Summary of Study ST004262

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 PR002689. The data can be accessed directly via it's Project DOI: 10.21228/M82R9H This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Study ID	ST004262
Study Title	The lipidome of drug-resistant glioblastoma persister cells.
Study Summary	This study aimed to determine mechanisms through which glioblastoma stem cells acquire a drug-resistant phenotype. A small proportion of glioblastoma stem cells survive chemotherapy and radiotherapy, creating a drug-resistant persister cell population that resumes proliferation after the cessation of drug treatment. The specific experiment profiled the lipidome of glioblastoma stem cells that survive treatment with the anti-microtubule agent CMPD1. Glioblastoma stem cell line RKI1 was treated for 14 days with 25 micromolar CMPD1 to generate drug-resistant persister cells, replacing the cell culture medium every 3 days (n = 3). At day 14 of treatment, the CMPD1-treated cells were collected for lipid extraction and lipidomic analysis. The drug-resistant persister cells were compared to control cells grown in RKI1 growth medium and collected prior to drug-treatment (n = 3). The drug-resistant persister cells displayed significantly decreased levels of ceramide and cholesterol, and increased sphingomyelin and diacylgylcerol, indicative of membrane remodelling that may allow the cells to survive chemotherapy. Further investigation indicated that the reduced cholesterol content provides a point of metabolic vulnerability to eliminate the drug resistant persister cells.
Institute	University of Sydney
Department	School of Medical Sciences
Last Name	Don
First Name	Anthony
Address	Office 3210, D17 Charles Perkins Centre, Camperdown, NSW, 2006
Email	anthony.don@sydney.edu.au
Phone	+612 8627 5578
Submit Date	2025-09-29
Num Groups	2
Total Subjects	6
Study Comments	Control and drug-treated RKI1 glioblastoma cells
Publications	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-10-10
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002689
Project DOI:	doi: 10.21228/M82R9H
Project Title:	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma
Project Summary:	Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.
Institute:	The University of Sydney
Department:	School of Medical Sciences
Last Name:	Don
First Name:	Anthony
Address:	Office 3210, D17 Charles Perkins Centre, Camperdown, NSW, 2006, Australia
Email:	anthony.don@sydney.edu.au
Phone:	+612 8627 5578
Publications:	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma

Subject:

Subject ID:	SU004415
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Biosource Or Supplier:	QIMR Berghofer Institute
Cell Strain Details:	Patient-derived glioblastoma stem cell line RKI1

Factors:

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

mb_sample_id	local_sample_id	Sample source	Factor
SA497018	4	RKI1 cells	Control
SA497019	5	RKI1 cells	Control
SA497020	3	RKI1 cells	Control
SA497021	1	RKI1 cells	Drug Treated
SA497022	6	RKI1 cells	Drug Treated
SA497023	2	RKI1 cells	Drug Treated

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO004408
Collection Summary:	Glioblastoma stem cell line RKI1 is a patient-derived line described in: Stringer BW, et al. A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma. Scientific Reports 9, 4902 (2019). These cells are available at: https://www.qimrberghofer.edu.au/our-research/commercialisation/q-cell/. RKI1 cells are cultured in KnockOut DMEM/F-12 basal medium kit with neural supplement, EGF (20 ng/mL) and FGF-β (10 ng/mL) (ThermoFisher Scientific, Cat# 579 A1050901). GlutaMAX-ICTS (2 mM) (ThermoFisher Scientific, Cat# A1286001) and Antibiotic- Antimycotic (ThermoFisher Scientific, Cat# 15240112) were also added. Adherent cells were plated on flasks coated with 0.15% in PBS MatriGel Matrix (Corning Life Sciences, Cat# BDAA356237), incubated at 37 °C, 5% CO2. Specific treatment conditions are described under "treatment".
Sample Type:	Glioma cells

Treatment:

Treatment ID:	TR004424
Treatment Summary:	RKI1 cells (1.5 × 10^4 cells/cm2) were grown in 10 cm2 dishes and treated with CMPD1 (25 μM) for 14 days. Every 3 days, the media containing CMPD1 was replaced. At Day 14, three independent cultures of drug tolerant persister (DTP) cells were collected for lipidomic analysis. Control cells were RKI1 cells grown in culture medium without CMPD1 (1.5 x 10^5 cells in 10 cm2 dishes). Three independent cultures of the cells were used. RKI1 cells are cultured in KnockOut DMEM/F-12 basal medium kit with neural supplement, EGF (20 ng/mL) and FGF-β (10 ng/mL) (ThermoFisher Scientific, Cat# 579 A1050901). GlutaMAX-ICTS (2 mM) (ThermoFisher Scientific, Cat# A1286001) and Antibiotic- Antimycotic (ThermoFisher Scientific, Cat# 15240112) were also added. Adherent cells were plated on flasks coated with 0.15% in PBS MatriGel Matrix (Corning Life Sciences, Cat# BDAA356237), incubated at 37 °C, 5% CO2.

