#METABOLOMICS WORKBENCH AnthonyDon_20250929_182902 DATATRACK_ID:6506 STUDY_ID:ST004262 ANALYSIS_ID:AN007093 PROJECT_ID:PR002689
VERSION             	1
CREATED_ON             	October 5, 2025, 12:46 am
#PROJECT
PR:PROJECT_TITLE                 	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister
PR:PROJECT_TITLE                 	cells in glioblastoma
PR:PROJECT_SUMMARY               	Chemotherapy often kills a large fraction of cancer cells but leaves behind a
PR:PROJECT_SUMMARY               	small population of drug tolerant persister cells. These persister cells survive
PR:PROJECT_SUMMARY               	drug treatments through reversible, non-genetic mechanisms and cause tumour
PR:PROJECT_SUMMARY               	recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism
PR:PROJECT_SUMMARY               	regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through
PR:PROJECT_SUMMARY               	histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies
PR:PROJECT_SUMMARY               	combined with CRISPR knockout and phenotypic drug screen, we identified that
PR:PROJECT_SUMMARY               	chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in
PR:PROJECT_SUMMARY               	glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically,
PR:PROJECT_SUMMARY               	PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol
PR:PROJECT_SUMMARY               	biosynthesis, which persister cells rely on to maintain homeostasis under
PR:PROJECT_SUMMARY               	chemotherapy induced oxidative stress and lipid peroxidation. PRDM9 inhibition,
PR:PROJECT_SUMMARY               	combined with chemotherapy, resulted in strong anti-cancer efficacy in
PR:PROJECT_SUMMARY               	preclinical glioblastoma models, significantly enhancing the magnitude and
PR:PROJECT_SUMMARY               	duration of the antitumor response by eliminating persisters. These findings
PR:PROJECT_SUMMARY               	demonstrate a previously unknown role of PRDM9 in promoting metabolic
PR:PROJECT_SUMMARY               	reprogramming that enables the survival of drug-tolerant persister cells.
PR:INSTITUTE                     	The University of Sydney
PR:DEPARTMENT                    	School of Medical Sciences
PR:LAST_NAME                     	Don
PR:FIRST_NAME                    	Anthony
PR:ADDRESS                       	Office 3210, D17 Charles Perkins Centre, Camperdown, NSW, 2006, Australia
PR:EMAIL                         	anthony.don@sydney.edu.au
PR:PHONE                         	+612 8627 5578
PR:PUBLICATIONS                  	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister
PR:PUBLICATIONS                  	cells in glioblastoma
#STUDY
ST:STUDY_TITLE                   	The lipidome of drug-resistant glioblastoma persister cells.
ST:STUDY_SUMMARY                 	This study aimed to determine mechanisms through which glioblastoma stem cells
ST:STUDY_SUMMARY                 	acquire a drug-resistant phenotype. A small proportion of glioblastoma stem
ST:STUDY_SUMMARY                 	cells survive chemotherapy and radiotherapy, creating a drug-resistant persister
ST:STUDY_SUMMARY                 	cell population that resumes proliferation after the cessation of drug
ST:STUDY_SUMMARY                 	treatment. The specific experiment profiled the lipidome of glioblastoma stem
ST:STUDY_SUMMARY                 	cells that survive treatment with the anti-microtubule agent CMPD1. Glioblastoma
ST:STUDY_SUMMARY                 	stem cell line RKI1 was treated for 14 days with 25 micromolar CMPD1 to generate
ST:STUDY_SUMMARY                 	drug-resistant persister cells, replacing the cell culture medium every 3 days
ST:STUDY_SUMMARY                 	(n = 3). At day 14 of treatment, the CMPD1-treated cells were collected for
ST:STUDY_SUMMARY                 	lipid extraction and lipidomic analysis. The drug-resistant persister cells were
ST:STUDY_SUMMARY                 	compared to control cells grown in RKI1 growth medium and collected prior to
ST:STUDY_SUMMARY                 	drug-treatment (n = 3). The drug-resistant persister cells displayed
ST:STUDY_SUMMARY                 	significantly decreased levels of ceramide and cholesterol, and increased
ST:STUDY_SUMMARY                 	sphingomyelin and diacylgylcerol, indicative of membrane remodelling that may
ST:STUDY_SUMMARY                 	allow the cells to survive chemotherapy. Further investigation indicated that
ST:STUDY_SUMMARY                 	the reduced cholesterol content provides a point of metabolic vulnerability to
ST:STUDY_SUMMARY                 	eliminate the drug resistant persister cells.
