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.
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 | ST002709 |
| Study Title | FH variant pathogenicity promotes purine salvage pathway dependence in kidney cancer |
| Study 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 |
| nmatulionis@mednet.ucla.edu | |
| Phone | (310) 206-0163 |
| Submit Date | 2023-05-15 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-06-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | - |
Factors:
Subject type: Cultured cells; Subject species: NCCFH1; UOK262; UTFHC1 (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Tracers used |
|---|---|---|---|
| SA272370 | Sample_22 | FH | 3,3,3',3'-D4 Cystine |
| SA272371 | Sample_23 | FH | 3,3,3',3'-D4 Cystine |
| SA272372 | Sample_24 | FH | 3,3,3',3'-D4 Cystine |
| SA272379 | Sample_15 | FH | none |
| SA272380 | Sample_14 | FH | none |
| SA272381 | Sample_13 | FH | none |
| SA272373 | Sample_17 | FH | U-C13 glucose |
| SA272374 | Sample_16 | FH | U-C13 glucose |
| SA272375 | Sample_18 | FH | U-C13 glucose |
| SA272376 | Sample_21 | FH | U-C13 glutamine |
| SA272377 | Sample_19 | FH | U-C13 glutamine |
| SA272378 | Sample_20 | FH | U-C13 glutamine |
| SA272382 | Sample_10 | WT | 3,3,3',3'-D4 Cystine |
| SA272383 | Sample_11 | WT | 3,3,3',3'-D4 Cystine |
| SA272384 | Sample_12 | WT | 3,3,3',3'-D4 Cystine |
| SA272391 | Sample_2 | WT | none |
| SA272392 | Sample_3 | WT | none |
| SA272393 | Sample_1 | WT | none |
| SA272385 | Sample_5 | WT | U-C13 glucose |
| SA272386 | Sample_4 | WT | U-C13 glucose |
| SA272387 | Sample_6 | WT | U-C13 glucose |
| SA272388 | Sample_8 | WT | U-C13 glutamine |
| SA272389 | Sample_7 | WT | U-C13 glutamine |
| SA272390 | Sample_9 | WT | 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 |
