#METABOLOMICS WORKBENCH wanglgene_20241206_112213 DATATRACK_ID:5437 STUDY_ID:ST003624 ANALYSIS_ID:AN005954 PROJECT_ID:PR002208
VERSION             	1
CREATED_ON             	December 12, 2024, 6:32 pm
#PROJECT
PR:PROJECT_TITLE                 	NRF2 supports non-small cell lung cancer growth independently of
PR:PROJECT_TITLE                 	CBP/p300-enhanced glutathione synthesis
PR:PROJECT_TYPE                  	MS quantitative analysis
PR:PROJECT_SUMMARY               	Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress responsive
PR:PROJECT_SUMMARY               	transcription factor that is mutationally activated in a subset (~25%) of
PR:PROJECT_SUMMARY               	clinically-aggressive non-small cell lung cancers (NSCLC). Mechanistic insight
PR:PROJECT_SUMMARY               	into drivers of the NRF2 dependency remains poorly understood. Here, we defined
PR:PROJECT_SUMMARY               	a novel NRF2 target gene set linked to NRF2-dependency in cancer cell lines, and
PR:PROJECT_SUMMARY               	observed that a significant portion of these genes is devoid of
PR:PROJECT_SUMMARY               	promoter-proximal NRF2. Using integrated genomic analyses, we characterized
PR:PROJECT_SUMMARY               	extensive NRF2-dependent enhancer RNA (eRNA) synthesis and NRF2-mediated H3K27ac
PR:PROJECT_SUMMARY               	deposition at proximal and distal enhancer regions regulating these genes. While
PR:PROJECT_SUMMARY               	CBP/p300 is a well-validated direct interaction partner of NRF2 with prominent
PR:PROJECT_SUMMARY               	functions at enhancers, we report that this interaction is not required for
PR:PROJECT_SUMMARY               	NRF2-dependent NSCLC cell growth, indicating that NRF2 can sustain sufficient
PR:PROJECT_SUMMARY               	transcriptional activity in the absence of CBP/p300 coactivation. Broad
PR:PROJECT_SUMMARY               	metabolic profiling established a primary role for CBP/p300 in NRF2-dependent
PR:PROJECT_SUMMARY               	accumulation of glutathione and glutathione-related metabolites. While redox
PR:PROJECT_SUMMARY               	homeostasis via enhanced glutathione production is commonly associated with the
PR:PROJECT_SUMMARY               	normal physiological role of NRF2, collectively our results suggest that
PR:PROJECT_SUMMARY               	NRF2-dependent cancer cell growth does not require this enhanced glutathione
PR:PROJECT_SUMMARY               	production.
PR:INSTITUTE                     	Genentech Inc.
PR:LAST_NAME                     	Wang
PR:FIRST_NAME                    	Mike Lingjue
PR:ADDRESS                       	1 DNA Way, South San Francisco, CA 94404
PR:EMAIL                         	wang.mike@gene.com
PR:PHONE                         	650-245-7991
PR:CONTRIBUTORS                  	Ryan J. Conrad, James A. Mondo, Mike Lingjue Wang, Peter S. Liu, Zijuan Lai,
PR:CONTRIBUTORS                  	Feroza K Choudhury, Qingling Li, Weng Ruh Wong, James Lee, Frances Shanahan, Eva
PR:CONTRIBUTORS                  	Lin, Scott Martin, Joachim Rudolph, John G. Moffat, Dewakar Sangaraju, Wendy
PR:CONTRIBUTORS                  	Sandoval, Timothy Sterne-Weiler, Scott A. Foster
#STUDY
ST:STUDY_TITLE                   	NRF2 supports non-small cell lung cancer growth independently of
ST:STUDY_TITLE                   	CBP/p300-enhanced glutathione synthesis: Absolute Quantification of NADP+ and
ST:STUDY_TITLE                   	NADPH in A549 cells (Part 2 of 3)
ST:STUDY_SUMMARY                 	This study aims at directly quantifying NADPH, NADP+, and ratios of NADPH/NADP+
ST:STUDY_SUMMARY                 	and its association with NRF2 to reveal NRF2 function in redox homeostasis.
ST:STUDY_SUMMARY                 	Briefly, cells were extracted by using acidified ACN/MeOH/water mixture with
ST:STUDY_SUMMARY                 	15N-labeled ADP followed by adjusting pH to 8. Standard curve was established to
ST:STUDY_SUMMARY                 	quantify NADPH and NADP+ by using LC-HRMS. The result shows that when NRF2 is
ST:STUDY_SUMMARY                 	absent, both NADPH and NADP+ levels decrease.
