Summary of Study ST003820

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	ST003820
Study Title	Polar metabolite profiling of SLC31A1 knockout cells with or without rescue with over-expression of SLC31A1, and control intergenic cells
Study Summary	To investigate whether the effects of SLC31A1 knockout on steady state metabolite levels is specific to SLC31A1 knockout, SEM cells containing a SLC31A1-targeting sgRNA (guide #2) 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 eGFP (control) or a CRISPR-resistant form of SLC31A1. 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 SLC31A1.
Institute	Boston Childrens Hospital
Last Name	Wong
First Name	Alan
Address	300 Longwood Avenue
Email	alan.wong@childrens.harvard.edu
Phone	(617) 355-7433
Submit Date	2025-03-21
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:	SU004630
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	sgRNA	overexpression
SA530453	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW961	SEM leukemia cells	Intergenic1	GFP
SA530454	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW962	SEM leukemia cells	Intergenic1	GFP
SA530455	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW963	SEM leukemia cells	Intergenic1	GFP
SA530456	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW964	SEM leukemia cells	Intergenic1	SLC31A1
SA530457	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW965	SEM leukemia cells	Intergenic1	SLC31A1
SA530458	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW966_rerun	SEM leukemia cells	Intergenic1	SLC31A1
SA530459	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW973	SEM leukemia cells	SLC31A1_#2	GFP
SA530460	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW974	SEM leukemia cells	SLC31A1_#2	GFP
SA530461	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW975	SEM leukemia cells	SLC31A1_#2	GFP
SA530462	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW976	SEM leukemia cells	SLC31A1_#2	SLC31A1
SA530463	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW977	SEM leukemia cells	SLC31A1_#2	SLC31A1
SA530464	20230712_QE1_HILIC_SEM_sgSLC31A1KOandRescue_AYW978	SEM leukemia cells	SLC31A1_#2	SLC31A1

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO004623
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:	TR004639
Treatment Summary:	Cells were seeded 1million/mL into 2 mL of RPMI-1640 + 10%FBS + penicillin/streptomycin the day prior to experiment in 6 well plates.

Sample Preparation:

Sampleprep ID:	SP004636
Sampleprep Summary:	Cell pellet was resuspended in 160uL 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, 40uL 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 20uL and 2uL was injected.

Sampleprep ID:

SP004636

Sampleprep Summary:

Cell pellet was resuspended in 160uL 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, 40uL 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 20uL and 2uL was injected.

Combined analysis:

Analysis ID	AN007471
Chromatography ID	CH005663
MS ID	MS007167
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:	CH005663
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 acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH adjustment. Gradient conditions used were: 0-20 min: linear gradient from 20% to 80% B; 20-24 min: hold at 80% B; 24-24.1 min: from 80% to 20% 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:	Gradient conditions were as follows: linear gradient from 20 to 80% B; 20–20.5 min: from 80 to 20% B; 20.5–28 min: hold at 20% B.
Flow Rate:	0.15 mL/min
Solvent A:	100% acetonitrile
Solvent B:	100% water; 20mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS007167
Analysis ID:	AN007471
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