#METABOLOMICS WORKBENCH nwajapey_20250723_100439 DATATRACK_ID:6206 STUDY_ID:ST004077 ANALYSIS_ID:AN006748 PROJECT_ID:PR002559
VERSION             	1
CREATED_ON             	July 25, 2025, 4:09 pm
#PROJECT
PR:PROJECT_TITLE                 	PDE7A Inhibition Suppresses Triple-Negative Breast Cancer by Attenuating de novo
PR:PROJECT_TITLE                 	Pyrimidine Biosynthesis
PR:PROJECT_TYPE                  	CE-TOFMS Analysis
PR:PROJECT_SUMMARY               	Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer,
PR:PROJECT_SUMMARY               	associated with poor response to therapies and high mortality. We identified
PR:PROJECT_SUMMARY               	that phosphodiesterase 7A (PDE7A) is overexpressed in the majority of TNBC, and
PR:PROJECT_SUMMARY               	higher level of PDE7A is associated with poor prognosis. The PI3K/AKT pathway,
PR:PROJECT_SUMMARY               	via the transcription factor IRF1, stimulated the expression of PDE7A in TNBC
PR:PROJECT_SUMMARY               	cells. PDE7A inhibition attenuated TNBC growth in both cell culture and in mouse
PR:PROJECT_SUMMARY               	models of TNBC. Inhibition of PDE7A suppressed de novo pyrimidine biosynthesis,
PR:PROJECT_SUMMARY               	in part through downregulation of the enzyme dihydroorotate dehydrogenase
PR:PROJECT_SUMMARY               	(DHODH). DHODH suppression attenuated TNBC tumor growth, mirroring the effects
PR:PROJECT_SUMMARY               	of PDE7A inhibition, and ectopic DHODH expression rescued PDE7A
PR:PROJECT_SUMMARY               	inhibition-induced tumor suppression. Pharmacological co-targeting of PDE7A and
PR:PROJECT_SUMMARY               	DHODH potently inhibited TNBC tumor growth and metastasis. These findings
PR:PROJECT_SUMMARY               	identify the PDE7A→ DHODH→ de novo pyrimidine biosynthesis pathway as a key
PR:PROJECT_SUMMARY               	driver of TNBC, offering additional therapeutic opportunities for TNBC patients.
PR:INSTITUTE                     	University of Alabama, Birmingham
PR:DEPARTMENT                    	Department of Biochemistry and Molecular Genetics
PR:LAST_NAME                     	Wajapeyee
PR:FIRST_NAME                    	Narendra
PR:ADDRESS                       	Kaul 540A, Kaul Human Genetics Building, University of Alabama at Birmingham,
PR:ADDRESS                       	AL, 35233
PR:EMAIL                         	nwajapey@uab.edu
PR:PHONE                         	205-934-5331
#STUDY
ST:STUDY_TITLE                   	PDE7A Inhibition Suppresses Triple-Negative Breast Cancer by Attenuating de novo
ST:STUDY_TITLE                   	Pyrimidine Biosynthesis
ST:STUDY_SUMMARY                 	The primary goal of this study was to identify metabolic alteration following
ST:STUDY_SUMMARY                 	treatment with BRL-50481(a PDE7A inhibition) using untargeted metabolomics
ST:STUDY_SUMMARY                 	approach. Using the Triple-Negative Breast Cancer cell line, MDA-MB-231, six
ST:STUDY_SUMMARY                 	human cell samples were analyzed – 3 control (MDA-MB-231 cells treated with
ST:STUDY_SUMMARY                 	DMSO (MDA-CTRL) and 3 treated (MDA-MB-231 cells treated with BRL-50481
ST:STUDY_SUMMARY                 	(MDA-Treat). Samples were sent frozen, extracted using methanol with internal
ST:STUDY_SUMMARY                 	standards, filtered, and concentrated prior to analysis using CE-TOFMS. All
ST:STUDY_SUMMARY                 	metabolite concentrations were calculated by normalizing the peak area of each
ST:STUDY_SUMMARY                 	metabolite with respect to the area of the internal standard and by using
ST:STUDY_SUMMARY                 	standard curves, which were obtained by single-point (100 μm) calibrations.
ST:STUDY_SUMMARY                 	The profile of peaks of putative metabolites was represented on metabolic
ST:STUDY_SUMMARY                 	pathway maps using Visualization and Analysis of Networks containing
ST:STUDY_SUMMARY                 	Experimental Data (VANTED) software.
ST:INSTITUTE                     	University of Alabama, Birmingham
ST:DEPARTMENT                    	Department of Biochemistry and Molecular Genetics
ST:LAST_NAME                     	Wajapeyee
ST:FIRST_NAME                    	Narendra
ST:ADDRESS                       	Kaul 540A, Kaul Human Genetics Building, University of Alabama at Birmingham,
ST:ADDRESS                       	AL, 35233
ST:EMAIL                         	nwajapey@uab.edu
ST:PHONE                         	205-934-5331
ST:NUM_GROUPS                    	2
ST:STUDY_COMMENTS                	MDA-MB-231 cells were treated with either BRL-50481(50mM) and control DMSO for
ST:STUDY_COMMENTS                	72 hours.