ST:INSTITUTE                     	University of Sydney
ST:DEPARTMENT                    	School of Medical Sciences
ST:LAST_NAME                     	Don
ST:FIRST_NAME                    	Anthony
ST:ADDRESS                       	Office 3210, D17 Charles Perkins Centre, Camperdown, NSW, 2006
ST:EMAIL                         	anthony.don@sydney.edu.au
ST:PHONE                         	+612 8627 5578
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	6
ST:STUDY_COMMENTS                	Control and drug-treated RKI1 glioblastoma cells
ST:PUBLICATIONS                  	Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister
ST:PUBLICATIONS                  	cells in glioblastoma
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_BIOSOURCE_OR_SUPPLIER    	QIMR Berghofer Institute
SU:CELL_STRAIN_DETAILS           	Patient-derived glioblastoma stem cell line RKI1
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	4	Sample source:RKI1 cells | Factor:Control	RAW_FILE_NAME(Raw file name)=4
SUBJECT_SAMPLE_FACTORS           	-	5	Sample source:RKI1 cells | Factor:Control	RAW_FILE_NAME(Raw file name)=5
SUBJECT_SAMPLE_FACTORS           	-	3	Sample source:RKI1 cells | Factor:Control	RAW_FILE_NAME(Raw file name)=3
SUBJECT_SAMPLE_FACTORS           	-	1	Sample source:RKI1 cells | Factor:Drug Treated	RAW_FILE_NAME(Raw file name)=1
SUBJECT_SAMPLE_FACTORS           	-	6	Sample source:RKI1 cells | Factor:Drug Treated	RAW_FILE_NAME(Raw file name)=6
SUBJECT_SAMPLE_FACTORS           	-	2	Sample source:RKI1 cells | Factor:Drug Treated	RAW_FILE_NAME(Raw file name)=2
#COLLECTION
CO:COLLECTION_SUMMARY            	Glioblastoma stem cell line RKI1 is a patient-derived line described in:
CO:COLLECTION_SUMMARY            	Stringer BW, et al. A reference collection of patient-derived cell line and
CO:COLLECTION_SUMMARY            	xenograft models of proneural, classical and mesenchymal glioblastoma.
CO:COLLECTION_SUMMARY            	Scientific Reports 9, 4902 (2019). These cells are available at:
CO:COLLECTION_SUMMARY            	https://www.qimrberghofer.edu.au/our-research/commercialisation/q-cell/. RKI1
CO:COLLECTION_SUMMARY            	cells are cultured in KnockOut DMEM/F-12 basal medium kit with neural
CO:COLLECTION_SUMMARY            	supplement, EGF (20 ng/mL) and FGF-β (10 ng/mL) (ThermoFisher Scientific, Cat#
CO:COLLECTION_SUMMARY            	579 A1050901). GlutaMAX-ICTS (2 mM) (Thermofisher Scientific, Cat# A1286001) and
CO:COLLECTION_SUMMARY            	Antibiotic- Antimycotic (Thermofisher Scientific, Cat# 15240112) were also
CO:COLLECTION_SUMMARY            	added. Adherent cells were plated on flasks coated with 0.15% in PBS MatriGel
CO:COLLECTION_SUMMARY            	Matrix (Corning Life Sciences, Cat# BDAA356237), incubated at 37 °C, 5% CO2.
CO:COLLECTION_SUMMARY            	Specific treatment conditions are described under "treatment".
