Summary of Study ST002709

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 PR001679. The data can be accessed directly via it's Project DOI: 10.21228/M8P71W 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 IDST002709
Study TitleFH variant pathogenicity promotes purine salvage pathway dependence in kidney cancer
Study SummaryThe tricarboxylic citric acid cycle enzyme fumarate hydratase (FH) is a tumor suppressor. When lost in cells, its substrate fumarate accumulates to mM levels and drives oncogenic signaling and transformation. Germline alterations lead to an autosomal dominant condition known as hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to various benign tumors and an aggressive form of kidney cancer. FH alterations of unclear significance are frequently observed with germline testing; thus, there is an unmet need to classify FH variants by their cancer-associated risk, allowing for screening, early diagnosis and treatment. Here we quantify catalytic efficiency of 74 FH variants of uncertain significance. Over half were enzymatically inactive which is strong evidence of pathogenicity. We generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks purine biosynthesis, rendering FH-deficient cells more sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. Together, these findings suggest pathogenicity of many patientassociated FH variants and reveal nucleotide salvage as a targetable vulnerability in FHdeficient cancer cells.
Institute
University of California, Los Angeles
DepartmentBiological Chemistry
LaboratoryHeather Christofk
Last NameMatulionis
First NameNedas
Address615 Charles E Young Dr S, BSRB 354-05
Emailnmatulionis@mednet.ucla.edu
Phone(310) 206-0163
Submit Date2023-05-15
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2023-06-12
Release Version1
Nedas Matulionis Nedas Matulionis
https://dx.doi.org/10.21228/M8P71W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001679
Project DOI:doi: 10.21228/M8P71W
Project Title:FH variant pathogenicity promotes salvage pathway dependence in kidney cancer
Project Summary:The tricarboxylic citric acid cycle enzyme fumarate hydratase (FH) is a tumor suppressor. When lost in cells, its substrate fumarate accumulates to mM levels and drives oncogenic signaling and transformation. Germline alterations lead to an autosomal dominant condition known as hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to various benign tumors and an aggressive form of kidney cancer. FH alterations of unclear significance are frequently observed with germline testing; thus, there is an unmet need to classify FH variants by their cancer-associated risk, allowing for screening, early diagnosis and treatment. Here we quantify catalytic efficiency of 74 FH variants of uncertain significance. Over half were enzymatically inactive which is strong evidence of pathogenicity. We generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks purine biosynthesis, rendering FH-deficient cells more sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. Together, these findings suggest pathogenicity of many patientassociated FH variants and reveal nucleotide salvage as a targetable vulnerability in FHdeficient cancer cells.
Institute:University of California, Los Angeles
Department:Biological Chemistry
Laboratory:Heather Christofk
Last Name:Matulionis
First Name:Nedas
Address:615 Charles E Young Dr S, BSRB 354-05
Email:nmatulionis@mednet.ucla.edu
Phone:(310) 206-0163

Subject:

Subject ID:SU002814
Subject Type:Cultured cells
Subject Species:NCCFH1; UOK262; UTFHC1
Taxonomy ID:-
Species Group:Other

Factors:

Subject type: Cultured cells; Subject species: NCCFH1; UOK262; UTFHC1 (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Tracers used
SA272370Sample_22FH 3,3,3',3'-D4 Cystine
SA272371Sample_23FH 3,3,3',3'-D4 Cystine
SA272372Sample_24FH 3,3,3',3'-D4 Cystine
SA272379Sample_15FH none
SA272380Sample_14FH none
SA272381Sample_13FH none
SA272373Sample_17FH U-C13 glucose
SA272374Sample_16FH U-C13 glucose
SA272375Sample_18FH U-C13 glucose
SA272376Sample_21FH U-C13 glutamine
SA272377Sample_19FH U-C13 glutamine
SA272378Sample_20FH U-C13 glutamine
SA272382Sample_10WT 3,3,3',3'-D4 Cystine
SA272383Sample_11WT 3,3,3',3'-D4 Cystine
SA272384Sample_12WT 3,3,3',3'-D4 Cystine
SA272391Sample_2WT none
SA272392Sample_3WT none
SA272393Sample_1WT none
SA272385Sample_5WT U-C13 glucose
SA272386Sample_4WT U-C13 glucose
SA272387Sample_6WT U-C13 glucose
SA272388Sample_8WT U-C13 glutamine
SA272389Sample_7WT U-C13 glutamine
SA272390Sample_9WT U-C13 glutamine
Showing results 1 to 24 of 24