ST:INSTITUTE                     	Genentech Inc.
ST:LAST_NAME                     	Wang
ST:FIRST_NAME                    	Mike
ST:ADDRESS                       	1 DNA Way, South San Francisco, CA 94080, USA
ST:EMAIL                         	wang.mike@gene.com
ST:PHONE                         	6502457991
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	A549
SU:SPECIES_GROUP                 	Mammals
#FACTORS
#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           	-	Sample01	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Cell count (1E6)=23.23; RAW_FILE_NAME(Raw data)=20231005_017_neg_LunaNH2_CellG2_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample02	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Cell count (1E6)=23.23; RAW_FILE_NAME(Raw data)=20231005_024_neg_LunaNH2_CellG2_r3
SUBJECT_SAMPLE_FACTORS           	-	Sample03	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Cell count (1E6)=23.23; RAW_FILE_NAME(Raw data)=20231005_031_neg_LunaNH2_CellG2_r4
SUBJECT_SAMPLE_FACTORS           	-	Sample04	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Cell count (1E6)=23.23; RAW_FILE_NAME(Raw data)=20231005_038_neg_LunaNH2_CellG2_r5
SUBJECT_SAMPLE_FACTORS           	-	Sample05	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Cell count (1E6)=23.23; RAW_FILE_NAME(Raw data)=20231005_045_neg_LunaNH2_CellG2_r1
SUBJECT_SAMPLE_FACTORS           	-	Sample06	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Cell count (1E6)=28.5; RAW_FILE_NAME(Raw data)=20231005_020_neg_LunaNH2_CellG5_r5
SUBJECT_SAMPLE_FACTORS           	-	Sample07	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Cell count (1E6)=28.5; RAW_FILE_NAME(Raw data)=20231005_027_neg_LunaNH2_CellG5_r1
SUBJECT_SAMPLE_FACTORS           	-	Sample08	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Cell count (1E6)=28.5; RAW_FILE_NAME(Raw data)=20231005_034_neg_LunaNH2_CellG5_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample09	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Cell count (1E6)=28.5; RAW_FILE_NAME(Raw data)=20231005_041_neg_LunaNH2_CellG5_r3
SUBJECT_SAMPLE_FACTORS           	-	Sample10	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Cell count (1E6)=28.5; RAW_FILE_NAME(Raw data)=20231005_048_neg_LunaNH2_CellG5_r4
SUBJECT_SAMPLE_FACTORS           	-	Sample11	Sample source:STD | Genotype:STD | Treatment:STD4 | Sample Label:39ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_054_neg_LunaNH2_STD4_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample12	Sample source:STD | Genotype:STD | Treatment:STD5 | Sample Label:78ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_055_neg_LunaNH2_STD5_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample13	Sample source:STD | Genotype:STD | Treatment:STD6 | Sample Label:156ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_056_neg_LunaNH2_STD6_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample14	Sample source:STD | Genotype:STD | Treatment:STD7 | Sample Label:312ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_057_neg_LunaNH2_STD7_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample15	Sample source:STD | Genotype:STD | Treatment:STD8 | Sample Label:625ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_058_neg_LunaNH2_STD8_r2
SUBJECT_SAMPLE_FACTORS           	-	Sample16	Sample source:STD | Genotype:STD | Treatment:STD9 | Sample Label:1250ng	Cell count (1E6)=0; RAW_FILE_NAME(Raw data)=20231005_059_neg_LunaNH2_STD9_r2
#COLLECTION
CO:COLLECTION_SUMMARY            	All cell lines were obtained from the Genentech cell bank (Yu et al, 2015) and
CO:COLLECTION_SUMMARY            	grown in RPMI or DMEM supplemented with 10% FBS, 1% glutamine, and 1%
CO:COLLECTION_SUMMARY            	penicillin-streptomycin at 37°C, 5% CO2. Cell lines were confirmed via STR
CO:COLLECTION_SUMMARY            	profiling and were mycoplasma free. A549 and A549shNRF2-BIND rescue cell lines
CO:COLLECTION_SUMMARY            	were generated using the Piggybac system. Donor constructs were co-transfected
CO:COLLECTION_SUMMARY            	with transposase (Ding et al, 2005) at a 4:1 ratio using Lipofetamine 3000
CO:COLLECTION_SUMMARY            	(Thermo) according to manufacturer’s protocol. 3-days post-transfection,
CO:COLLECTION_SUMMARY            	puromycin selection (1µg/mL) was initiated and was performed for ~2 weeks prior
CO:COLLECTION_SUMMARY            	to experimentation. A549shNRF2-BIND rescue cells represent polyclonal pools.
