Summary of Study ST003823

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	ST003823
Study Title	Polar metabolite profiling of BCS-treated SEM cells with or without genetic overexpression of AOX.
Study Summary	To investigate whether the effects of bathocuproinedisulfonic acid (BCS) treatment (a copper chelator) on steady state metabolite levels can be rescued by overexpression of alternative oxidase (AOX), SEM cells were engineered to overexpress red fluorescent protein (RFP) or AOX and treated for 14 days with vehicle or 50uM BCS. Cells were seeded at 0.25 million/mL and counted and passaged every two days. This experiment revealed that AOX overexpression significantly rescues levels of nucleotides and reduces ureidosuccinic acid/carbamoyl aspartic acid to near normal.
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	2025-03-26
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

Select appropriate tab below to view additional metadata details:

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:	SU004634
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
SA530513	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1194	SEM leukemia cells	BCS	AOX
SA530514	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1193	SEM leukemia cells	BCS	AOX
SA530515	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1195	SEM leukemia cells	BCS	AOX
SA530516	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1187	SEM leukemia cells	BCS	RFP
SA530517	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1188	SEM leukemia cells	BCS	RFP
SA530518	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1189	SEM leukemia cells	BCS	RFP
SA530519	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1190	SEM leukemia cells	Vehicle	AOX
SA530520	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1191	SEM leukemia cells	Vehicle	AOX
SA530521	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1192	SEM leukemia cells	Vehicle	AOX
SA530522	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1184	SEM leukemia cells	Vehicle	RFP
SA530523	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1185	SEM leukemia cells	Vehicle	RFP
SA530524	20240812_QE2_HILIC_BCSPU_BCSAOX_AYW1186	SEM leukemia cells	Vehicle	RFP

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO004627
Collection Summary:	One 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:	TR004643
Treatment Summary:	Cells were treated for 14 days with vehicle or 50uM BCS. 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:	SP004640
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:

SP004640

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	AN007475
Chromatography ID	CH005667
MS ID	MS007171
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:	CH005667
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:	MS007171
Analysis ID:	AN007475
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 × 10e6, 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