Treatment ID:

TR004424

Treatment Summary:

RKI1 cells (1.5 × 10^4 cells/cm2) were grown in 10 cm2 dishes and treated with CMPD1 (25 μM) for 14 days. Every 3 days, the media containing CMPD1 was replaced. At Day 14, three independent cultures of drug tolerant persister (DTP) cells were collected for lipidomic analysis. Control cells were RKI1 cells grown in culture medium without CMPD1 (1.5 x 10^5 cells in 10 cm2 dishes). Three independent cultures of the cells were used. RKI1 cells are cultured in KnockOut DMEM/F-12 basal medium kit with neural supplement, EGF (20 ng/mL) and FGF-β (10 ng/mL) (ThermoFisher Scientific, Cat# 579 A1050901). GlutaMAX-ICTS (2 mM) (ThermoFisher Scientific, Cat# A1286001) and Antibiotic- Antimycotic (ThermoFisher Scientific, Cat# 15240112) were also added. Adherent cells were plated on flasks coated with 0.15% in PBS MatriGel Matrix (Corning Life Sciences, Cat# BDAA356237), incubated at 37 °C, 5% CO2.

Sample Preparation:

Sampleprep ID:	SP004421
Sampleprep Summary:	RKI1 parent and CMPD1-derived drug-tolerant persister cells were collected, homogenized in sample extraction buffer (50 mM Hepes pH 7.4, 25 mM KCl, Protease Inhibitor Cocktail) by sonicating for 5 min (30 s on/30 s off) at 4ºC with a Qsonica Q800R2 sonicating bath. Protein concentration was determined with the BCA assay. Lipids were extracted from 200 µL lysate (~200 µg protein) using the methyl-tert-butyl ether (MTBE)/methanol/water protocol. Cell homogenate was combined with 250 µL methanol containing 0.01% 3,5-di-tert-4-butylhydroxyltoluene (BHT), internal standards and 850 µL MTBE, sonicated in a 4°C water bath for 30 min, and phase separation was induced by centrifuging at 2000g for 5 min. The upper organic phase was transferred to a 5 mL glass tube, and the aqueous phase was re-extracted with 500 µL MTBE and 150 µL methanol. The organic phase from the second extraction was combined with the first, after which the extracts were dried in a Savant SC210 SpeedVac desiccator (ThermoFisher Scientific). Lipids were reconstituted in 400 µL of 80% (v/v) methanol:20% water containing 0.01% BHT, 1 mM ammonium formate, and 0.1% formic acid, and stored at -80 degrees C.

Sampleprep ID:

SP004421

Sampleprep Summary:

RKI1 parent and CMPD1-derived drug-tolerant persister cells were collected, homogenized in sample extraction buffer (50 mM Hepes pH 7.4, 25 mM KCl, Protease Inhibitor Cocktail) by sonicating for 5 min (30 s on/30 s off) at 4ºC with a Qsonica Q800R2 sonicating bath. Protein concentration was determined with the BCA assay. Lipids were extracted from 200 µL lysate (~200 µg protein) using the methyl-tert-butyl ether (MTBE)/methanol/water protocol. Cell homogenate was combined with 250 µL methanol containing 0.01% 3,5-di-tert-4-butylhydroxyltoluene (BHT), internal standards and 850 µL MTBE, sonicated in a 4°C water bath for 30 min, and phase separation was induced by centrifuging at 2000g for 5 min. The upper organic phase was transferred to a 5 mL glass tube, and the aqueous phase was re-extracted with 500 µL MTBE and 150 µL methanol. The organic phase from the second extraction was combined with the first, after which the extracts were dried in a Savant SC210 SpeedVac desiccator (ThermoFisher Scientific). Lipids were reconstituted in 400 µL of 80% (v/v) methanol:20% water containing 0.01% BHT, 1 mM ammonium formate, and 0.1% formic acid, and stored at -80 degrees C.

Chromatography:

Chromatography ID:	CH005388
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1 mm, 1.7 µm)
Column Temperature:	45°C
Flow Gradient:	0-3 min, 20% B; 3-5.5 min, ramp to 45% B; 5.5-8 min, ramp to 65% B; 8-13 min, ramp to 85% B; 13-14 min, ramp to 100% B; 14-20 min, hold at 100% B; 20-25 min, decrease to 20% B and hold to 25 min
Flow Rate:	0.28 mL/min
Solvent A:	60% Acetonitrile/40% water; 10 mM Ammonium formate; 0.1% Formic acid,
Solvent B:	10% Acetonitrile/90% Isopropanol; 10 mM Ammonium formate, 0.1% Formic acid,
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN007092
Analysis Type:	MS
Chromatography ID:	CH005388
Num Factors:	2
Num Metabolites:	70
Units:	pmoles/mg protein

Analysis ID:	AN007093
Analysis Type:	MS
Chromatography ID:	CH005388
Num Factors:	2
Num Metabolites:	56
Units:	pmoles/mg protein