Summary of Study ST003822
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 PR002390. The data can be accessed directly via it's Project DOI: 10.21228/M8Q250 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003822 |
| Study Title | Polar metabolite profiling of SLC31A1 knockout or control cells with or without rescue with over-expression of AOX |
| Study Summary | To investigate whether the effects of SLC31A1 knockout can be rescued with alternative oxidase (AOX) overexpression, SEM cells containing a SLC31A1-targeting sgRNA (guide #2 or guide #7) or a intergenic -targetting sgRNA (i.e. a control sgRNA, "Intergenic1") and Cas9 were FACS-purified based on SLC31A1 expression, and then engineered to stably over-express either red fluorescent protein (RFP, as a control) or AOX from Ciona Intestinalis. Cells were then cultured in RPMI-1640 containing 10% FBS and penicillin and streptomycin in 6-well plates, and then collected for metabolomics. This demonstrated that the changes in aspartate, ureidosuccinic acid/carbamoyl aspartate and nucleotides were largely reverted by genetic overexpression of AOX. |
| Institute | Boston Childrens Hospital |
| Last Name | Wong |
| First Name | Alan |
| Address | 300 Longwood Avenue |
| alan.wong@childrens.harvard.edu | |
| Phone | (617) 355-7433 |
| Submit Date | 2025-03-26 |
| Num Groups | 4 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-12-22 |
| Release Version | 1 |
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Project:
| Project ID: | PR002390 |
| Project DOI: | doi: 10.21228/M8Q250 |
| Project Title: | In vivo CRISPR screen identifies copper metabolism as a vulnerability in acute lymphoblastic leukemia |
| Project Summary: | The nutrient-sparse cerebrospinal fluid (CSF) poses a significant challenge to spreading cancer cells. Despite this challenge, leukemia often spreads to the CSF and represents a significant clinical complication. To uncover nutritional dependencies of leukemia cells in the CSF that could be targeted therapeutically, we conducted an in vivo targeted CRISPR screen in a xenograft model of leukemia. We found that SLC31A1, the primary cell surface copper importer, is a genetic dependency of leukemia in both the central nervous system as well as in the hematopoietic organs. Perturbation of copper metabolism leads to complex IV deficiency, perturbed nucleotide metabolism and slowed leukemia cell proliferation. Furthermore, nutritional copper depletion reduced cancer progression in cell line based and patient-derived xenograft models of leukemia. Copper thus appears to be an actionable micronutrient in leukemia. |
| Institute: | Boston Children's Hospital |
| Department: | Pathology |
| Laboratory: | Naama Kanarek |
| Last Name: | Wong |
| First Name: | Alan |
| Address: | 300 Longwood Avenue, Boston, MA, 02115, USA |
| Email: | alan.wong@childrens.harvard.edu |
| Phone: | (617) 355-7433 |
| Funding Source: | NCI 1R01CA282477-01A1 |
Subject:
| Subject ID: | SU004633 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | SEM leukemia cells |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | guideRNA | overexpression |
|---|---|---|---|---|
| SA530495 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1314 | SEM leukemia cells | Intergenic1 | RFP |
| SA530496 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1310 | SEM leukemia cells | Intergenic1 | RFP |
| SA530497 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1309 | SEM leukemia cells | Intergenic1 | RFP |
| SA530498 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1313 | SEM leukemia cells | Intergenic1 | RFP |
| SA530499 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1312 | SEM leukemia cells | Intergenic1 | RFP |
| SA530500 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1311 | SEM leukemia cells | Intergenic1 | RFP |
| SA530501 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1319 | SEM leukemia cells | SLC31A1 guide #2 | AOX |
| SA530502 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1320 | SEM leukemia cells | SLC31A1 guide #2 | AOX |
| SA530503 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1318 | SEM leukemia cells | SLC31A1 guide #2 | AOX |
| SA530504 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1315 | SEM leukemia cells | SLC31A1 guide #2 | RFP |
| SA530505 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1316 | SEM leukemia cells | SLC31A1 guide #2 | RFP |
