Summary of Study ST004144

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 PR002607. The data can be accessed directly via it's Project DOI: 10.21228/M8NZ6Q 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	ST004144
Study Title	Metabolic rewiring in isogenic SW48 colorectal cancer cells with different oncogenic KRAS G12 point mutations
Study Summary	Transcriptomics data and phenotypic characterization(including mitochondrial stress assays) in SW48 isogenic cell lines expressing oncogenic KRAS mutations (G12A, G12C, G12D and G12V) have revealed a differential metabolic features in these mutants. We have for that reason performed Glucose labelling and Nitrogen labelling experiments to assess more in detail the metabolic differences in these mutants. Coupled with transcriptomics data we have seen changes in FOXO1 signalling which is well documented in its involvement in glutamine metabolism and ammonia recycling pathway. We have repeated those labelling experiments in the presence of FOXO1 inhibitor to understand whether these differential metabolic traits are controlled by FOXO1 pathway.
Institute	Brunel University of London
Last Name	Esposito
First Name	Alessandro
Address	Brunel University of London, Kingston Lane, UB8 3PH, London, United Kingdom
Email	alessandro.esposito@brunel.ac.uk
Phone	07954383731
Submit Date	2025-08-17
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-08-25
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002607
Project DOI:	doi: 10.21228/M8NZ6Q
Project Title:	FOXO1 links KRAS G12D and G12V alleles to glutamine and nitrogen metabolism in colorectal cancer
Project Summary:	Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming are unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we characterise distinctive features of each allele. We demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy.
Institute:	Brunel University of London
Department:	Biosciences
Laboratory:	Laboratory of Quantitative Biology (Esposito group)
Last Name:	Esposito
First Name:	Alessandro
Address:	Brunel University of London, Kingston Lane, UB8 3PH, London, United Kingdom
Email:	alessandro.esposito@brunel.ac.uk
Phone:	07954383731

Subject:

Subject ID:	SU004294
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Biosource Or Supplier:	Isogenic cell line SW48 (Horizon Discoveries)
Cell Strain Details:	SW48 WT, G12A, G12C, G12D, G12V

Factors:

