Summary of Study ST002876

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 PR001798. The data can be accessed directly via it's Project DOI: 10.21228/M89D97 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST002876
Study TitleHigh Level Expression of NSD2 Creates a Metabolic Dependency in Multiple Myeloma
Study SummaryMultiple myeloma (MM) is a malignancy of plasma cells with several molecular subtypes and variable prognosis. Despite therapeutic advances, most patients ultimately relapse due to drug resistance. Frontline treatments for MM target malignant cells based on their differentiated B cell nature, but not the underlying genetic lesions. Chromosomal translocation t(4;14), observed in 15% of MM patients, results in overexpression of the histone methyltransferase NSD2, which contributes to MM pathogenesis by promoting an oncogenic transcriptional program and is associated with a worse prognosis. A genome-wide CRISPR based functional screen in isogenic MM cell lines with high and low NSD2 expression identified the mitochondrial enzyme adenylate kinase 2 (AK2) as a NSD2-driven MM cell dependency. AK2 loss in t(4;14) MM cells induced apoptosis and inhibited cell growth in vitro and in vivo. Consistent with a defect in shuttling ATP from the mitochondria to intracellular utilization sites, AK2 depletion impaired ATP-dependent protein folding in the ER and increased MM cell sensitivity to the proteasome inhibitor bortezomib. Furthermore, AK2 suppression decreased intracellular NAD(H) phosphorylation resulting in lower NADP(H) levels. Cytosolic NADPH is necessary for reducing thioredoxin, an essential cofactor for ribonucleotide reductase which is critical for deoxyribonucleotides (dNTP) synthesis. Consequently, AK2 deficiency in MM cells resulted in dNTP pool depletion and induced DNA replication stress. Creatine phosphorylation by mitochondrial creatine kinase is an alternative route for shuttling ATP from the mitochondria to the cytosol. Metabolomics analysis revealed decreased levels of creatine and accumulation of its precursor guanadoacetate in NSD2 high cells, in association with elevated levels of S-adenosyl homocysteine (SAH) indicating consumption of the carbon donor S-Adenosyl methionine (SAM). This along with the 6-fold increase in genome wide H3K36me2 levels and 40% increase in DNA methylation levels in NSD2 high cells suggested that overexpression of NSD2 redirected one-carbon metabolism to the epigenome and away from the SAM-dependent creatine synthesis. Therefore, decreased creatine levels in NSD2 overexpressing cells underlie the increased reliance on AK2. Correspondingly, supplementation with exogenous creatine restored NADP(H) levels and rescued AK2 deficient cells from apoptosis. These findings revealed a novel metabolic susceptibility in t(4;14) MM and provided insight into a novel therapeutic strategy to improve patient prognosis. We performed metabolomic analysis of multiple myeloma cell lines with either high or low NSD2 levels with or without NSD2 knockdown.
Institute
University of Florida
Last NameLicht
First NameJonathan
Address2033 MOWRY RD STE 145 GAINESVILLE FL 32610
Emailjdlicht@ufl.edu
Phone(352) 273-8143
Submit Date2023-09-07
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2023-10-30
Release Version1
Jonathan Licht Jonathan Licht
https://dx.doi.org/10.21228/M89D97
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001798
Project DOI:doi: 10.21228/M89D97
Project Title:High Level Expression of NSD2 Creates a Metabolic Dependency in Multiple Myeloma
Project Summary:Multiple myeloma (MM) is a malignancy of plasma cells with several molecular subtypes and variable prognosis. Despite therapeutic advances, most patients ultimately relapse due to drug resistance. Frontline treatments for MM target malignant cells based on their differentiated B cell nature, but not the underlying genetic lesions. Chromosomal translocation t(4;14), observed in 15% of MM patients, results in overexpression of the histone methyltransferase NSD2, which contributes to MM pathogenesis by promoting an oncogenic transcriptional program and is associated with a worse prognosis. A genome-wide CRISPR based functional screen in isogenic MM cell lines with high and low NSD2 expression identified the mitochondrial enzyme adenylate kinase 2 (AK2) as a NSD2-driven MM cell dependency. AK2 loss in t(4;14) MM cells induced apoptosis and inhibited cell growth in vitro and in vivo. Consistent with a defect in shuttling ATP from the mitochondria to intracellular utilization sites, AK2 depletion impaired ATP-dependent protein folding in the ER and increased MM cell sensitivity to the proteasome inhibitor bortezomib. Furthermore, AK2 suppression decreased intracellular NAD(H) phosphorylation resulting in lower NADP(H) levels. Cytosolic NADPH is necessary for reducing thioredoxin, an essential cofactor for ribonucleotide reductase which is critical for deoxyribonucleotides (dNTP) synthesis. Consequently, AK2 deficiency in MM cells resulted in dNTP pool depletion and induced DNA replication stress. Creatine phosphorylation by mitochondrial creatine kinase is an alternative route for shuttling ATP from the mitochondria to the cytosol. Metabolomics analysis revealed decreased levels of creatine and accumulation of its precursor guanadoacetate in NSD2 high cells, in association with elevated levels of S-adenosyl homocysteine (SAH) indicating consumption of the carbon donor S-Adenosyl methionine (SAM). This along with the 6-fold increase in genome wide H3K36me2 levels and 40% increase in DNA methylation levels in NSD2 high cells suggested that overexpression of NSD2 redirected one-carbon metabolism to the epigenome and away from the SAM-dependent creatine synthesis. Therefore, decreased creatine levels in NSD2 overexpressing cells underlie the increased reliance on AK2. Correspondingly, supplementation with exogenous creatine restored NADP(H) levels and rescued AK2 deficient cells from apoptosis. These findings revealed a novel metabolic susceptibility in t(4;14) MM and provided insight into a novel therapeutic strategy to improve patient prognosis.
Institute:Northwestern University
Last Name:Licht
First Name:Jonathan
Address:2033 MOWRY RD STE 145 GAINESVILLE FL 32610
Email:jdlicht@ufl.edu
Phone:(352) 273-8143

