#METABOLOMICS WORKBENCH dk3039_20231205_020157 DATATRACK_ID:4488 STUDY_ID:ST002999 ANALYSIS_ID:AN004926 PROJECT_ID:PR001869
VERSION             	1
CREATED_ON             	December 7, 2023, 9:55 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolic Reprogramming by Histone Deacetylase Inhibition Preferentially Targets
PR:PROJECT_TITLE                 	NRF2-activated tumors
PR:PROJECT_SUMMARY               	Interplay between metabolism and chromatin signaling are implicated in cancer
PR:PROJECT_SUMMARY               	progression. However, whether and how metabolic reprogramming in tumors
PR:PROJECT_SUMMARY               	generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD)
PR:PROJECT_SUMMARY               	tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway
PR:PROJECT_SUMMARY               	which drives aggressive and chemo-resistant disease. Using a chromatin-focused
PR:PROJECT_SUMMARY               	CRISPR screen we report that NRF2 activation sensitizes LUAD cells to genetic
PR:PROJECT_SUMMARY               	and chemical inhibition of class I histone deacetylases (HDAC). This association
PR:PROJECT_SUMMARY               	is observed across cultured cells, mouse models and patient-derived xenografts.
PR:PROJECT_SUMMARY               	Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates
PR:PROJECT_SUMMARY               	that HDAC inhibition causes widespread redistribution of H4ac and its reader
PR:PROJECT_SUMMARY               	protein, which transcriptionally downregulates metabolic enzymes. This results
PR:PROJECT_SUMMARY               	in reduced flux into amino acid metabolism and de novo nucleotide synthesis
PR:PROJECT_SUMMARY               	pathways that are preferentially required for the survival of NRF2-active cancer
PR:PROJECT_SUMMARY               	cells. Together, our findings suggest NRF2 activation as a potential biomarker
PR:PROJECT_SUMMARY               	for effective repurposing of HDAC inhibitors to treat solid tumors.
PR:INSTITUTE                     	Columbia University Medical Center
PR:DEPARTMENT                    	Genetics and Development
PR:LABORATORY                    	Chao Lu
PR:LAST_NAME                     	Karagiannis
PR:FIRST_NAME                    	Dimitris
PR:ADDRESS                       	630 West 168th Street, NEW YORK, NY, 10032, USA
PR:EMAIL                         	karagiannis_dimitrios@yahoo.gr
PR:PHONE                         	+30 6982804931
#STUDY
ST:STUDY_TITLE                   	Metabolomics and glucose and glutamine labeled isotope tracing analysis in
ST:STUDY_TITLE                   	murine lung adenocarcinoma cells in the context of normal or active NRF2 pathway
ST:STUDY_TITLE                   	as well as HDAC and glutaminase inhibitor treatment.
ST:STUDY_SUMMARY                 	Interplay between metabolism and chromatin signaling are implicated in cancer
ST:STUDY_SUMMARY                 	progression. However, whether and how metabolic reprogramming in tumors
ST:STUDY_SUMMARY                 	generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD)
ST:STUDY_SUMMARY                 	tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway
ST:STUDY_SUMMARY                 	which drives aggressive and chemo-resistant disease. Using a chromatin-focused
ST:STUDY_SUMMARY                 	CRISPR screen we report that NRF2 activation sensitizes LUAD cells to genetic
ST:STUDY_SUMMARY                 	and chemical inhibition of class I histone deacetylases (HDAC). This association
ST:STUDY_SUMMARY                 	is observed across cultured cells, mouse models and patient-derived xenografts.
ST:STUDY_SUMMARY                 	Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates
ST:STUDY_SUMMARY                 	that HDAC inhibition causes widespread redistribution of H4ac and its reader
ST:STUDY_SUMMARY                 	protein, which transcriptionally downregulates metabolic enzymes. This results
ST:STUDY_SUMMARY                 	in reduced flux into amino acid metabolism and de novo nucleotide synthesis
ST:STUDY_SUMMARY                 	pathways that are preferentially required for the survival of NRF2-active cancer
ST:STUDY_SUMMARY                 	cells. Together, our findings suggest NRF2 activation as a potential biomarker
ST:STUDY_SUMMARY                 	for effective repurposing of HDAC inhibitors to treat solid tumors. In this
ST:STUDY_SUMMARY                 	metabolomics experiment we characterize the changes in metabolic pathway flux in
ST:STUDY_SUMMARY                 	KP LUAD cells in response to HDAC and glutaminase inhibition. This dataset
ST:STUDY_SUMMARY                 	includes metabolomics of U-C13 glucose tracing (1h and 24h) and U-C13 glutamine
ST:STUDY_SUMMARY                 	(8h) of mouse LUAD cell lines with Kras overexpression and p53 knock-out (KP),
ST:STUDY_SUMMARY                 	carrying empty vector (EV) or overexpression of NRF2dNeh2 (NRF2) and treated
ST:STUDY_SUMMARY                 	with DMSO, Romidepsin or CB-839. 3 technical replicates were done per condition
ST:STUDY_SUMMARY                 	in 2 separate experiments, 1: glucose tracing and 2: glutamine tracing.