CO:COLLECTION_SUMMARY            	Cell viability was measured using CellTiter-Glo® Luminescent Cell Viability
CO:COLLECTION_SUMMARY            	Assay (Promega) using the Envision 2103 Plate Reader (Promega). Cell growth
CO:COLLECTION_SUMMARY            	curves were generated using the IncuCyte System (Sartorius) according to the
CO:COLLECTION_SUMMARY            	manufacturer’s protocol. Curve fits were performed in Prism. Clonogenic assays
CO:COLLECTION_SUMMARY            	were performed by washing cells in PBS, staining with 0.5% crystal violet
CO:COLLECTION_SUMMARY            	solution (Sigma HT90132) for 5 min, and washing 3x with PBS. Four replicates of
CO:COLLECTION_SUMMARY            	20-30 million A549shNRF2-BIND-luc, WT NRF2 or Neh4/5mut cells were treated with
CO:COLLECTION_SUMMARY            	2.5ng/mL dox for 48h.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment was administered and this is a study based on genetic variants.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Briefly, cells were harvested by washing with PBS twice and quenched on dry-ice
SP:SAMPLEPREP_SUMMARY            	by adding acidified 2:2:1 ACN/MeOH/water with spiked-in 15N5-ADP (Sigma, St.
SP:SAMPLEPREP_SUMMARY            	Louis, MO, USA) as an internal standard. 2M ammonium bicarbonate was then added
SP:SAMPLEPREP_SUMMARY            	to adjust the pH to 8 (22:3 v/v). The extract was freeze-thawed by liquid
SP:SAMPLEPREP_SUMMARY            	nitrogen and sonicated three times and centrifuged at 15,000 rpm for 15 minutes.
SP:SAMPLEPREP_SUMMARY            	The supernatant was transferred and subjected to LC-MS analysis. The extract was
SP:SAMPLEPREP_SUMMARY            	kept on ice in all steps.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Shimadzu Nexera HPLC
CH:COLUMN_NAME                   	Phenomenex Luna-NH2 (50 x 2.1mm x 3um, 100Å)
CH:SOLVENT_A                     	5% acetonitrile/95% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
CH:SOLVENT_B                     	95% acetonitrile/5% water
CH:FLOW_GRADIENT                 	0-0.5 min 60% B, 3-10 min 0% B, 11.5 60% B
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	25
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Samples were run in negative mode. The Q Exactive Plus Mass Spectrometer was
MS:MS_COMMENTS                   	operated with the following parameters: Sheath gas flow rate, 50 units; Aux gas
MS:MS_COMMENTS                   	flow rate, 13 units; Aux gas temperature, 425 °C; Capillary temperature,
MS:MS_COMMENTS                   	263°C; Spray voltage, -2600V; Scan mode, MS2 scan with data-dependent MS/MS
MS:MS_COMMENTS                   	acquisition. In Full MS scan, scan range is 60-900 m/z; resolution is 70,000;
MS:MS_COMMENTS                   	AGC target, 1×e^6; Maximum IT, 100 ms. In ddMS2 scan, top 5 ions are selected
MS:MS_COMMENTS                   	with an isolation window of 1.5 m/z; resolution is 17,500; AGC target, 5×e^4;
MS:MS_COMMENTS                   	Maximum IT, 50 ms.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	ng/mL
MS_METABOLITE_DATA_START
Samples	Sample01	Sample02	Sample03	Sample04	Sample05	Sample06	Sample07	Sample08	Sample09	Sample10
Factors	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox- | Sample Label:Ctrl	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox	Sample source:A549 Cell | Genotype:shNRF2-Luciferase | Treatment:Dox+ | Sample Label:Dox
NADP+	16.64757804	15.78449946	14.7285253	18.05020452	14.46820237	23.81487719	32.68421053	23.40870175	22.06673684	21.65224561
NADPH	45.77395048	52.91354144	47.33024758	53.15044133	46.33860065	62.95550877	62.77238596	62.58550877	55.60164912	64.98252632
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Ion Adduct	Formula	Exact mass	Super class	Main class	Sub class
NADP+	[M-H]-	C21H28N7O17P3	743.0755	Nucleic acids	Nicotinamides	Nicotinamide dinucleotides
NADPH	[M-2H]2-	C21H30N7O17P3	745.0911	Nucleic acids	Nicotinamides	Nicotinamide dinucleotides
METABOLITES_END
#END