| SA530506 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1317 | SEM leukemia cells | SLC31A1 guide #2 | RFP |
| SA530507 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1321 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| SA530508 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1322 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| SA530509 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1323 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| SA530510 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1324 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| SA530511 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1325 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| SA530512 | 20241122_QE2_HILIC_KO_AOX_repeat_AYW1326 | SEM leukemia cells | SLC31A1 guide #7 | AOX |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO004626 |
| Collection Summary: | One and a half million cells from culture were collected via centrifugation for 20 seconds at 18,000xG at 4C, washed once with ice-cold 0.9% NaCl, and collected via centrifugation for 20 seconds at 18,000xG at 4C. Cells were cultured in RPMI-1640 with 10% FBS and penicillin/streptomycin in a 37C incubator with 5% CO2. |
| Sample Type: | Leukemia cells |
Treatment:
| Treatment ID: | TR004642 |
| Treatment Summary: | Cells were seeded 0.5 million/mL into 4 mL of RPMI-1640 + 10%FBS + penicillin/streptomycin 48 hours prior to collection in 6 well plates. |
Sample Preparation:
| Sampleprep ID: | SP004639 |
| Sampleprep Summary: | Cell pellet was resuspended in 400uL of 100% LC-MS grade methanol supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). with repeated pipetting up and down and vortexing for 10 seconds. Then, 100uL of LCMS-grade water containing 125 mM Ammonium Acetate, 10 mM Na-Ascorbate, and 7.9 mg/mL 5,5-dithio-bis-(2-nitrobenzoic acid (Ellman's reagent) was added, and the sample vortex for another 10 seconds. Samples were then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was transferred to a new tube and dried on ice using a liquid nitrogen dryer. The dried metabolites were then resuspended in 30uL and 2uL was injected. |
Combined analysis:
| Analysis ID | AN007474 |
|---|---|
| Chromatography ID | CH005666 |
| MS ID | MS007170 |
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Normalized peak area |
Chromatography:
| Chromatography ID: | CH005666 |
| Chromatography Summary: | 2 μL of each sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore) operated on a Vanquish™ Flex UHPLC system (Thermo Fisher Scientific). Chromatographic separation was achieved using the following conditions: buffer A was 95% acetonitrile and 5% 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; buffer B was 95% 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water and 5% acetonitrile. Gradient conditions used were: 0-20 min: linear gradient from 16.6% to 83.4% B; 20-24 min: hold at 83.4% B; 24-24.1 min: from 83.4% to 16.6% B; 24.1-32 min: hold at 20% B at 0.150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0-20 min: linear gradient from 16.6% to 83.4% B; 20-24 min: hold at 83.4% B; 24-24.1 min: from 83.4% to 16.6% B; 24.1-32 min: hold at 20% B at 0.150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. |
| Flow Rate: | 0.15 mL/min |
| Solvent A: | 95% acetonitrile/5% water; 20mM ammonium carbonate; 0.1% ammonium hydroxide |
| Solvent B: | 95% water/5% acetonitrile; 20mM ammonium carbonate; 0.1% ammonium hydroxide |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS007170 |
| Analysis ID: | AN007474 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS data acquisition was performed using a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo Fisher Scientific) and polarity switching was used. Four scans were used: full scans in both positive and negative ionization mode in a range of m/z = 70–1000, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 20 msec from 0-20 minutes. A third scan in the negative mode was used with range of m/z = 220-700 from 0-20 minutes and the same resolution, AGC settings with 30ms Max IT. Lastly, a targeted-SIM scan was added with a resolution of 35k, AGC target 1e5, and max IT 20ms, isolation window = 1.0 m/z, with an inclusion m/z of 503.0552 (corresponding to Ellman-derivatized glutathione). Tune file parameters were: spray voltage = 3.5kV, capillary temperature = 320C, S-lens RF = 50, auxillary gas temperature = 350C. |
| Ion Mode: | UNSPECIFIED |