ST:INSTITUTE                     	Columbia University - Medical Center
ST:DEPARTMENT                    	Genetics and Development
ST:LABORATORY                    	Chao Lu
ST:LAST_NAME                     	Dimitris
ST:FIRST_NAME                    	Karagiannis
ST:ADDRESS                       	622 W 168th St, New York, NY 10032
ST:EMAIL                         	karagiannis_dimitrios@yahoo.gr
ST:PHONE                         	+30 6982804931
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#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           	NA	blank1	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=blank1.mzML
SUBJECT_SAMPLE_FACTORS           	NA	blank2	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=blank2.mzML
SUBJECT_SAMPLE_FACTORS           	NA	blank3	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=blank3.mzML
SUBJECT_SAMPLE_FACTORS           	NA	blank4	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=blank4.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_CB839_Gln_1	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_CB839_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_CB839_Gln_2	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_CB839_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_CB839_Gln_3	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_CB839_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_1h_1	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_1h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_1h_2	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_1h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_1h_3	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_1h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_24h_1	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_24h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_24h_2	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_24h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_24h_3	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_DMSO_24h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_Gln_1	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_DMSO_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_Gln_2	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_DMSO_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_DMSO_Gln_3	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_DMSO_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_1h_1	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_1h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_1h_2	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_1h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_1h_3	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_1h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_24h_1	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_24h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_24h_2	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_24h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_Rom_24h_3	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=EV_Rom_24h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_ROM_Gln_1	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_ROM_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_ROM_Gln_2	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_ROM_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_EV	EV_ROM_Gln_3	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=EV_ROM_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_CB839_Gln_1	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_CB839_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_CB839_Gln_2	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_CB839_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_CB839_Gln_3	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_CB839_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_1h_1	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_1h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_1h_2	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_1h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_1h_3	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_1h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_24h_1	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_24h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_24h_2	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_24h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_24h_3	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_DMSO_24h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_Gln_1	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_DMSO_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_Gln_2	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_DMSO_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_DMSO_Gln_3	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_DMSO_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_1h_1	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_1h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_1h_2	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_1h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_1h_3	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_1h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_24h_1	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_24h_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_24h_2	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_24h_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_Rom_24h_3	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Experiment=1_20211110; RAW_FILE_NAME=NRF2_Rom_24h_3.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_ROM_Gln_1	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_ROM_Gln_1.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_ROM_Gln_2	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_ROM_Gln_2.mzML
SUBJECT_SAMPLE_FACTORS           	KP_NRF2	NRF2_ROM_Gln_3	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Experiment=2_20220428; RAW_FILE_NAME=NRF2_ROM_Gln_3.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC_1	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=2_20220428; RAW_FILE_NAME=QC_1.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC_2	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=2_20220428; RAW_FILE_NAME=QC_2.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC_3	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=2_20220428; RAW_FILE_NAME=QC_3.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC_4	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=2_20220428; RAW_FILE_NAME=QC_4.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC1	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=QC1.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC2	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=QC2.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC3	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=QC3.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC4	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=QC4.mzML
SUBJECT_SAMPLE_FACTORS           	NA	QC5	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=1_20211110; RAW_FILE_NAME=QC5.mzML
SUBJECT_SAMPLE_FACTORS           	NA	Blank_1	Genotype:NA | Treatment:NA | Timepoint:NA | Tracer:NA	Experiment=2_20220428; RAW_FILE_NAME=Blank_1.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Mouse lung adenocarcinoma (LUAD) cell lines were established as in a previous
CO:COLLECTION_SUMMARY            	study by the Papagiannakopoulos lab (Romero et al. Nature Medicine 2017).
