Summary of Study ST004440
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 PR002804. The data can be accessed directly via it's Project DOI: 10.21228/M86V71 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004440 |
| Study Title | Glutamine and Glucose stable-isotope tracing in NAMPT Inhibition |
| Study Type | SIL Tracing Metabolomics by HPLC/MS |
| Study Summary | Using cell media-supplemented heavy carbon-labeled glucose and glutamine, samples (N=4 per group) were collected at baseline and 3 hours (glucose) or 12 hours (glutamine) after OT82 or DMSO exposure. We observed major shifts in metabolic intermediates across multiple pathways with OT82 treatment including TCA cycle and glycolysis. |
| Institute | University of Colorado School of Medicine |
| Department | Hematology |
| Laboratory | Jordan / Pietras |
| Last Name | Anderson |
| First Name | Colin |
| Address | 13001 East 17th Place |
| Colin.c.anderson@cuanschutz.edu | |
| Phone | 303-724-3339 |
| Submit Date | 2025-10-28 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2026-01-06 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002804 |
| Project DOI: | doi: 10.21228/M86V71 |
| Project Title: | The Nicotinamide Salvage Pathway is a Unique Metabolic target in High-Risk MDS Stem Cells |
| Project Type: | Primary Research Article |
| Project Summary: | High-risk myelodysplastic syndrome (HR-MDS) is an incurable malignant clonal disorder originating in the hematopoietic stem and progenitor cells (HSPC). The current standard of care for HR-MDS patients is hypomethylating agents, like azacitidine; however, the response rate is poor, usually with remission of less than two years. Thus, to improve HR-MDS patient outcomes, an unmet clinical need, it is essential to identify better therapeutic targets by exploring vulnerabilities of HR-MDS HSPCs. We have previously demonstrated that inhibiting protein synthesis is a vulnerability for HR-MDS. Subsequently, we identified that HR-MDS HSPCs have a significant upregulation of metabolic proteins required for glycolysis, TCA cycle and oxidative phosphorylation. Consistently, we see reduced glycolytic and TCA cycle metabolites, but an increased oxygen consumption rate in HR-MDS HSPCs compared to healthy, suggesting an increased metabolic rate. Most of the enriched proteins in HR-MDS either use NAD for energy production, maintain NAD(H) redox balance or use NAD as a cofactor for their function. Therefore, we inhibited NAMPT, the rate-limiting enzyme in the nicotinamide salvage pathway of NAD anabolism, using small molecule inhibitors and genetic approaches. NAMPT inhibition significantly decreased NAD levels, reducing the oxygen consuming capacity of HR-MDS-HSPCs compared to normal. Consequently, to compensate, HR-MDS HSPCs, upon NAMPT inhibition positively enriched metabolic proteins associated with amino acid metabolism, lipid metabolism, and the citric acid cycle increasing carbon flux into the aspartate-malate shuttle and the pentose phosphate pathway, while reducing flux through glycolysis and citric acid cycle. Finally, from a functional perspective, NAMPT significantly impaired self-renewal and colony forming potential of HR-MDS HSPCs compared to normal. Importantly, we observed increased cell death of HR-MDS HSPCs compared to normal in both in vitro cultures and in vivo xenograft studies, indicating NAMPT as a tractable target for therapeutic inhibition in HR- MDS patients. Collectively, our data suggest that NAMPT is uniquely required for the function and survival of HR-MDS HSPCs compared to normal and thus can serve as a promising therapeutic target. |
| Institute: | University of Colorado School of Medicine |
| Department: | Hematology |
| Laboratory: | Jordan / Pietras |
| Last Name: | Anderson |
| First Name: | Colin |
| Address: | 12801 East 17th Avenue, Aurora, CO 80045 |
| Email: | Colin.c.anderson@cuanschutz.edu |
| Phone: | 303-724-3339 |
| Funding Source: | NIH |
| Contributors: | Sweta B. Patel, Daniel Moskop, Steven Moriera, Stephanie Gipson, Colin C. Anderson, Alexendra Crook, Maxwell McCabe, Daniel Stephenson, Hannah E. Terry, Andrew Kent, Tracy Young, Anna Krug, Caitlin Price, Monica Ransom, Regan Miller, Ana Vujovic, Mohammad Minhajuddin, Mark J. Althoff, The CUIJBP Consortium, Anthony Saviola, Brett M. Stevens, Robert S. Welner, Ekaterina L. Andrianova, Andrei V. Gudkov, Travis Nemkov, Angelo D’Alessandro, Austin E. Gillen, Craig T. Jordan*, Eric M. Pietras* |
Subject:
| Subject ID: | SU004602 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment | Timepoint | Sample source |
|---|---|---|---|---|
| SA526344 | CA24_52-2 | DMSO | 12H | MDS-L Cells |
| SA526345 | CA24_52-1 | DMSO | 12H | MDS-L Cells |
| SA526346 | CA24_52-4 | DMSO | 12H | MDS-L Cells |
| SA526347 | CA24_52-3 | DMSO | 12H | MDS-L Cells |
| SA526348 | CA24_52-18 | DMSO | 3H | MDS-L Cells |
| SA526349 | CA24_52-19 | DMSO | 3H | MDS-L Cells |
