Summary of Study ST003623

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002208. The data can be accessed directly via it's Project DOI: 10.21228/M86Z6P This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003623
Study TitleNRF2 supports non-small cell lung cancer growth independently of CBP/p300-enhanced glutathione synthesis: Global metabolomics analysis on A549 cells at different NRF2 status (Part 1 of 3)
Study TypeUntargeted Metabolomics
Study SummaryThis study aims at discovering metabolic changes in A549 cancer cells in the presence and absence of NRF2, and a mutant NRF2 genotypes. Metabolites were extracted from cell pellets by using an LLE method with Methanol/Chloroform/water. The aqueous layer was analyzed by HILIC-HRMS. An in-house RT library was used to identify metabolites. Statistical analyses was performed to identify statistically significant changes in the metabolism. 35 metabolites presented differential abundance between NRF2 knockdown and wildtype conditions. In particular, GSH and several glutamate dipeptides were significantly depleted upon NRF2 knockdown, in line with the prevailing role of NRF2 in controlling GSH biosynthesis. Disruption of additional metabolites involved in the PPP (sedoheptulose-7-phosphate, S7P), nucleotide (CMP, IMP) and amino acid (kynurenine, homocitrulline) metabolism were also observed upon NRF2 knockdown.
Institute
Genentech Inc.
Last NameWang
First NameMike
Address1 DNA Way, South San Francisco, CA 94080, USA
Emailwang.mike@gene.com
Phone6502457991
Submit Date2024-12-06
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-01-02
Release Version1
Mike Wang Mike Wang
https://dx.doi.org/10.21228/M86Z6P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002208
Project DOI:doi: 10.21228/M86Z6P
Project Title:NRF2 supports non-small cell lung cancer growth independently of CBP/p300-enhanced glutathione synthesis
Project Type:MS quantitative analysis
Project Summary:Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress responsive transcription factor that is mutationally activated in a subset (~25%) of clinically-aggressive non-small cell lung cancers (NSCLC). Mechanistic insight into drivers of the NRF2 dependency remains poorly understood. Here, we defined a novel NRF2 target gene set linked to NRF2-dependency in cancer cell lines, and observed that a significant portion of these genes is devoid of promoter-proximal NRF2. Using integrated genomic analyses, we characterized extensive NRF2-dependent enhancer RNA (eRNA) synthesis and NRF2-mediated H3K27ac deposition at proximal and distal enhancer regions regulating these genes. While CBP/p300 is a well-validated direct interaction partner of NRF2 with prominent functions at enhancers, we report that this interaction is not required for NRF2-dependent NSCLC cell growth, indicating that NRF2 can sustain sufficient transcriptional activity in the absence of CBP/p300 coactivation. Broad metabolic profiling established a primary role for CBP/p300 in NRF2-dependent accumulation of glutathione and glutathione-related metabolites. While redox homeostasis via enhanced glutathione production is commonly associated with the normal physiological role of NRF2, collectively our results suggest that NRF2-dependent cancer cell growth does not require this enhanced glutathione production.
Institute:Genentech Inc.
Last Name:Wong
First Name:Weng Ruh
Address:1 DNA Way, South San Francisco, CA 94080, USA
Email:wongw24@gene.com
Phone:4089048962
Contributors:Ryan J. Conrad, James A. Mondo, Mike Lingjue Wang, Peter S. Liu, Zijuan Lai, Feroza K Choudhury, Qingling Li, Weng Ruh Wong, James Lee, Frances Shanahan, Eva Lin, Scott Martin, Joachim Rudolph, John G. Moffat, Dewakar Sangaraju, Wendy Sandoval, Timothy Sterne-Weiler, Scott A. Foster

Subject:

Subject ID:SU003753
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:A549
Species Group:Mammals

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Genotype Treatment Sample Label
SA393310Sample21A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox+ FL+1
SA393314Sample05A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox- FL-1
SA393311Sample22A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox+ FL+2
SA393315Sample06A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox- FL-2
SA393312Sample23A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox+ FL+3
SA393316Sample07A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox- FL-3
SA393313Sample24A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox+ FL+4
SA393317Sample08A549 human lung epithelial cells shNRF2-Ful length NRF2 Dox- FL-4
SA393318Sample41A549 human lung epithelial cells shNRF2-Luciferase Dox+A485 A485+1
SA393319Sample42A549 human lung epithelial cells shNRF2-Luciferase Dox+A485 A485+2
SA393320Sample43A549 human lung epithelial cells shNRF2-Luciferase Dox+A485 A485+3
SA393321Sample44A549 human lung epithelial cells shNRF2-Luciferase Dox+A485 A485+4
SA393322Sample33A549 human lung epithelial cells shNRF2-Luciferase Dox+DMSO DMSO+1
SA393323Sample34A549 human lung epithelial cells shNRF2-Luciferase Dox+DMSO DMSO+2
SA393324Sample35A549 human lung epithelial cells shNRF2-Luciferase Dox+DMSO DMSO+3
SA393325Sample36A549 human lung epithelial cells shNRF2-Luciferase Dox+DMSO DMSO+4
SA393326Sample17A549 human lung epithelial cells shNRF2-Luciferase Dox+ luc+1
SA393330Sample01A549 human lung epithelial cells shNRF2-Luciferase Dox- luc-1
SA393327Sample18A549 human lung epithelial cells shNRF2-Luciferase Dox+ luc+2
SA393331Sample02A549 human lung epithelial cells shNRF2-Luciferase Dox- luc-2
SA393328Sample19A549 human lung epithelial cells shNRF2-Luciferase Dox+ luc+3
SA393332Sample03A549 human lung epithelial cells shNRF2-Luciferase Dox- luc-3
SA393329Sample20A549 human lung epithelial cells shNRF2-Luciferase Dox+ luc+4
SA393333Sample04A549 human lung epithelial cells shNRF2-Luciferase Dox- luc-4
SA393334Sample25A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox+ 45+1
SA393338Sample09A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox- 45-1
SA393335Sample26A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox+ 45+2
SA393339Sample10A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox- 45-2
SA393336Sample27A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox+ 45+3
SA393340Sample11A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox- 45-3
SA393337Sample28A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox+ 45+4
SA393341Sample12A549 human lung epithelial cells shNRF2-NRF2 Neh4,5 mut Dox- 45-4
Showing results 1 to 32 of 32