CO:COLLECTION_SUMMARY            	Briefly, KrasLSL-G12D/+; p53flox/flox genetically engineered mice were
CO:COLLECTION_SUMMARY            	intratracheally infected with pSECC lentiviral vectors expressing sgRNAs against
CO:COLLECTION_SUMMARY            	Keap1 or tdTomato as a control. The mice developed LUAD tumors and cell lines
CO:COLLECTION_SUMMARY            	were derived from them. In this experiment we used a cell line derived from
CO:COLLECTION_SUMMARY            	control tumors (KP: Kras-mutant, p53-null). In this cell line we overexpressed
CO:COLLECTION_SUMMARY            	an active form of NRF2 (NRF2) or introduced the empty vector (EV).
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	For glucose tracing analysis, 2x105 KP cells (n=3) were plated in 6-well plates
TR:TREATMENT_SUMMARY             	overnight in RPMI medium (Sigma). After 24 hours, the media was replaced with
TR:TREATMENT_SUMMARY             	fresh RPMI medium containing DMSO or Romidepsin. At 48 hours the media was
TR:TREATMENT_SUMMARY             	replaced with fresh glucose-free RPMI medium (Sigma) containing 10% dialyzed
TR:TREATMENT_SUMMARY             	fetal bovine serum (Gibco), 2.0 g/L 13C6-glucose (Sigma) and DMSO or 5nM
TR:TREATMENT_SUMMARY             	Romidepsin. Cells were harvested at 49 and 72 hours and processed as described
TR:TREATMENT_SUMMARY             	below. For glutamine tracing, 2x105 KP cells (n=3) were plated in 6-well plates
TR:TREATMENT_SUMMARY             	overnight in RPMI medium (Sigma). After 24 hours, the media was replaced with
TR:TREATMENT_SUMMARY             	fresh RPMI medium containing DMSO, 1nM Romidepsin or 150nM CB-839. At 48 hours
TR:TREATMENT_SUMMARY             	the media was replaced with fresh glutamine-free RPMI medium (Sigma) containing
TR:TREATMENT_SUMMARY             	10% dialyzed fetal bovine serum (Gibco), 2.0 g/L 13C6-glutamine (Cambridge
TR:TREATMENT_SUMMARY             	Isotope Laboratories) and DMSO, 1nM Romidepsin or 150nM CB-839
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells were washed with cold PBS, lysed in 80% Ultra LC-MS acetonitrile (Thermo
SP:SAMPLEPREP_SUMMARY            	Scientific) supplemented with 20 µM deuterated 2-hydroxyglutarate
SP:SAMPLEPREP_SUMMARY            	(D-2-hydroxyglutaric-2,3,3,4,4-d5 acid (d5-2HG), Cambridge Isotope Laboratories)
SP:SAMPLEPREP_SUMMARY            	as an internal standard on ice for 15 minutes, and centrifuged for 10 minutes at
SP:SAMPLEPREP_SUMMARY            	20,000 x g at 4 °C. 200 µL of supernatants were subjected to mass spectrometry
SP:SAMPLEPREP_SUMMARY            	analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
CH:SOLVENT_A                     	25 mM ammonium carbonate in water
CH:SOLVENT_B                     	acetonitrile
CH:FLOW_GRADIENT                 	0-1.5 min, 80% B; 1.5-7 min, 80% B to 50% B, 7-8.5 min, 50% B; 8.5-8.7 min, 50%
CH:FLOW_GRADIENT                 	B to 80% B, 8.7-13 min, 80% B
CH:FLOW_RATE                     	0.3 mL/min
CH:COLUMN_TEMPERATURE            	35
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6545 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The overall runtime was 13 minutes, and the injection volume was 5 µL. The
MS:MS_COMMENTS                   	Agilent Q-TOF was operated in negative mode and the relevant parameters were as
MS:MS_COMMENTS                   	listed: ion spray voltage, 3500 V; nozzle voltage, 1000 V; fragmentor voltage,
MS:MS_COMMENTS                   	125 V; drying gas flow, 11 L/min; capillary temperature, 325 °C; drying gas
MS:MS_COMMENTS                   	temperature, 350 °C; and nebulizer pressure, 40 psi. A full scan range was set
MS:MS_COMMENTS                   	at 50 to 1600 (m/z). The reference masses were 119.0363 and 980.0164. The
