Summary of Study ST003821

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	ST003821
Study Title	Polar metabolite profiling of BCS-treated SEM cells with or without rescue with copper (ii) chloride
Study Summary	To investigate the effects of bathocuproinedisulfonic acid (BCS) treatment (a copper chelator) on steady state metabolite levels, SEM cells were treated for 14 days with vehicle, 50uM BCS, 50uM CuCl2, or 50uM of both. Cells were seeded at 0.25 million/mL and counted and passaged every two days. This revealed that copper depletion with BCS lead to electron transport chain dysfunction, resulting in decreased aspartate levels, increased levels of ureidosuccinic acid/carbamoyl aspartic acid, and changes in nucleotide levels.
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:	SU004631
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	Treatment1
SA530465	20240717_QE2_HILIC_BCSd14_AYW1160	SEM leukemia cells	BCS
SA530466	20240717_QE2_HILIC_BCSd14_AYW1161	SEM leukemia cells	BCS
SA530467	20240717_QE2_HILIC_BCSd14_AYW1162	SEM leukemia cells	BCS
SA530468	20240717_QE2_HILIC_BCSd14_AYW1166	SEM leukemia cells	BCS+CuCl2
SA530469	20240717_QE2_HILIC_BCSd14_AYW1167	SEM leukemia cells	BCS+CuCl2
SA530470	20240717_QE2_HILIC_BCSd14_AYW1168	SEM leukemia cells	BCS+CuCl2
SA530471	20240717_QE2_HILIC_BCSd14_AYW1163	SEM leukemia cells	CuCl2
SA530472	20240717_QE2_HILIC_BCSd14_AYW1164	SEM leukemia cells	CuCl2
SA530473	20240717_QE2_HILIC_BCSd14_AYW1165	SEM leukemia cells	CuCl2
SA530474	20240717_QE2_HILIC_BCSd14_AYW1157	SEM leukemia cells	Vehicle
SA530475	20240717_QE2_HILIC_BCSd14_AYW1158	SEM leukemia cells	Vehicle
SA530476	20240717_QE2_HILIC_BCSd14_AYW1159	SEM leukemia cells	Vehicle

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO004624
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:	TR004640
Treatment Summary:	Cells were treated for 14 days with vehicle, 50uM BCS, 50uM CuCl2, or 50uM of both. Culture media was RPMI-1640 containing 10% FBS and penicillin and streptomycin in 6-well plates. Cells were seeded at 0.25 million/mL in 4mL and counted and passaged every two days.

Sample Preparation:

Sampleprep ID:	SP004637
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.

Sampleprep ID:

SP004637

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	AN007472
Chromatography ID	CH005664
MS ID	MS007168
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:	CH005664
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
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:	MS007168
Analysis ID:	AN007472
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