Collection:

Collection ID:CO003746
Collection Summary:All cell lines were obtained from the Genentech cell bank (Yu et al, 2015) and grown in RPMI or DMEM supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin at 37°C, 5% CO2. Cell lines were confirmed via STR profiling and were mycoplasma free. A549 and A549shNRF2-BIND rescue cell lines were generated using the Piggybac system. Donor constructs were co-transfected with transposase (Ding et al, 2005) at a 4:1 ratio using Lipofetamine 3000 (Thermo) according to manufacturer’s protocol. 3-days post-transfection, puromycin selection (1µg/mL) was initiated and was performed for ~2 weeks prior to experimentation. A549shNRF2-BIND rescue cells represent polyclonal pools. Cell viability was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using the Envision 2103 Plate Reader (Promega). Cell growth curves were generated using the IncuCyte System (Sartorius) according to the manufacturer’s protocol. Curve fits were performed in Prism. Clonogenic assays were performed by washing cells in PBS, staining with 0.5% crystal violet solution (Sigma HT90132) for 5 min, and washing 3x with PBS. Four replicates of 20-30 million A549shNRF2-BIND-luc, WT NRF2 or Neh4/5mut cells were treated with 2.5ng/mL dox for 48h. For A485 testing, cells were treated 1µM A-485 or DMSO for 24h before harvesting. After treatment, cells were trypsinized, washed 3x with ice cold PBS and flash frozen in liquid nitrogen for metabolic quenching.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003762
Treatment Summary:No treatment was administered and this is a study based on genetic variants.

Sample Preparation:

Sampleprep ID:SP003760
Sampleprep Summary:Cells were trypsinized, washed 3x with ice cold PBS and flash frozen in liquid nitrogen for metabolic quenching. All metabolite extraction procedures were kept on ice. Briefly, 350μL of cold methanol containing in-house metabolomics Recovery IS (Stable Isotope Labeled Internal Standards) mixture and 200μL of cold chloroform were added to each sample. Samples were vortexed for 10 seconds, homogenized with beads for 2 minutes, and centrifuged at 4000 RPM for 5 minutes. Next, supernatants were transferred and 200μl cold water was added to perform liquid–liquid phase separation. Samples were mixed and centrifuged again. The top layer aliquots were transferred, dried and reconstituted in 100μl acetonitrile:water (8:2, v/v) containing the in-house metabolomics Global IS (Stable Isotope Labeled Internal Standards) mixture and submitted for metabolomics analysis.

Combined analysis:

Analysis ID AN005953
Analysis type MS
Chromatography type HILIC
Chromatography system Shimadzu Nexera HPLC
Column Waters ACQUITY UPLC BEH Amide (150 x 2.1mm × 1.7μm, 130Å)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode UNSPECIFIED
Units Ion Count

Chromatography:

Chromatography ID:CH004523
Instrument Name:Shimadzu Nexera HPLC
Column Name:Waters ACQUITY UPLC BEH Amide (150 x 2.1mm × 1.7μm, 130Å)
Column Temperature:40
Flow Gradient:0-2min, 100% B; 7.7min, 70% B; 9.5min, 40%B; 10.25min, 30%B; 12.75min, 100%B; 16.75min, 100%B;
Flow Rate:0.4 mL/min
Solvent A:100% water; 10mM ammonium formate; 0.125% formic acid
Solvent B:95% acetonitrile/5% water; 10mM ammonium formate; 0.125% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS005668
Analysis ID:AN005953
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Samples were run in polarity switching mode for both positive and negative acquisitions. The Q Exactive Plus Mass Spectrometer was operated with the following parameters: Sheath gas flow rate, 50 units; Aux gas flow rate, 13 units; Aux gas temperature, 425‎ °C; Capillary temperature, 263‎°C; Spray voltage, 3500V for pos and -2500V for neg; Scan mode, Full MS scan with data-dependent MS/MS acquisition. In Full MS scan, scan range is 60-900 m/z; resolution is 70,000; AGC target, 1×e^6; Maximum IT, 200 ms. In ddMS2 scan, top 5 ions are selected with an isolation window of 1.5 m/z; resolution is 17,500; AGC target, 5×e^4; Maximum IT, 20 ms.
Ion Mode:UNSPECIFIED
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