| SA526350 | CA24_52-20 | DMSO | 3H | MDS-L Cells |
| SA526351 | CA24_52-17 | DMSO | 3H | MDS-L Cells |
| SA526352 | CA24_52-5 | OT | 12H | MDS-L Cells |
| SA526353 | CA24_52-6 | OT | 12H | MDS-L Cells |
| SA526354 | CA24_52-7 | OT | 12H | MDS-L Cells |
| SA526355 | CA24_52-8 | OT | 12H | MDS-L Cells |
| SA526356 | CA24_52-21 | OT | 3H | MDS-L Cells |
| SA526357 | CA24_52-22 | OT | 3H | MDS-L Cells |
| SA526358 | CA24_52-23 | OT | 3H | MDS-L Cells |
| SA526359 | CA24_52-24 | OT | 3H | MDS-L Cells |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO004595 |
| Collection Summary: | Cells were pelleted by gentle centrifugation and media was removed. The cells were washed several times with PBS before finally being pelleted. The remaining PBS was aspirated and the cell pellet was flash frozen in liquid nitrogen and stored at -80C. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR004611 |
| Treatment Summary: | 500,000 MDS-L cells were treated with DMSO and 100nM OT82 for 24h. At the final 3h and 12h, 2.5mM heavy labeled glucose (13C6; Cambridge Isotope Laboratories, CLM-1396-1) and 0.5mM heavy labeled glutamine (13C6,15N2; Cambridge Isotope Laboratories, CNLM-1275-H-0.1) were spiked into the culture respectively. |
Sample Preparation:
| Sampleprep ID: | SP004608 |
| Sampleprep Summary: | Cells were extracted to a final concentration of 2E06 per mL of cold MeOH:ACN:H2O (5:3:2, v:v:v). Samples were then vortexed at 4 °C for 30 minutes. Following vortexing, samples were centrifuged at 12700 RPM for 10 minutes at 4 °C and supernatant was transferred to a new autosampler vial for analysis. |
Combined analysis:
| Analysis ID | AN007435 | AN007436 |
|---|---|---|
| Chromatography ID | CH005628 | CH005629 |
| MS ID | MS007127 | MS007128 |
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) | Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Orbitrap Exploris 120 | Thermo Orbitrap Exploris 120 |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | A.U. | A.U. |
Chromatography:
| Chromatography ID: | CH005628 |
| Chromatography Summary: | 5 Min Negative: After sample randomization, 10 μL of extracts were injected into a Thermo Vanquish UHPLC system (San Jose, CA, USA) and resolved on a Waters Acquity BEH C18 column (2.1 x 100 mm, 1.7 um) at 450 μL/min using a 5 min gradient in negative and positive modes (separate runs). The negative polarity gradient utilized mobile phases: A = water, 10 mM ammonium acetate; B = 50% acetonitrile, 50% methanol, 10 mM ammonium acetate. Solvent gradient: 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) |
| Column Temperature: | 45°C |
| Flow Gradient: | 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B |
| Flow Rate: | 450 μL/min |
| Solvent A: | 100% Water; 10 mM Ammonium acetate |
| Solvent B: | 50% Acetonitrile/50% Methanol; 10 mM Ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH005629 |
| Chromatography Summary: | 5 Min Positive: After sample randomization, 10 μL of extracts were injected into a Thermo Vanquish UHPLC system (San Jose, CA, USA) and resolved on a Waters Acquity BEH C18 column (2.1 x 100 mm, 1.7 um) at 450 μL/min through a 5 min gradient from 5 to 95% organic solvent B (mobile phases: A = water, 0.1% formic acid; B = acetonitrile, 0.1% formic acid) in positive ion mode. Solvent gradient: 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) |
| Column Temperature: | 45°C |
| Flow Gradient: | 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B |
| Flow Rate: | 450 μL/min |
| Solvent A: | 100% Water; 0.1% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1% Formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS007127 |
| Analysis ID: | AN007435 |
| Instrument Name: | Thermo Orbitrap Exploris 120 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Following data acquisition, .raw files were converted to .mzXML using RawConverter. Metabolites were then annotated based on intact mass, 13C natural isotope pattern and retention times in conjunction with the KEGG database and an in-house standard library. Peaks were integrated using El-Maven (Elucidata). Quality control was assessed as using technical replicates run at the beginning, end, and middle of each sequence as previously described. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS007128 |
| Analysis ID: | AN007436 |
| Instrument Name: | Thermo Orbitrap Exploris 120 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Following data acquisition, .raw files were converted to .mzXML using RawConverter. Metabolites were then annotated based on intact mass, 13C natural isotope pattern and retention times in conjunction with the KEGG database and an in-house standard library. Peaks were integrated using El-Maven (Elucidata). Quality control was assessed as using technical replicates run at the beginning, end, and middle of each sequence as previously described. |
| Ion Mode: | POSITIVE |
