Summary of Study ST004120
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 PR002590. The data can be accessed directly via it's Project DOI: 10.21228/M8VN87 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004120 |
| Study Title | Assessing the dependence of murine WT and Crebbp-/- tumour B cells on glucose to fuel Oxidative Phosphorylation. |
| Study Summary | The objective of this experiment was to compare the oxidation of glucose by murine WT and Crebbp-/- tumour B cells by examining the relative incorporation of glucose into TCA cycle-derived intermediates in these cells. To perform this experiment we cultured CD19+ splenocytes isolated from WT and Crebbp-/- tumour mice for 4 hours in Seahorse RPMI with U13C-labelled glucose stable isotope tracer. Data were generated from 5 independent cultures. |
| Institute | Cambridge Stem Cell Institute |
| Last Name | Horton |
| First Name | Sarah |
| Address | Puddicombe Way, Cambridge, Cambridgeshire, CB2 0AW, United Kingdom |
| sjh244@cam.ac.uk | |
| Phone | +44 1223 763368. |
| Submit Date | 2025-07-31 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-09-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002590 |
| Project DOI: | doi: 10.21228/M8VN87 |
| Project Title: | CREBBP-loss confers metabolic and epigenetic vulnerabilities upon lymphoma cells. |
| Project Summary: | Inactivating-mutations of CREBBP are common in diffuse large B-cell lymphoma (DLBCL). We previously demonstrated that Crebbp-/- mice develop mature B-cell malignancies preceded by a pre-malignant phase. Using single-cell RNA-seq of WT and Crebbp-/- B-cells from distinct stages of disease evolution, we discovered expansion of aberrant germinal center B-cells during disease progression. Both Crebbp-/- murine and human CRISPR-engineered isogenic CREBBP-null cells displayed reduced expression of BCR-signaling genes and increased OXPHOS transcriptional programs, a finding recapitulated in DLBCL patients with low CREBBP expression. Mechanistically, BCR-signaling decreased upon CREBBP loss, alleviating p65-mediated repression of the transcriptional coactivator PGC1β, resulting in enhanced OXPHOS transcriptional programs. This metabolic vulnerability could be targeted therapeutically. Furthermore, CREBBP-loss also conferred an epigenetic vulnerability by altering the landscape of acetylated transcription factors, including BET proteins, at super-enhancers. Combined inhibition of complex I and BET proteins exploited the metabolic and epigenetic vulnerabilities of Crebbp-/- tumors, extending lymphoma survival in vivo. |
| Institute: | Cambridge Stem Cell Institute |
| Department: | Haematology |
| Laboratory: | Huntly / Frezza |
| Last Name: | Horton |
| First Name: | Sarah |
| Address: | Puddicombe Way, Cambridge, Cambridgeshire, CB2 0AW, United Kingdom |
| Email: | sjh244@cam.ac.uk |
| Phone: | +44 1223 763368 |
Subject:
| Subject ID: | SU004269 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Sample source |
|---|---|---|---|
| SA476350 | SH_248 | Crebbp-/- Tumour | Spleen cells |
| SA476351 | SH_247 | Crebbp-/- Tumour | Spleen cells |
| SA476352 | SH_249 | Crebbp-/- Tumour | Spleen cells |
| SA476353 | SH_250 | Crebbp-/- Tumour | Spleen cells |
| SA476354 | SH_251 | Crebbp-/- Tumour | Spleen cells |
| SA476349 | - | - | - |
| SA476355 | SH_237 | WT | Spleen cells |
| SA476356 | SH_238 | WT | Spleen cells |
| SA476357 | SH_239 | WT | Spleen cells |
| SA476358 | SH_240 | WT | Spleen cells |
| SA476359 | SH_241 | WT | Spleen cells |
| Showing results 1 to 11 of 11 |
Collection:
| Collection ID: | CO004262 |
| Collection Summary: | 1x106 CD19+ splenocytes were plated in 24-well plates in Seahorse RPMI media with U13C-labelled glucose stable isotope tracer (5 replicates for each condition). Before extraction, cells were counted using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept on ice prior to adding the metabolite extraction solution. |
| Collection Protocol Filename: | Sample_and_treatment_preparation.pdf |
| Sample Type: | Splenocytes |
Treatment:
| Treatment ID: | TR004278 |
| Treatment Summary: | CD19+ splenocytes were cultured for 4 hours in Seahorse RPMI media supplemented with 10% dialyzed FCS, 2mM glutamine with 1:1000 CD40Ab at a concentration of 1 x 106/mL. For the glucose tracing experiments 50 μM unlabeled palmitate and 11.1 mM D-Glucose tracer were added. |
Sample Preparation:
| Sampleprep ID: | SP004275 |
| Sampleprep Summary: | After the PBS washes, 100 μL metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 μM final concentration valine-d8) was added to each sample. The samples were vortexed for 20 seconds and incubated for 5 minutes in a dry ice / methanol bath and stored at -80°C overnight. The next day the samples were incubated at 4°C with shaking for 15 minutes and then centrifuged for 20 minutes at maximum speed at 4°C. The top 80% of supernatant was then transferred to individual autosampler vials and stored at -80°C prior to analysis. |
Combined analysis:
| Analysis ID | AN006832 |
|---|---|
| Chromatography ID | CH005188 |
| MS ID | MS006531 |
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Orbitrap Exploris 240 |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH005188 |
| Chromatography Summary: | Chromatographic separation of polar metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4°C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. Samples were randomized and analyzed with LC–MS in a blinded manner with an injection volume was 5 µL. Pooled samples were generated from an equal mixture of all individual samples and analyzed interspersed at regular intervals within sample sequence as a quality control. |
| Methods Filename: | Chromatography_and_MS.pdf |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm) |
| Column Temperature: | 40°C |
| Flow Gradient: | 0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 100% Water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% Acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS006531 |
| Analysis ID: | AN006832 |
| Instrument Name: | Thermo Orbitrap Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were measured with Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 240 mass spectrometer (both Thermo Fisher Scientific) via a heated electrospray ionization source. The spray voltages were set to +3.5kV/-2.8 kV, RF lens value at 70, the heated capillary held at 320°C, and the auxiliary gas heater held at 280°C. The flow rate for sheath gas, aux gas and sweep gas were set to 40, 15 and 0, respectively. For MS1 scans, mass range was set to m/z=70-900, AGC target set to standard and maximum injection time (IT) set to auto. Data acquisition for experimental samples used full scan mode with polarity switching at an Orbitrap resolution of 120000. Data acquisition for untargeted metabolite identification was performed using the AcquireX Deep Scan workflow, an iterative data-dependent acquisition (DDA) strategy using multiple injections of the pooled sample. In brief, sample was first injected in full scan-only mode in single polarity to create an automated inclusion list. MS2 acquisition was then carried out in triplicate, where ions on the inclusion list were prioritized for fragmentation in each run, after which both the exclusion and inclusion lists were updated in a manner where fragmented ions from the inclusion list were moved to exclusion list for the next run. DDA full scan-ddMS2 method for AcquireX workflow used the following parameters: full scan resolution was set to 60000, fragmentation resolution to 30000, fragmentation intensity threshold to 5.0e3. Dynamic exclusion was enabled after 1 time and exclusion duration was 10s. Mass tolerance was set to 5 ppm. Isolation window was set to 1.2 m/z. Normalized HCD collision energies were set to stepped mode with values at 30, 50, 150. Fragmentation scan range was set to auto, AGC target at standard and max IT at auto. Xcalibur AcquireX method modification was on. Mild trapping was enabled. |
| Ion Mode: | UNSPECIFIED |
