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 |
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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℃ |