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

mb_sample_id	local_sample_id	Sample source	Media	Inhibitor Treatment	Labelling
SA479514	SB006B	Media only SW48_G12A	Low Nutrient	-	13C-glucose
SA479515	SB010B	Media only SW48_G12A	Low Nutrient	-	13C-glucose
SA479516	SB009B	Media only SW48_G12A	Low Nutrient	-	13C-glucose
SA479517	SB008B	Media only SW48_G12A	Low Nutrient	-	13C-glucose
SA479518	SB007B	Media only SW48_G12A	Low Nutrient	-	13C-glucose
SA479519	SB015B	Media only SW48_G12C	Low Nutrient	-	13C-glucose
SA479520	SB014B	Media only SW48_G12C	Low Nutrient	-	13C-glucose
SA479521	SB013B	Media only SW48_G12C	Low Nutrient	-	13C-glucose
SA479522	SB011B	Media only SW48_G12C	Low Nutrient	-	13C-glucose
SA479523	SB012B	Media only SW48_G12C	Low Nutrient	-	13C-glucose
SA479544	SB07-070	Media only SW48_G12D	Low Nutrient	DMSO	13C-glucose
SA479545	SB07-069	Media only SW48_G12D	Low Nutrient	DMSO	13C-glucose
SA479546	SB07-068	Media only SW48_G12D	Low Nutrient	DMSO	13C-glucose
SA479547	SB07-067	Media only SW48_G12D	Low Nutrient	DMSO	13C-glucose
SA479548	SB07-066	Media only SW48_G12D	Low Nutrient	DMSO	13C-glucose
SA479549	SB07-097	Media only SW48_G12D	Low Nutrient	DMSO	15N-Ammonia
SA479550	SB07-100	Media only SW48_G12D	Low Nutrient	DMSO	15N-Ammonia
SA479551	SB07-098	Media only SW48_G12D	Low Nutrient	DMSO	15N-Ammonia
SA479552	SB07-096	Media only SW48_G12D	Low Nutrient	DMSO	15N-Ammonia
SA479553	SB07-099	Media only SW48_G12D	Low Nutrient	DMSO	15N-Ammonia
SA479554	SB07-085	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479555	SB07-084	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479556	SB07-083	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479557	SB07-082	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479558	SB07-081	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479559	SB07-111	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479560	SB07-113	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479561	SB07-112	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479562	SB07-114	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479563	SB07-115	Media only SW48_G12D	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479524	SB020B	Media only SW48_G12D	Low Nutrient	-	13C-glucose
SA479525	SB018B	Media only SW48_G12D	Low Nutrient	-	13C-glucose
SA479526	SB019B	Media only SW48_G12D	Low Nutrient	-	13C-glucose
SA479527	SB017B	Media only SW48_G12D	Low Nutrient	-	13C-glucose
SA479528	SB016B	Media only SW48_G12D	Low Nutrient	-	13C-glucose
SA479529	SB05-039	Media only SW48_G12D	Low Nutrient	-	15N-alpha-glutamine
SA479530	SB05-038	Media only SW48_G12D	Low Nutrient	-	15N-alpha-glutamine
SA479531	SB05-037	Media only SW48_G12D	Low Nutrient	-	15N-alpha-glutamine
SA479532	SB05-036	Media only SW48_G12D	Low Nutrient	-	15N-alpha-glutamine
SA479533	SB05-040	Media only SW48_G12D	Low Nutrient	-	15N-alpha-glutamine
SA479534	SB05-010	Media only SW48_G12D	Low Nutrient	-	15N-amide-glutamine
SA479535	SB05-009	Media only SW48_G12D	Low Nutrient	-	15N-amide-glutamine
SA479536	SB05-008	Media only SW48_G12D	Low Nutrient	-	15N-amide-glutamine
SA479537	SB05-007	Media only SW48_G12D	Low Nutrient	-	15N-amide-glutamine
SA479538	SB05-006	Media only SW48_G12D	Low Nutrient	-	15N-amide-glutamine
SA479539	SB05-021	Media only SW48_G12D	Low Nutrient	-	15N-ammonia
SA479540	SB05-022	Media only SW48_G12D	Low Nutrient	-	15N-ammonia
SA479541	SB05-025	Media only SW48_G12D	Low Nutrient	-	15N-ammonia
SA479542	SB05-024	Media only SW48_G12D	Low Nutrient	-	15N-ammonia
SA479543	SB05-023	Media only SW48_G12D	Low Nutrient	-	15N-ammonia
SA479584	SB07-072	Media only SW48_G12V	Low Nutrient	DMSO	13C-glucose
SA479585	SB07-071	Media only SW48_G12V	Low Nutrient	DMSO	13C-glucose
SA479586	SB07-075	Media only SW48_G12V	Low Nutrient	DMSO	13C-glucose
SA479587	SB07-073	Media only SW48_G12V	Low Nutrient	DMSO	13C-glucose
SA479588	SB07-074	Media only SW48_G12V	Low Nutrient	DMSO	13C-glucose
SA479589	SB07-104	Media only SW48_G12V	Low Nutrient	DMSO	15N-Ammonia
SA479590	SB07-105	Media only SW48_G12V	Low Nutrient	DMSO	15N-Ammonia
SA479591	SB07-101	Media only SW48_G12V	Low Nutrient	DMSO	15N-Ammonia
SA479592	SB07-102	Media only SW48_G12V	Low Nutrient	DMSO	15N-Ammonia
SA479593	SB07-103	Media only SW48_G12V	Low Nutrient	DMSO	15N-Ammonia
SA479594	SB07-089	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479595	SB07-090	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479596	SB07-088	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479597	SB07-087	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479598	SB07-086	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	13C-glucose
SA479599	SB07-116	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479600	SB07-117	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479601	SB07-118	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479602	SB07-119	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479603	SB07-120	Media only SW48_G12V	Low Nutrient	FOXO1 inhibitor (AS1842856), 1µM	15N-Ammonia
SA479564	SB023B	Media only SW48_G12V	Low Nutrient	-	13C-glucose
SA479565	SB024B	Media only SW48_G12V	Low Nutrient	-	13C-glucose
SA479566	SB025B	Media only SW48_G12V	Low Nutrient	-	13C-glucose
SA479567	SB022B	Media only SW48_G12V	Low Nutrient	-	13C-glucose
SA479568	SB021B	Media only SW48_G12V	Low Nutrient	-	13C-glucose
SA479569	SB05-041	Media only SW48_G12V	Low Nutrient	-	15N-alpha-glutamine
SA479570	SB05-042	Media only SW48_G12V	Low Nutrient	-	15N-alpha-glutamine
SA479571	SB05-043	Media only SW48_G12V	Low Nutrient	-	15N-alpha-glutamine
SA479572	SB05-044	Media only SW48_G12V	Low Nutrient	-	15N-alpha-glutamine
SA479573	SB05-045	Media only SW48_G12V	Low Nutrient	-	15N-alpha-glutamine
SA479574	SB05-011	Media only SW48_G12V	Low Nutrient	-	15N-amide-glutamine
SA479575	SB05-012	Media only SW48_G12V	Low Nutrient	-	15N-amide-glutamine
SA479576	SB05-013	Media only SW48_G12V	Low Nutrient	-	15N-amide-glutamine
SA479577	SB05-014	Media only SW48_G12V	Low Nutrient	-	15N-amide-glutamine
SA479578	SB05-015	Media only SW48_G12V	Low Nutrient	-	15N-amide-glutamine
SA479579	SB05-030	Media only SW48_G12V	Low Nutrient	-	15N-ammonia
SA479580	SB05-026	Media only SW48_G12V	Low Nutrient	-	15N-ammonia
SA479581	SB05-027	Media only SW48_G12V	Low Nutrient	-	15N-ammonia
SA479582	SB05-028	Media only SW48_G12V	Low Nutrient	-	15N-ammonia
SA479583	SB05-029	Media only SW48_G12V	Low Nutrient	-	15N-ammonia
SA479624	SB07-061	Media only SW48_WT	Low Nutrient	DMSO	13C-glucose
SA479625	SB07-063	Media only SW48_WT	Low Nutrient	DMSO	13C-glucose
SA479626	SB07-064	Media only SW48_WT	Low Nutrient	DMSO	13C-glucose
SA479627	SB07-065	Media only SW48_WT	Low Nutrient	DMSO	13C-glucose
SA479628	SB07-062	Media only SW48_WT	Low Nutrient	DMSO	13C-glucose
SA479629	SB07-095	Media only SW48_WT	Low Nutrient	DMSO	15N-Ammonia
SA479630	SB07-094	Media only SW48_WT	Low Nutrient	DMSO	15N-Ammonia
SA479631	SB07-093	Media only SW48_WT	Low Nutrient	DMSO	15N-Ammonia
SA479632	SB07-092	Media only SW48_WT	Low Nutrient	DMSO	15N-Ammonia
SA479633	SB07-091	Media only SW48_WT	Low Nutrient	DMSO	15N-Ammonia