MS:MS_COMMENTS                   	acquisition rate was 2 spectra/s. Targeted analysis, isotopologues extraction
MS:MS_COMMENTS                   	(for the metabolic tracing study), and natural isotope abundance correction were
MS:MS_COMMENTS                   	performed by the Agilent Profinder B.10.00 Software (Agilent Technologies).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Abundance (Extracted ion chromatogram core area)
MS_METABOLITE_DATA_START
Samples	EV_DMSO_1h_1	EV_DMSO_1h_2	EV_DMSO_1h_3	EV_DMSO_24h_1	EV_DMSO_24h_2	EV_DMSO_24h_3	EV_Rom_1h_1	EV_Rom_1h_2	EV_Rom_1h_3	EV_Rom_24h_1	EV_Rom_24h_2	EV_Rom_24h_3	NRF2_DMSO_1h_1	NRF2_DMSO_1h_2	NRF2_DMSO_1h_3	NRF2_DMSO_24h_1	NRF2_DMSO_24h_2	NRF2_DMSO_24h_3	NRF2_Rom_1h_1	NRF2_Rom_1h_2	NRF2_Rom_1h_3	NRF2_Rom_24h_1	NRF2_Rom_24h_2	NRF2_Rom_24h_3	EV_DMSO_Gln_1	NRF2_DMSO_Gln_2	NRF2_ROM_Gln_2	NRF2_CB839_Gln_2	EV_DMSO_Gln_3	EV_ROM_Gln_3	EV_CB839_Gln_3	NRF2_DMSO_Gln_3	NRF2_ROM_Gln_3	NRF2_CB839_Gln_3	EV_ROM_Gln_1	EV_CB839_Gln_1	NRF2_DMSO_Gln_1	NRF2_ROM_Gln_1	NRF2_CB839_Gln_1	EV_DMSO_Gln_2	EV_ROM_Gln_2	EV_CB839_Gln_2
Factors	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:1 hour | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:24 hours | Tracer:U-C13 Glucose	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:NRF2 overexpression | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:DMSO | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:Romidepsin | Timepoint:8 hours | Tracer:U-C13 Glutamine	Genotype:Empty vector | Treatment:CB-839 | Timepoint:8 hours | Tracer:U-C13 Glutamine
Citric acid	9703905	10039190	9652787	8420485	6811671	4818766	6808011	7691035	5227506	8053279	5772542	4342688	3889894	7875880	4962037	7117551	10829799	10436017	6232296	4985864	4194055	11887838	7853048	7718016	12885419	257312556	141140527	208661898	43477044	22275043	119551134	251083503	205948538	164621532	78314898	69337020	111780432	171547484	194973395	112768460	76179663	226479367
L-Glutamate	27580302	34924121	42269286	20403174	24189441	23169393	21178370	16109699	18053176	10563561	10977803	10672864	42826057	26932363	41977337	29386744	23991138	23300901	14757804	17017996	17816602	9739633	12721216	9670175	39152359	46612569	22722229	8609921	30513774	9129717	11353092	47533420	26605608	11196291	21197592	11440457	32438715	23102962	8204356	44307632	22645190	10922776
Fumaric acid	642596	1709828	104030	1317369	4428931	6804722	1794772	551730	1185672	1425147	2014536	2198168	5774811	652261	5812514	5782279	4714044	5621889	1611579	2275146	3199192	2030483	5137740	3923433	14075758	11131200	9322436	6678911	11641135	4493464	7118564	11476304	11248255	5632047	13433437	6299782	7820186	9723789	5822880	15622235	13743383	9599693
L-Malic acid	3913594	10642969	12890667	7336755	27663119	43445675	10687873	5719968	7301193	13512797	12365603	16772018	36184458	4027600	36673395	35630793	29011455	34719814	9631199	13737965	19532159	12052406	31913230	24739320	109088072	82557008	69526076	46824585	24572	30547	22837	84558619	86210019	38447792	33688	31260	56384809	7770	39460415	119394474	31843	68248699
Succinic acid	407875	1422455	852042	421880	800136	1440530	185115	105048	519157	406838	335430	331316	1360502	161437	1203175	1185709	721214	1264793	430900	485047	553542	557299	528968	487259	1597227	2460234	1133449	1588093	1343245	259970	1138781	2998654	1761766	1389822	1088730	840976	1180991	1268274	1473839	2247608	1007333	1819938
Oxoglutaric acid	199673	295422	346119	97615	161455	193479	321356	122868	218548	105339	143696	180263	302716	174103	324775	189807	141360	156321	116651	140220	125161	100478	167928	105455	245622	77055	429023	38210	554193	273248	49735	134180	242528	29335	444540	26325	57649	91219	37674	349536	678960	33751
Pyruvate	489856	298172	615141	657208	179892	103606	54821	96016	68874	65392	52552	35636	328110	544544	268335	301731	213165	123067	122762	141394	138917	124923	108432	80489	636689	1510292	1145225	184651	828377	318621	334714	1952020	1201095	453953	270525	356075	1504387	1047664	193099	560887	383137	213887
