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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
Study ID | ST003623 |
Study Title | NRF2 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 Type | Untargeted Metabolomics |
Study Summary | This 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 Name | Wang |
First Name | Mike |
Address | 1 DNA Way, South San Francisco, CA 94080, USA |
wang.mike@gene.com | |
Phone | 6502457991 |
Submit Date | 2024-12-06 |
Raw Data Available | Yes |
Raw Data File Type(s) | mzML |
Analysis Type Detail | LC-MS |
Release Date | 2025-01-02 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|---|
SA393310 | Sample21 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox+ | FL+1 |
SA393314 | Sample05 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox- | FL-1 |
SA393311 | Sample22 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox+ | FL+2 |
SA393315 | Sample06 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox- | FL-2 |
SA393312 | Sample23 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox+ | FL+3 |
SA393316 | Sample07 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox- | FL-3 |
SA393313 | Sample24 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox+ | FL+4 |
SA393317 | Sample08 | A549 human lung epithelial cells | shNRF2-Ful length NRF2 | Dox- | FL-4 |
SA393318 | Sample41 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+A485 | A485+1 |
SA393319 | Sample42 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+A485 | A485+2 |
SA393320 | Sample43 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+A485 | A485+3 |
SA393321 | Sample44 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+A485 | A485+4 |
SA393322 | Sample33 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+DMSO | DMSO+1 |
SA393323 | Sample34 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+DMSO | DMSO+2 |
SA393324 | Sample35 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+DMSO | DMSO+3 |
SA393325 | Sample36 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+DMSO | DMSO+4 |
SA393326 | Sample17 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+ | luc+1 |
SA393330 | Sample01 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox- | luc-1 |
SA393327 | Sample18 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+ | luc+2 |
SA393331 | Sample02 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox- | luc-2 |
SA393328 | Sample19 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+ | luc+3 |
SA393332 | Sample03 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox- | luc-3 |
SA393329 | Sample20 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox+ | luc+4 |
SA393333 | Sample04 | A549 human lung epithelial cells | shNRF2-Luciferase | Dox- | luc-4 |
SA393334 | Sample25 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox+ | 45+1 |
SA393338 | Sample09 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox- | 45-1 |
SA393335 | Sample26 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox+ | 45+2 |
SA393339 | Sample10 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox- | 45-2 |
SA393336 | Sample27 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox+ | 45+3 |
SA393340 | Sample11 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox- | 45-3 |
SA393337 | Sample28 | A549 human lung epithelial cells | shNRF2-NRF2 Neh4,5 mut | Dox+ | 45+4 |
SA393341 | Sample12 | A549 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 |