CO:SAMPLE_TYPE                   	Glioma cells
#TREATMENT
TR:TREATMENT_SUMMARY             	RKI1 cells (1.5 × 10^4 cells/cm2) were grown in 10 cm2 dishes and treated with
TR:TREATMENT_SUMMARY             	CMPD1 (25 μM) for 14 days. Every 3 days, the media containing CMPD1 was
TR:TREATMENT_SUMMARY             	replaced. At Day 14, three independent cultures of drug tolerant persister (DTP)
TR:TREATMENT_SUMMARY             	cells were collected for lipidomic analysis. Control cells were RKI1 cells grown
TR:TREATMENT_SUMMARY             	in culture medium without CMPD1 (1.5 x 10^5 cells in 10 cm2 dishes). Three
TR:TREATMENT_SUMMARY             	independent cultures of the cells were used. RKI1 cells are cultured in KnockOut
TR:TREATMENT_SUMMARY             	DMEM/F-12 basal medium kit with neural supplement, EGF (20 ng/mL) and FGF-β (10
TR:TREATMENT_SUMMARY             	ng/mL) (ThermoFisher Scientific, Cat# 579 A1050901). GlutaMAX-ICTS (2 mM)
TR:TREATMENT_SUMMARY             	(Thermofisher Scientific, Cat# A1286001) and Antibiotic- Antimycotic
TR:TREATMENT_SUMMARY             	(Thermofisher Scientific, Cat# 15240112) were also added. Adherent cells were
TR:TREATMENT_SUMMARY             	plated on flasks coated with 0.15% in PBS MatriGel Matrix (Corning Life
TR:TREATMENT_SUMMARY             	Sciences, Cat# BDAA356237), incubated at 37 °C, 5% CO2.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	RKI1 parent and CMPD1-derived drug-tolerant persister cells were collected,
SP:SAMPLEPREP_SUMMARY            	homogenized in sample extraction buffer (50 mM Hepes pH 7.4, 25 mM KCl, Protease
SP:SAMPLEPREP_SUMMARY            	Inhibitor Cocktail) by sonicating for 5 min (30 s on/30 s off) at 4ºC with a
SP:SAMPLEPREP_SUMMARY            	Qsonica Q800R2 sonicating bath. Protein concentration was determined with the
SP:SAMPLEPREP_SUMMARY            	BCA assay. Lipids were extracted from 200 µL lysate (~200 µg protein) using
SP:SAMPLEPREP_SUMMARY            	the methyl-tert-butyl ether (MTBE)/methanol/water protocol. Cell homogenate was
SP:SAMPLEPREP_SUMMARY            	combined with 250 µL methanol containing 0.01%
SP:SAMPLEPREP_SUMMARY            	3,5-di-tert-4-butylhydroxyltoluene (BHT), internal standards and 850 µL MTBE,
SP:SAMPLEPREP_SUMMARY            	sonicated in a 4°C water bath for 30 min, and phase separation was induced by
SP:SAMPLEPREP_SUMMARY            	centrifuging at 2000g for 5 min. The upper organic phase was transferred to a 5
SP:SAMPLEPREP_SUMMARY            	mL glass tube, and the aqueous phase was re-extracted with 500 µL MTBE and 150
SP:SAMPLEPREP_SUMMARY            	µL methanol. The organic phase from the second extraction was combined with the
SP:SAMPLEPREP_SUMMARY            	first, after which the extracts were dried in a Savant SC210 SpeedVac dessicator
SP:SAMPLEPREP_SUMMARY            	(ThermoFisher Scientific). Lipids were reconstituted in 400 µL of 80% (v/v)
SP:SAMPLEPREP_SUMMARY            	methanol:20% water containing 0.01% BHT, 1 mM ammonium formate, and 0.1% formic