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Collection:

Collection ID:	CO004287
Collection Summary:	Cells were washed twice in PBS and incubated in PBS on cold bath (dry ice and methanol). PBS was then replaced with metabolite extraction buffer (MEB, 50% LC–MS grade methanol (Fisher Scientific), 30% LC–MS grade acetonitrile (Fisher Scientific) and 20% ultrapure water), 1 mL extraction buffer for 1x106cells; after a couple of minutes on the cold bath, cells were stored at −80°C overnight. The next day, extracts were scraped and mixed agitating for 15 minutes at 4°C at maximum speed (3000rpm). Finally, extracts were centrifuged for 10 minutes at maximum speed (14000rpm) at 4°C and transferred into LC–MS vials for analysis. Valine-d8 5 μM (CK isotopes) was used as internal standard for the MEB.
Sample Type:	Cultured cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR004303
Treatment Summary:	SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 2 mM 13C-labelled glucose (D-glucose, CK Isotopes) and cultured for another 24 hours before extraction. For 13C-glucose labelling experiments with the FOXO1 inhibitor, cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). 24 hours later cells were pre-treated with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for 12 hours in full media conditions, then washed with PBS and media replaced with low nutrient media 2 mM 13C-labelled glucose (D-glucose, CK Isotopes) with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for further 12 hours. For Glutamine (nitrogen) labelling experiments, SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 2mM 15N-amide glutamine or 2mM 15N-alpha glutamine for 4 hours before extraction. For ammonia labelling experiments, SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 3 mM of 15N-labelled ammonium chloride (CK Isotopes) for 4 hours before extraction. For ammonia labelling experiments with the FOXO1 inhibitor, cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). 24 hours later cells were pre-treated with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for 12 hours in full media conditions, then washed with PBS and media replaced with low nutrient media with 3mM of 15N-labelled ammonium chloride (CK Isotopes) with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for for 4 hours before extraction.

Treatment ID:

TR004303

Treatment Summary:

SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 2 mM 13C-labelled glucose (D-glucose, CK Isotopes) and cultured for another 24 hours before extraction. For 13C-glucose labelling experiments with the FOXO1 inhibitor, cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). 24 hours later cells were pre-treated with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for 12 hours in full media conditions, then washed with PBS and media replaced with low nutrient media 2 mM 13C-labelled glucose (D-glucose, CK Isotopes) with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for further 12 hours. For Glutamine (nitrogen) labelling experiments, SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 2mM 15N-amide glutamine or 2mM 15N-alpha glutamine for 4 hours before extraction. For ammonia labelling experiments, SW48 isogenic cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). The next day, after a PBS wash, the media was changed into low nutrient media with 3 mM of 15N-labelled ammonium chloride (CK Isotopes) for 4 hours before extraction. For ammonia labelling experiments with the FOXO1 inhibitor, cells were plated on 6-well plates at a density of 8x105 cells/well in full media conditions (5 replicates for each cell line). 24 hours later cells were pre-treated with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for 12 hours in full media conditions, then washed with PBS and media replaced with low nutrient media with 3mM of 15N-labelled ammonium chloride (CK Isotopes) with DMSO (0.003% v/v) control or FOXO1 inhibitor (1 μM) for for 4 hours before extraction.

Sample Preparation:

Sampleprep ID:	SP004300
Sampleprep Summary:	Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well after the washes in PBS following the proportion of 1ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials.

Sampleprep ID:

SP004300

Sampleprep Summary:

Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well after the washes in PBS following the proportion of 1ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials.

Chromatography:

Chromatography ID:	CH005221
Chromatography Summary:	Chromatographic separation of polar metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. Samples were randomized and analysed with LC–MS in a blinded manner with an injection volume was 5 µl. Pooled samples were generated from an equal mixture of all individual samples and analysed interspersed at regular intervals within sample sequence as a quality control.
Instrument Name:	Dionex Ultimate 3000 UHPLC
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm)
Column Temperature:	40°C
Flow Gradient:	0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B.
Flow Rate:	0.200 mL/min
Solvent A:	100% Water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN006869
Analysis Type:	MS
Chromatography ID:	CH005221
Num Factors:	59
Num Metabolites:	885
Units:	peak area