Summary of Study ST004169

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 PR002629. The data can be accessed directly via it's Project DOI: 10.21228/M8TK1K This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004169
Study Title	Alanine catabolism as a targetable vulnerability for MYC-driven liver cancer
Study Summary	Liver cancer remains a leading cause of cancer-related death in part due to the shortage of effective therapies, and MYC overexpression defines an aggressive and especially difficult to treat subset of patients. Given MYC’s ability to reprogram cancer cell metabolism, and the liver’s role as a coordinator of systemic metabolism, we hypothesized that MYC could induce metabolic dependencies that could be targeted to attenuate tumor growth. We discovered that MYC-driven liver cancers catabolize alanine in a GPT2-dependent manner to fuel their growth. We observed that GPT2 was the predominate alanine enzyme expressed in MYC-driven liver tumors and that genetic ablation of GPT2 limited MYC-driven liver tumorigenesis. In vivo isotope tracing studies uncovered a role for alanine in fueling a repertoire of pathways including the tricarboxylic acid cycle, nucleotide production, and amino acid synthesis in liver tumors and non-tumor liver, and also examined the kidney as an internal positive control, since it is a known site of alanine metabolism. Treating transgenic MYC-driven liver tumor mouse models with L-Cycloserine, a compound that inhibits GPT2, was sufficient to diminish the frequency of mouse tumor formation and attenuate growth of established human liver tumors. Thus, we identify a new targetable metabolic dependency that MYC-driven liver tumors usurp to fuel their survival.
Institute	University of California, San Francisco
Department	Cell & Tissue Biology
Laboratory	Andrei Goga
Last Name	Montoya
First Name	Tonatiuh
Address	513 Parnassus Ave
Email	Tonatiuh.Montoya@ucsf.edu
Phone	415-476-4187
Submit Date	2025-07-11
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2026-01-12
Release Version	1

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Project:

Project ID:	PR002629
Project DOI:	doi: 10.21228/M8TK1K
Project Title:	Alanine catabolism as a targetable vulnerability for MYC-driven liver cancer
Project Summary:	The aim was to compare how alanine is metabolized in MYC-driven liver tumors versus non-tumor liver tissue. Liver tumor bearing mice were infused with C13/N15 alanine, and kidney, liver tumor, and non tumor liver tissue were harvested. Kidney was used as an internal positive control, since the kidney is a known site of alanine metabolism. We found that on average ~35% of the tumor alanine pool had at least one heavy isotope label, thus confirming the efficacy of our tracing strategy. We uncovered a repertoire of pathways downstream of alanine, with multiple metabolites being preferentially labelled in tumor versus non-tumor tissue. Among the pathways fed by alanine in tumors were metabolites relating to cellular bioenergetics, biosynthesis, and the oxidative stress response. Thus our study identified how alanine is a substrate for metabolism in MYC-driven liver tumors.
Institute:	University of California, San Francisco
Department:	Cell & Tissue Biology
Last Name:	Montoya
First Name:	Tonatiuh
Address:	513 Parnassus Ave
Email:	Tonatiuh.Montoya@ucsf.edu
Phone:	415-476-4187

Subject:

Subject ID:	SU004320
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Sample type
SA481687	Kidney 6	kidney	no MYC overexpression
SA481688	Kidney 2	kidney	no MYC overexpression
SA481689	Kidney 7	kidney	no MYC overexpression
SA481690	Kidney 1	kidney	no MYC overexpression
SA481691	Kidney 3	kidney	no MYC overexpression
SA481692	Kidney 5	kidney	no MYC overexpression
SA481693	Kidney 4	kidney	no MYC overexpression
SA481694	Tumor 2	liver	MYC overexpression
SA481695	Tumor 7	liver	MYC overexpression
SA481696	Tumor 6	liver	MYC overexpression
SA481697	Tumor 5	liver	MYC overexpression
SA481698	Tumor 4	liver	MYC overexpression
SA481699	Tumor 3	liver	MYC overexpression
SA481700	Tumor 1	liver	MYC overexpression
SA481701	Non-tumor 7	liver	no MYC overexpression
SA481702	Non-tumor 6	liver	no MYC overexpression
SA481703	Non-tumor 4	liver	no MYC overexpression
SA481704	Non-tumor 3	liver	no MYC overexpression
SA481705	Non-tumor 2	liver	no MYC overexpression
SA481706	Non-tumor 1	liver	no MYC overexpression
SA481707	Non-tumor 5	liver	no MYC overexpression