SP:SAMPLEPREP_SUMMARY            	acid, and stored at -80 degrees C.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	10 mM ammonium formate, 0.1% formic acid, 60% acetonitrile and 40% water
CH:SOLVENT_B                     	10 mM ammonium formate, 0.1% formic acid, 10% acetonitrile and 90% isopropanol
CH: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,
CH:FLOW_GRADIENT                 	ramp to 85% B; 13-14 min, ramp to 100% B; 14-20 min, hold at 100% B; 20-25 min,
CH:FLOW_GRADIENT                 	decrease to 20% B and hold to 25 min
CH:FLOW_RATE                     	0.28 ml/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo TSQ Altis
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Phosphatidylethanolamine (PE) and phosphatidylinositol (PI) species were
MS:MS_COMMENTS                   	detected as the [M-H]- precursor ion, with product ions corresponding to the
MS:MS_COMMENTS                   	fatty acyl anion. PS was identified through neutral loss of the serine headgroup
MS:MS_COMMENTS                   	(PS) (precursor m/z - 87). Note that PE species were also identified through
MS:MS_COMMENTS                   	neutral loss of the ethanolamine phosphate headgroup.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmoles/mg protein
MS_METABOLITE_DATA_START
Samples	4	5	3	1	6	2
Factors	Sample source:RKI1 cells | Factor:Control	Sample source:RKI1 cells | Factor:Control	Sample source:RKI1 cells | Factor:Control	Sample source:RKI1 cells | Factor:Drug Treated	Sample source:RKI1 cells | Factor:Drug Treated	Sample source:RKI1 cells | Factor:Drug Treated
PE(16:0/16:0)	160.7	142.2	117.2	38.0	95.7	67.6
PE(16:0/16:1)	1049.1	860.5	1067.0	213.5	530.5	543.5
PE(16:0/18:0)	141.7	102.9	112.3	53.5	86.9	57.2
PE(16:0/18:1)	5308.5	4177.4	4855.2	2211.9	4089.9	3321.1
PE(16:0/20:4)	676.0	260.0	588.8	116.7	288.6	248.0
PE(16:0/22:5)	1166.8	595.8	1231.9	92.1	225.6	188.0
PE(16:0/22:6)	262.8	133.7	319.6	35.0	30.6	87.1
PE(16:1/18:1)	1512.0	1366.1	1749.0	485.9	885.5	709.9
PE(16:1/20:4)	88.2	47.1	73.3	15.7	36.8	18.8
PE(16:1/22:6)	43.7	23.2	59.5	4.0	0.7	3.1
PE(18:0/ 20:1)	563.6	790.1	719.0	1742.3	2197.1	1811.4
PE(18:0/16:0)	448.6	282.3	303.3	150.9	213.0	205.4
PE(18:0/18:0)	884.9	754.1	829.7	772.8	1185.6	1105.0
PE(18:0/18:1)	11791.0	14914.3	13464.4	10349.6	23865.5	19435.6
PE(18:0/20:0)	19.8	20.8	24.5	53.3	64.5	75.1
PE(18:0/20:4)	7443.7	3668.1	5612.4	2918.0	4716.6	5820.4
PE(18:0/22:4)	3441.9	1495.4	2828.7	2518.4	3988.4	3936.1
PE(18:0/22:6)	866.0	500.2	753.4	476.3	619.2	903.8
PE(18:1/18:1)	33280.5	34183.1	31652.4	40218.1	48042.3	39013.6
PE(18:1/18:2)	1645.0	2957.0	1929.6	8055.2	14763.3	10551.4
PE(18:1/20:4)	120.8	73.2	113.9	57.0	78.9	89.0
PE(18:1/22:6)	489.5	359.7	577.2	250.1	278.1	392.8
PI(16:0/16:0)	22.8	10.8	24.6	8.9	9.6	14.1
PI(16:0/16:1)	21.8	19.2	30.3	12.9	6.1	9.6
PI(16:0/18:0)	383.5	166.8	362.8	111.1	64.1	120.9
PI(16:0/18:1)	215.0	169.3	235.3	326.9	199.8	278.2
PI(16:0/20:4)	107.3	57.9	76.2	32.9	28.8	31.3
PI(16:1/18:1)	182.4	138.6	182.2	140.3	82.2	114.0
PI(18:0/16:0)	15.5	11.1	14.5	19.3	12.9	21.4