Uridine triphosphate (UTP)	2911005	6924621	9750239	3858198	7154539	7280015	3807056	1577897	2945001	2666096	2603648	2389098	10184891	3577607	10804949	7828739	5931815	6292197	3343093	3876284	4983912	2918176	4569828	3543491	6889865	2035183	882649	1519069	1053194	0	787451	1691383	1641694	1252310	1089288	618912	958075	907314	1346420	9519081	716843	4004845
Adenosine triphosphate (ATP)	3678369	6257982	8276401	5510498	7364648	8318932	4853018	2493014	3845170	3505903	3750675	3459381	9770730	4247836	9580605	9668827	7527519	7740248	4108880	4788171	5744060	3806198	5816213	4693883	12079107	9652165	3335399	4833439	3521621	371716	3267167	6708823	5630610	3394128	5185846	3013594	4025183	3512965	4040674	16035117	5082101	10267957
L-Lactic acid	1946456	3297617	4174776	17001197	6098693	5815201	865522	877521	1165572	2838265	1236981	1168822	3036472	3444470	3897287	13628433	12189742	10936151	1336141	1416869	1426377	4928059	3976615	3595176	7691051	11334385	2891500	5880207	8618764	1029898	7829069	17558543	3387568	8925523	2117346	5976271	11299610	3907286	6847800	7455194	1463890	6700648
L-Serine	889786	715881	655647	783442	537733	581583	698695	1006549	915777	1231198	1059878	1142206	906402	1023761	1020077	390501	390650	318251	668718	606318	569865	700623	578473	641631	3563555	4523773	3938506	3017024	2470788	1481760	2592042	4689678	5565337	2636979	3694906	2451136	3049999	4498432	2929148	4201648	3654164	4011151
D-Glyceraldehyde 3-phosphate	858244	1417326	1497022	840583	1345582	2798547	2379558	923864	1893131	817670	1068142	1361819	3382849	1150765	3932061	4546297	2445008	3208523	2466562	3189516	4073610	1529885	3320734	2491184	1236388	4574771	816393	3830392	1428032	85860	2664605	4471471	1505219	3224025	288779	1850966	1900182	710281	2473425	2540416	352980	4450000
D-Glycerate 3-phosphate	762198	475130	946732	668163	2618382	5914696	2118407	625663	1654717	1460226	1801425	2092514	2108396	740883	3997815	6862568	5654822	5865637	1266563	2366357	4545001	2218395	4616142	3727382	2293608	7382590	2532055	4508962	1998889	339350	3127777	6201416	3447689	3541502	2141944	3116629	4136849	3339555	4307548	3840600	2233782	5157699
D-Glucose 6-phosphate	168324	343370	405874	139116	182370	231204	381752	204168	364481	156021	174839	185392	663468	275180	723576	332683	234737	297755	395581	488859	545685	209420	307887	234702	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Guanosine triphosphate (GTP)	156263	331977	477581	220075	337378	352511	215954	102308	158734	128978	122859	99061	490300	182931	520143	463800	373620	386397	199924	207882	284404	169111	259983	198333	422673	691423	229757	521037	245500	51870	415412	558039	364302	345334	284478	349392	289973	290969	457730	697032	256306	970948
L-Glutamine	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	12830452	18362521	16345281	63330202	6726325	4797556	44739765	15587779	21171916	56525455	13201020	40368680	14644411	19885154	59531398	11767287	12154844	66452150
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PubChem	KEGG	Tgt RT	Expected m/z
Citric acid	311	C00158	12.5	191.0197
L-Glutamate	33032	C00025	7.55	NA
Fumaric acid	444972	C00122	9	115.0037
L-Malic acid	222656	C00149	9	NA
Succinic acid	1110	C00042	7	117.0193
Oxoglutaric acid	51	C00026	7.2	145.01373
Pyruvate	1060	C00022	1.5	87.00088
Uridine triphosphate (UTP)	6133	C00075	12.6	NA
Adenosine triphosphate (ATP)	5957	C00002	12.5	505.9885
L-Lactic acid	107689	C00186	2.44	89.0244
L-Serine	5951	C00065	7.6	NA
D-Glyceraldehyde 3-phosphate	3418	C00118	10.873	NA
D-Glycerate 3-phosphate	439183	C00197	11	NA
D-Glucose 6-phosphate	5958	C00092	11.5	NA
Guanosine triphosphate (GTP)	6830	C00044	13.5	521.9834
L-Glutamine	5961	C00064	8.44	145.0619
METABOLITES_END
#END