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENDER                        	Not applicable
#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	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_001_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_001_d1.mzML
SUBJECT_SAMPLE_FACTORS           	-	2	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_002_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_002_d1.mzML
SUBJECT_SAMPLE_FACTORS           	-	3	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_003_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_003_d1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_004_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_004_d1.mzML
SUBJECT_SAMPLE_FACTORS           	-	5	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_005_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_005_d1.mzML
SUBJECT_SAMPLE_FACTORS           	-	6	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line	RAW_FILE_NAME(Raw file name anions)=ALAUR007-1_A_20201208_006_d2.mzML; RAW_FILE_NAME(Raw file name cations)=ALAUR007-1_C_20201204_006_d1.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Aspirate medium from the culture plate, add 10 ml of wash buffer (5% (w/w)
CO:COLLECTION_SUMMARY            	mannitol solution) gently from the edge of the plate, and wash the cells by
CO:COLLECTION_SUMMARY            	slightly tilting the plate. Aspirate the wash buffer, wash the culture plate
CO:COLLECTION_SUMMARY            	again with the appropriate amount of wash buffer. The wash buffer then needs to
CO:COLLECTION_SUMMARY            	be aspired completely from the edge of the culture plate. Scape the cells using
CO:COLLECTION_SUMMARY            	cell scraper and snap freeze and ship for metabolomics analysis.
CO:SAMPLE_TYPE                   	MDA-MB-231 TNBC cell line
#TREATMENT
TR:TREATMENT_SUMMARY             	For metabolomics analysis, MDA-MB-231 cells were treated with either
TR:TREATMENT_SUMMARY             	BRL-50481(50mM) and control DMSO for 72 hours.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The cell pellet (300 µL) were mixed with 436 µL of methanol containing
SP:SAMPLEPREP_SUMMARY            	internal standards (10 µM) and centrifuged (2,300 x g, 4℃, 5min). The
SP:SAMPLEPREP_SUMMARY            	supernatant (350 µL) was filtrated through 5-kDa cut-off filter
SP:SAMPLEPREP_SUMMARY            	(ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove
SP:SAMPLEPREP_SUMMARY            	macromolecules. The filtrate was centrifugally concentrated and resuspended in
SP:SAMPLEPREP_SUMMARY            	50 µL of ultrapure water immediately before the measurement.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Anionic Metabolites (Anion Mode): Device Agilent CE-TOFMS system (Agilent
CH:CHROMATOGRAPHY_SUMMARY        	Technologies Inc.); Capillary: Fused silica capillary i.d. 50 μm × 80 cm;
CH:CHROMATOGRAPHY_SUMMARY        	Analytical Condition Run buffer: Anion Buffer Solution (p/n : I3302-1023) Rinse
CH:CHROMATOGRAPHY_SUMMARY        	buffer: Anion Buffer Solution (p/n : I3302-1023); Sample injection: Pressure
CH:CHROMATOGRAPHY_SUMMARY        	injection 50 mbar, 10 sec CE voltage: Positive, 30 kV
CH:CHROMATOGRAPHY_TYPE           	CE
CH:INSTRUMENT_NAME               	Agilent G6230B CE-TOFMS system
CH:COLUMN_NAME                   	Agilent Fused silica capillary (80cm x 50um)
CH:SOLVENT_A                     	Anion Buffer Solution (p/n : I3302-1023)
CH:SOLVENT_B                     	N/A
CH:FLOW_GRADIENT                 	N/A
CH:FLOW_RATE                     	N/A
CH:COLUMN_TEMPERATURE            	N/A
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6210 TOF
MS:INSTRUMENT_TYPE               	TOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	CE-TOFMS was performed using Agilent CE-MS system. The system was composed of
MS:MS_COMMENTS                   	Agilent CE capillary electrophoresis units and Agilent TOF-MS. MS ionization:
MS:MS_COMMENTS                   	ESI Positive MS capillary voltage: 4,000 V MS scan range: m/z 50-1,000 Sheath
MS:MS_COMMENTS                   	liquid: HMT Sheath Liquid (p/n : H3301-1020) Machine No. 6 (Metabolon); The
MS:MS_COMMENTS                   	spectrometer was scanned from m/z 50 to 1000 in centroid mode. Raw data were
MS:MS_COMMENTS                   	analyzed using MasterHands software for peak detection, alignment, and
MS:MS_COMMENTS                   	quantification. Compounds were identified by matching m/z and migration time
MS:MS_COMMENTS                   	against the HMT metabolite library.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	micromole/gram
MS_METABOLITE_DATA_START
Samples	1	2	3	4	5	6
Factors	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	Treatment:control DMSO for 72 h | Sample source:MDA-MB-231 TNBC cell line	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line	Treatment:BRL-50481(50mM) | Sample source:MDA-MB-231 TNBC cell line
2-Hydroxybutyric acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
2-Oxoglutaric acid	1.0	1.0	1.0	1.7	1.6	1.9
2-Oxoisovaleric acid	0.5	0.6	0.5	0.7	0.6	0.7
2-Phosphoglyceric acid	0.5	0.6	0.5	0.5	0.6	0.6
3-Hydroxybutyric acid	0.2	0.4	N.D.	0.4	0.3	0.4
3-Phosphoglyceric acid	3.6	3.9	3.7	3.6	3.8	4.2
6-Phosphogluconic acid	0.3	0.5	1.3	0.9	0.4	0.5
Acetyl CoA_divalent	0.04	0.04	0.02	0.05	0.02	0.03
ADP	3.3	5.0	7.5	7.6	6.2	5.7
AMP	1.0	2.1	5.5	4.4	2.2	1.4
ATP	13	11	12	15	14	18
cAMP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CDP	0.4	0.6	0.8	0.8	0.6	0.6
cGMP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
cis-Aconitic acid	0.6	0.9	1.0	1.1	0.8	0.8
Citric acid	8.9	9.1	8.7	8.6	8.4	9.4
CMP	0.2	0.3	0.5	0.5	0.3	0.2
CoA_divalent	0.05	0.07	0.13	0.10	0.07	0.07
CTP	1.1	0.9	0.7	1.0	1.1	1.4
dATP	0.09	0.07	0.08	0.08	0.07	0.10
dCTP	0.04	N.D.	0.02	N.D.	0.04	N.D.