Collection:

Collection ID:CO002807
Collection Summary:Place the 6 well plates to be extracted on ice. Completely aspirate off the media from each well. Gently add 2mL of ice cold ammonium acetate to each and every well without disturbing the cells. Aspirate off the ammonium acetate. Repeat this wash step once more. Aspirate off as much ammonium acetate as possible. Add 500uL of the 80% methanol solution to each and every well. Place all the 6 well plates in a -80°C freezer for 15 min. Remove the plates from the freezer, place them back on ice, and use cell scrapers to scrape off the adherent cells into solution. Transfer the cell solution from each well into a new eppitube. Vortex all the eppitubes vigorously. Centrifuge the eppitubes at 17,000g for 10 min at 4°C. Transfer the top 250 uL of the supernatant into a new 2mL tube for evaporation. Move all the tubes to the N2 evaporator and open them all. Then adjust all the needles to point into the center of the 2mL tubes and lower them appropriately (slightly above the rim of the tube) to ensure efficient evaporation. Make sure all needles are open by turning the control valve above them to the on position with the red arrow pointing down. Very slowly and carefully open the nitrogen tank to allow gas to flow into the needles. Use the pressure gauge adjacent to the evaporator to make sure gas flow is in the appropriate range. Once the drying begins it usually takes about an hour to finish. Remove the tubes from the N2 evaporator post drying and individually inspect them to ensure that all the solvent has evaporated. Places the dried samples in the -80°C freezer for storage until they can be run.
Sample Type:Cultured cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002823
Treatment Summary:UOK-262 HLRCC cells expressing FH or GFP. Cells were treated for 6 hours with media containing labeled metabolites: None; U-13C-Glucose; U-13C-Glutamine and 3;3;3';3'-D4-Cystine.

Sample Preparation:

Sampleprep ID:SP002820
Sampleprep Summary:Place the 6 well plates to be extracted on ice. Completely aspirate off the media from each well. Gently add 2mL of ice cold ammonium acetate to each and every well without disturbing the cells. Aspirate off the ammonium acetate. Repeat this wash step once more. Aspirate off as much ammonium acetate as possible. Add 500uL of the 80% methanol solution to each and every well. Place all the 6 well plates in a -80°C freezer for 15 min. Remove the plates from the freezer, place them back on ice, and use cell scrapers to scrape off the adherent cells into solution. Transfer the cell solution from each well into a new eppitube. Vortex all the eppitubes vigorously. Centrifuge the eppitubes at 17,000g for 10 min at 4°C. Transfer the top 250 uL of the supernatant into a new 2mL tube for evaporation. Move all the tubes to the N2 evaporator and open them all. Then adjust all the needles to point into the center of the 2mL tubes and lower them appropriately (slightly above the rim of the tube) to ensure efficient evaporation. Make sure all needles are open by turning the control valve above them to the on position with the red arrow pointing down. Very slowly and carefully open the nitrogen tank to allow gas to flow into the needles. Use the pressure gauge adjacent to the evaporator to make sure gas flow is in the appropriate range. Once the drying begins it usually takes about an hour to finish. Remove the tubes from the N2 evaporator post drying and individually inspect them to ensure that all the solvent has evaporated. Places the dried samples in the -80°C freezer for storage until they can be run.
Processing Storage Conditions:Room temperature
Extract Storage:-80℃

Combined analysis:

Analysis ID AN004391
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units Peak Area

Chromatography:

Chromatography ID:CH003294
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:Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:35°C
Flow Gradient: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:20 mM Ammonium carbonate, pH 9.7
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS004140
Analysis ID:AN004391
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments: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:UNSPECIFIED
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