PI(18:0/18:0)	99.6	73.8	103.6	279.9	158.1	168.8
PI(18:0/18:1)	46.5	42.9	46.6	263.1	155.0	88.2
PI(18:0/20:3)	4504.0	5816.9	4348.0	4155.1	4284.7	5013.4
PI(18:0/20:4)	2502.3	1391.1	2037.1	1258.5	947.6	1313.0
PI(18:0/22:4)	222.9	98.9	242.8	466.5	242.3	356.6
PI(18:0/22:5)	154.8	68.9	123.9	201.0	129.5	176.3
PI(18:1/18:1)	626.2	542.4	542.7	3380.5	1159.2	1338.9
PI(18:1/18:2)	96.9	207.2	67.5	734.9	518.8	567.0
PI(18:1/20:4)	301.7	222.2	230.9	139.7	73.9	88.8
PS(32:0)	92.6	112.7	79.4	17.3	57.2	18.7
PS(32:1)	92.9	88.1	82.9	2.9	9.2	2.0
PS(34:1)	1956.4	1384.7	1943.2	209.5	362.1	341.1
PS(34:2)	182.3	322.1	189.2	31.3	80.9	71.9
PS(36:1)	9244.5	13088.0	7762.8	3738.9	4548.4	4039.1
PS(36:2)	1736.2	3458.5	1867.2	2172.4	3740.1	2836.6
PS(36:3)	246.9	593.9	263.0	355.4	501.7	415.1
PS(36:4)	56.7	108.7	49.4	40.1	34.2	30.1
PS(36:5)	11.9	15.8	10.3	0.8	4.4	0.0
PS(38:1)	64.8	120.9	73.2	63.4	53.2	43.8
PS(38:4)	1980.1	2366.7	2374.5	1502.2	2562.2	2004.8
PS(38:5)	209.0	223.0	191.0	64.5	111.2	91.5
PS(38:6)	88.5	79.3	120.4	6.9	12.0	15.9
PS(40:4)	2162.5	3561.9	2483.7	330.5	477.6	417.0
PS(40:5)	2599.2	1798.8	2799.3	716.3	861.9	856.2
PS(40:6)	2492.8	1615.8	2596.1	642.7	811.6	771.6
PS(40:7)	121.7	137.2	117.2	50.9	14.1	50.3
PS(42:10)	4.3	11.1	5.7	1.3	0.0	1.5
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Ion	Precursor m/z	Product m/z
PE(16:0/16:0)	[M-H]	690.5	255.2
PE(16:0/16:1)	[M-H]	688.5	253.2
PE(16:0/18:0)	[M-H]	718.5	283.3
PE(16:0/18:1)	[M-H]	716.5	281.2
PE(16:0/20:4)	[M-H]	738.5	303.2
PE(16:0/22:5)	[M-H]	764.5	329.2
PE(16:0/22:6)	[M-H]	762.5	327.2
PE(16:1/18:1)	[M-H]	714.5	281.2
PE(16:1/20:4)	[M-H]	736.5	303.2
PE(16:1/22:6)	[M-H]	760.5	327.2
PE(18:0/ 20:1)	[M-H]	772.6	309.3
PE(18:0/16:0)	[M-H]	718.5	255.2
PE(18:0/18:0)	[M-H]	746.6	283.3
PE(18:0/18:1)	[M-H]	744.6	281.2
PE(18:0/20:0)	[M-H]	774.6	311.3
PE(18:0/20:4)	[M-H]	766.5	303.2
PE(18:0/22:4)	[M-H]	794.6	331.3
PE(18:0/22:6)	[M-H]	790.5	327.2
PE(18:1/18:1)	[M-H]	742.5	281.2
PE(18:1/18:2)	[M-H]	740.5	281.2
PE(18:1/20:4)	[M-H]	764.5	303.2
PE(18:1/22:6)	[M-H]	788.5	327.2
PI(16:0/16:0)	[M-H]	809.5	255.2
PI(16:0/16:1)	[M-H]	807.5	253.2
PI(16:0/18:0)	[M-H]	837.6	283.3
PI(16:0/18:1)	[M-H]	835.5	281.2
PI(16:0/20:4)	[M-H]	857.5	303.2
PI(16:1/18:1)	[M-H]	833.5	281.2
PI(18:0/16:0)	[M-H]	837.6	255.2
PI(18:0/18:0)	[M-H]	865.6	283.3
PI(18:0/18:1)	[M-H]	863.6	281.2
PI(18:0/20:3)	[M-H]	887.6	305.2
PI(18:0/20:4)	[M-H]	885.6	303.2
PI(18:0/22:4)	[M-H]	913.6	331.3
PI(18:0/22:5)	[M-H]	911.6	329.2
PI(18:1/18:1)	[M-H]	861.6	281.2
PI(18:1/18:2)	[M-H]	859.5	279.2
PI(18:1/20:4)	[M-H]	883.5	303.2
PS(32:0)	[M-H]	734.5	647.5
PS(32:1)	[M-H]	732.5	645.5
PS(34:1)	[M-H]	760.5	673.5
PS(34:2)	[M-H]	758.5	671.5
PS(36:1)	[M-H]	788.5	701.5
PS(36:2)	[M-H]	786.5	699.5
PS(36:3)	[M-H]	784.5	697.5
PS(36:4)	[M-H]	782.5	695.5
PS(36:5)	[M-H]	780.5	693.5
PS(38:1)	[M-H]	818.6	731.6
PS(38:4)	[M-H]	812.5	725.5
PS(38:5)	[M-H]	808.5	721.5
PS(38:6)	[M-H]	806.5	719.5
PS(40:4)	[M-H]	840.6	753.5
PS(40:5)	[M-H]	836.5	749.5
PS(40:6)	[M-H]	836.5	749.5
PS(40:7)	[M-H]	832.5	745.5
PS(42:10)	[M-H]	854.6	767.5
METABOLITES_END
#END