Dihydroxyacetone phosphate	3.8	3.5	2.7	3.2	2.9	4.5
dTDP	0.06	0.08	0.13	0.14	0.09	0.07
dTMP	N.D.	N.D.	0.02	N.D.	N.D.	N.D.
dTTP	0.2	0.14	0.09	0.15	0.2	0.2
Erythrose 4-phosphate	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Fructose 1,6-diphosphate	6.7	8.0	8.5	8.2	8.3	7.5
Fructose 6-phosphate	0.2	0.3	0.2	0.3	0.3	0.3
Fumaric acid	0.8	1.0	0.9	1.2	1.2	1.3
GDP	0.7	1.1	1.7	1.7	1.3	1.1
Gluconic acid	0.7	0.8	0.9	0.8	0.8	0.9
Glucose 1-phosphate	0.6	1.0	1.0	1.2	1.1	0.7
Glucose 6-phosphate	1.1	1.6	1.6	1.9	1.7	1.3
Glyceraldehyde 3-phosphate	2.1	2.5	1.9	2.3	2.1	2.3
Glycerol 3-phosphate	8.2	6.7	6.6	7.4	6.8	9.9
Glycolic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Glyoxylic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
GMP	0.14	0.2	0.6	0.4	0.2	0.2
GTP	2.6	2.6	2.5	3.2	3.2	3.7
IMP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Isocitric acid	0.8	0.9	0.9	0.9	0.9	0.9
Lactic acid	482	492	469	549	554	629
Malic acid	6.8	7.1	6.8	8.7	8.8	9.4
Malonyl CoA_divalent	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
NAD+	11	11	12	15	13	15
NADP+	0.3	0.3	0.4	0.4	0.4	0.4
Phosphoenolpyruvic acid	0.6	0.9	0.9	1.0	0.9	0.9
PRPP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Pyruvic acid	11	11	13	17	13	15
Ribose 5-phosphate	0.6	0.5	0.4	0.6	0.4	0.6
Ribulose 5-phosphate	4.3	3.6	2.8	3.3	3.7	5.0
Sedoheptulose 7-phosphate	N.D.	N.D.	0.08	0.05	N.D.	N.D.
Succinic acid	0.7	0.8	0.8	0.9	0.9	0.9
UDP	0.6	1.1	1.6	1.4	1.2	0.9
UMP	0.2	0.3	0.7	0.5	0.3	0.3
UTP	2.1	1.9	1.7	1.9	2.1	2.6
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
2-Hydroxybutyric acid
2-Oxoglutaric acid
2-Oxoisovaleric acid
2-Phosphoglyceric acid
3-Hydroxybutyric acid
3-Phosphoglyceric acid
6-Phosphogluconic acid
Acetyl CoA_divalent
ADP
AMP
ATP
cAMP
CDP
cGMP
cis-Aconitic acid
Citric acid
CMP
CoA_divalent
CTP
dATP
dCTP
Dihydroxyacetone phosphate
dTDP
dTMP
dTTP
Erythrose 4-phosphate
Fructose 1,6-diphosphate
Fructose 6-phosphate
Fumaric acid
GDP
Gluconic acid
Glucose 1-phosphate
Glucose 6-phosphate
Glyceraldehyde 3-phosphate
Glycerol 3-phosphate
Glycolic acid
Glyoxylic acid
GMP
GTP
IMP
Isocitric acid
Lactic acid
Malic acid
Malonyl CoA_divalent
NAD+
NADP+
Phosphoenolpyruvic acid
PRPP
Pyruvic acid
Ribose 5-phosphate
Ribulose 5-phosphate
Sedoheptulose 7-phosphate
Succinic acid
UDP
UMP
UTP
METABOLITES_END
#END