Showing results 1 to 21 of 21

Showing results 1 to 21 of 21

Collection:

Collection ID:	CO004313
Collection Summary:	In vivo isotope tracing was performed on LT2-MYC mice that had been off DOX chow for 8-10 weeks. Tumor-bearing mice were anesthetized by isoflurane and kept on heating pads. Mice then had a catheter (Instech, C20PU-MJV2014) inserted into their jugular vein and tied in place using sutures (Surgical Specialties, SP117) and the catheter was then flushed with 50 μL heparin (McKesson, NDC 63739-931-14) to prevent coagulation. Mice were then infused with a 13C3 / 15N-alanine bolus of 0.114 mg per g body weight, followed by an infusion of 0.003 mg per g body weight per minute for 3 hours at a flow rate of 0.15 mL per hour using a Pump 11 Elite Dual Syringe Infusion Pump (Harvard apparatus, 70-4501). 13C3 / 15N-alanine was dissolved sterilely in normal saline at 48 mg/mL. After infusion, mice were sacrificed, perfused with saline, and tumor and non-tumor tissue dissected and snap frozen. After extraction, dried samples were then stored at -80°C until they were run on the LC/MS.
Sample Type:	Liver, Kidney
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR004329
Treatment Summary:	No treatment.

Sample Preparation:

Sampleprep ID:	SP004326
Sampleprep Summary:	Polar metabolite extraction from tissue samples was done by using a sharp blade to cut off a small piece of tissue 10-30 mg in weight. Each sample was placed in a 2 mL tube with a 5 mm stainless-steel bead (Qiagen, 69989) and 1 mL of methanol extraction solution composed of 80% methanol: 20% H2O with 100 nM trifluoromethanesulfonate (Fisher, A456-500; Fisher, W6-212; Sigma, 422843-5G). Samples were then extracted on a TissueLyser LT (Qiagen, 85600) by running samples for 1 minute at max speed, followed by a 1 minute break, three consecutive times. After extraction, samples were rested at -20°C for 10 minutes, and then vortexed and centrifuged at 17,000 x g for 10 minutes at 4°C. 700 µL of the supernatant was then transferred to new tubes and centrifuged in the same manner again. The top 500 µL of the supernatant was then transferred into a new tube and a volume of sample corresponding to a tissue equivalent of 4 mg was transferred into a new tube for evaporation. Samples were run on a DNA Vac-200 on high for 1-2 hours, or until the sample was completely evaporated. Dried samples were then stored at -80°C until they were run on the LC/MS.

Sampleprep ID:

SP004326

Sampleprep Summary:

Polar metabolite extraction from tissue samples was done by using a sharp blade to cut off a small piece of tissue 10-30 mg in weight. Each sample was placed in a 2 mL tube with a 5 mm stainless-steel bead (Qiagen, 69989) and 1 mL of methanol extraction solution composed of 80% methanol: 20% H2O with 100 nM trifluoromethanesulfonate (Fisher, A456-500; Fisher, W6-212; Sigma, 422843-5G). Samples were then extracted on a TissueLyser LT (Qiagen, 85600) by running samples for 1 minute at max speed, followed by a 1 minute break, three consecutive times. After extraction, samples were rested at -20°C for 10 minutes, and then vortexed and centrifuged at 17,000 x g for 10 minutes at 4°C. 700 µL of the supernatant was then transferred to new tubes and centrifuged in the same manner again. The top 500 µL of the supernatant was then transferred into a new tube and a volume of sample corresponding to a tissue equivalent of 4 mg was transferred into a new tube for evaporation. Samples were run on a DNA Vac-200 on high for 1-2 hours, or until the sample was completely evaporated. Dried samples were then stored at -80°C until they were run on the LC/MS.

Combined analysis:

Analysis ID	AN006920	AN006921
Chromatography ID	CH005256	CH005256
MS ID	MS006617	MS006618
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH005256
Chromatography Summary:	Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis. Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (20mM ammonium carbonate, pH 9.7) and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	35°C
Flow Gradient:	Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min.
Flow Rate:	150 µL/min
Solvent A:	100% water; 20mM ammonium carbonate, pH 9.7
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS006617
Analysis ID:	AN006920
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range = (70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor 2 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	POSITIVE

MS ID:	MS006618
Analysis ID:	AN006921
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range = (70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor 2 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	NEGATIVE