	99215.78
Linoleate	20670.14	33157.76	22990.34	24674.27	30690.86	8576.772
Dodecanedioic acid	2239.513	2909.635	4659.193	3219.257	6314.243	3302.674
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
ATP	C00002	505.9875	0.3576738
Phosphate	C00009	96.96812	0.2040902
Diphosphate	C00013	176.9349	0.18636
D-Glucose	C00031	179.0552	0.2114511
D-Fructose 1-6-bisphosphate	C00354	338.9886	0.1983463
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	C00118	168.9883	0.5055173
2/3-Phospho-D-glycerate	C00631	184.9847	0.2106258
Phosphoenolpyruvate	C00074	166.974	0.2086936
Pyruvate	C00022	87.00727	0.2537393
Lactate	C01432	89.0229	0.2570731
Mannitol	C00392	181.0704	0.2702953
Citrate	C00158	191.0189	0.2063361
2-Oxoglutarate	C00026	145.013	0.2116728
Succinate	C00042	117.0179	0.2344009
Fumarate	C00122	115.0023	0.2281322
Malate	C00149	133.0129	0.1987566
4-Acetamidobutanoate	C02946	144.0654	0.2938338
Octanoic acid (caprylate)	C06423	143.1065	0.5209751
Nonanoic acid (pelargonate)	C01601	157.1223	0.5523673
Decanoic acid (caprate)	C01571	171.138	0.5745994
Dodecanoic acid	C02679	199.1696	0.6075237
Tetradecanoic acid	C06424	227.2011	0.6438132
Hexadecanoic acid	C00249	255.2327	0.6944952
Hexadecenoic acid	C08362	253.2171	0.6539506
Octadecenoic acid	C00712	281.2485	0.7043023
Linoleate	C01595	279.2329	0.665722
Dodecanedioic acid	C02678	229.144	0.4839389
METABOLITES_END
#END