Summary of Study ST002734

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 PR001698. The data can be accessed directly via it's Project DOI: 10.21228/M8741T 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 IDST002734
Study TitleIntegrative multi-omics analysis of oncogenic EZH2 mutants: from epigenetic reprogramming to molecular signatures
Study SummaryThe Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable isogenic EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. A general dysregulation of mitochondrial processes, including TCA cycle and b-oxidation was common to all mutants. For EZH2(A677G) and EZH2(Y641F), alterations in methionine salvage pathway were predominant, while NAD+ pathways were highly disrupted in EZH2(H689A/F667I).
Institute
The Ohio State University
DepartmentChemistry and Biochemistry
Last NameAldana
First NameJulian
Address460 W 12th Ave, Columbus, OH 43210
Emailaldanaaroca.1@osu.edu
Phone+1 614-292-6136
Submit Date2023-06-08
Num Groups4
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-08-01
Release Version1
Julian Aldana Julian Aldana
https://dx.doi.org/10.21228/M8741T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001698
Project DOI:doi: 10.21228/M8741T
Project Title:Integrative multi-omics analysis of oncogenic EZH2 mutants: from epigenetic reprogramming to molecular signatures
Project Summary:Metabolic study of Enhancer of zeste homolog 2 (EZH2) isogenic mutants with gain-of-function (GOF) and loss-of-function (LOF) enzymatic activity in HEK-293T cell lines.
Institute:The Ohio State University
Department:Chemistry and Biochemistry
Last Name:Aldana
First Name:Julian
Address:460 W 12th Ave, Columbus, OH 43210
Email:aldanaaroca.1@osu.edu
Phone:+1 614-292-6136

Subject:

Subject ID:SU002840
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Female
Cell Biosource Or Supplier:American Tissue Culture Collection (ATCC)
Cell Strain Details:HEK-293T
Cell Passage Number:2

Factors:

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

mb_sample_id local_sample_id Genotype
SA288553A677G_2_negA677G-mutant
SA288554A677G_3_negA677G-mutant
SA288555A677G_1A677G-mutant
SA288556A677G_1_negA677G-mutant
SA288557A677G_2A677G-mutant
SA288558A677G_3A677G-mutant
SA288559DM_1F667I/H689A-mutant
SA288560DM_3F667I/H689A-mutant
SA288561DM_2_negF667I/H689A-mutant
SA288562DM_3_negF667I/H689A-mutant
SA288563DM_2F667I/H689A-mutant
SA288564DM_1_negF667I/H689A-mutant
SA288565QC_2_negQuality control
SA288566QC_3_negQuality control
SA288567QC_1_negQuality control
SA288568QC_4_negQuality control
SA288569QC_0_negQuality control
SA288570QC_0Quality control
SA288571QC_2Quality control
SA288572QC_3Quality control
SA288573QC_4Quality control
SA288574QC_1Quality control
SA288575WT_3_negWild-type
SA288576WT_1_negWild-type
SA288577WT_2_negWild-type
SA288578WT_1Wild-type
SA288579WT_3Wild-type
SA288580WT_2Wild-type
SA288581Y641F_1Y641F-mutant
SA288582Y641F_1_negY641F-mutant
SA288583Y641F_2Y641F-mutant
SA288584Y641F_3Y641F-mutant
SA288585Y641F_2_negY641F-mutant
SA288586Y641F_3_negY641F-mutant
Showing results 1 to 34 of 34

Collection:

Collection ID:CO002833
Collection Summary:HEK-293T cells were acquired from ATCC CRL-1573 and grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Cat No. 11965-118) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37 °C in 5 % CO2. Cells from cryopreservation were routinely tested following manufacturer’s instructions and found to be mycoplasma-free with MycoSensor PCR Assay Kit (Agilent, Cat No 302108). All cells utilized in experiments were early passage (fifth generation from initial ATCC vial) and were not passaged more than five times prior to disposal.
Sample Type:HEK cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002849
Treatment Summary:HEK-293T cells at 80% confluency were infected in the presence of polybrene for 16 h in DMEM with lentivirus constructs containing either wild-type (WT) EZH2, A677G-mutant (A677G) EZH2, Y641F-mutant (Y641F) EZH2, or H689A/F667I-mutant (DM) EZH2. Infected cells were selected for resistance to blasticidin (10 μg/mL) for 10 days to ensure monoclonal populations.
Cell Media:DMEM supplemented with 10%FBS and 1% Pen/Strep
Cell Pct Confluence:80

Sample Preparation:

Sampleprep ID:SP002846
Sampleprep Summary:Approximately 10 million cells per sample were washed with PBS at room tem-perature and harvested with a mixture of ice-cold methanol:water (80:20). Cell lysis was carried out by four freeze-and-thaw cycles with liquid nitrogen and dry-ice with vortexing between cycles. Supernatant was then dried on SpeedVac concentrator and samples stored at -80ºC. Dried extracts were resuspended in a mixture of Acetonitrile:Water (25:75).

Combined analysis:

Analysis ID AN004432 AN004433
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Agilent 6545 QTOF Agilent 6545 QTOF
Column Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um) Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6545 QTOF Agilent 6545 QTOF
Ion Mode POSITIVE NEGATIVE
Units Peak area Peak area

Chromatography:

Chromatography ID:CH003330
Instrument Name:Agilent 6545 QTOF
Column Name:Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um)
Column Temperature:30
Flow Gradient:98–55% B in 45 min; 55% B during 4 min; 55–98% in 2 min; and column re-equilibration for 15 min
Flow Rate:0.3 mL/min
Solvent A:100% water; 0.1 % formic acid; 10 mM ammonium formate
Solvent B:100% acetonitrile; 0.1 % formic acid;10 mM ammonium formate
Chromatography Type:HILIC

MS:

MS ID:MS004179
Analysis ID:AN004432
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:OpenMS (v2.6.0) was employed for feature detection, alignment, assembly, and de-convolution.
Ion Mode:POSITIVE
  
MS ID:MS004180
Analysis ID:AN004433
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:OpenMS (v2.6.0) was employed for feature detection, alignment, assembly, and de-convolution.
Ion Mode:NEGATIVE
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