Subject:

Subject ID:SU002989
Subject Type:Mammal
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Mammal; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Treatment
SA3144693High NSD2 Control
SA3144702High NSD2 Control
SA3144711High NSD2 Control
SA3144726High NSD2 Knockdown 1
SA3144735High NSD2 Knockdown 1
SA3144744High NSD2 Knockdown 1
SA3144759High NSD2 Knockdown 2
SA3144768High NSD2 Knockdown 2
SA3144777High NSD2 Knockdown 2
SA31447812Low NSD2 Control
SA31447910Low NSD2 Control
SA31448011Low NSD2 Control
SA31448113Low NSD2 Knockdown 1
SA31448215Low NSD2 Knockdown 1
SA31448314Low NSD2 Knockdown 1
SA31448418Low NSD2 Knockdown 2
SA31448516Low NSD2 Knockdown 2
SA31448617Low NSD2 Knockdown 2
Showing results 1 to 18 of 18

Collection:

Collection ID:CO002982
Collection Summary:Multiple myeloma cells were collected, spun down, and flash frozen.
Sample Type:B cells

Treatment:

Treatment ID:TR002998
Treatment Summary:Stable NSD2 knockdown cell lines and control cell lines were established using shRNA.

Sample Preparation:

Sampleprep ID:SP002995
Sampleprep Summary:Soluble metabolites were extracted directly from snap-frozen cell pellets using cold acetonitrile/water (80/20, vol/vol) at a dilution of 10,000 cells/ul solvent. Samples were vortexed and incubated at -80C overnight to precipitate proteins and subsequently thawed, vortexed, and centrifuged at 18,000 x g for 30 min at 4C to pellet debris. The supernatants were transferred to new vials and analyzed by high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry and tandem mass spectrometry (MS/MS).

Combined analysis:

Analysis ID AN004713
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000
Column Waters XBridge BEH Amide (100 x 4.6mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units Peak area

Chromatography:

Chromatography ID:CH003549
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters XBridge BEH Amide (100 x 4.6mm,3.5um)
Column Temperature:275
Flow Gradient:0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A
Flow Rate:150 μL/min
Solvent A:95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate, pH 9.0
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS004459
Analysis ID:AN004713
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The capillary of the ESI source was set to 275 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000.
Ion Mode:UNSPECIFIED
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