Summary of Study ST002586

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 PR001663. The data can be accessed directly via it's Project DOI: 10.21228/M8R72W 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	ST002586
Study Title	Stable isotope tracer analysis (SITA) using 13C6-glucose and 13C5-glutamine measured by GC/MS in BIN-67 cells ± SMARCA4 restoration
Study Summary	SMARCA4/2-loss drives the metabolic shift preferring glutamine than glucose to sustain the TCA cycle. SITA using 13C6-glucose confirmed that glucose uptake was increased in BIN-67 cells upon SMARCA4-restoration, leading to overall increased metabolic flux through glucose (lactate) and TCA cycle intermediates (citrate, fumarate, malate, aspartate) via pyruvate carboxylation.Conversely, the 13C5-glutamine SITA in BIN-67 cells revealed that SMARCA4-restoration decreased glutamine uptake and utilization via glutaminolysis to fuel the TCA cycle as indicated by reduced glutamine metabolites (glutamate, α-KG, m+5 metabolites) and TCA cycle intermediates (succinate, fumarate, malate, aspartate, m+4 metabolites)
Institute	McGill University
Department	Biochemistry
Laboratory	Sidong Huang Lab
Last Name	Fu
First Name	Zheng
Address	McIntyre Medical Sciences Building, 3655 promenade Sir-William-Osler
Email	zheng.fu2@mail.mcgill.ca
Phone	5143985446
Submit Date	2023-04-25
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	GC-MS
Release Date	2023-05-22
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001663
Project DOI:	doi: 10.21228/M8R72W
Project Title:	Alanine supplementation exploits glutamine dependency induced by SMARCA4/2-loss
Project Summary:	SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers.we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
Institute:	McGill University
Department:	Biochemistry
Laboratory:	Sidong Huang Lab
Last Name:	Fu
First Name:	Zheng
Address:	McIntyre Medical Sciences Building
Email:	zheng.fu2@mail.mcgill.ca
Phone:	5145869072

Subject:

Subject ID:	SU002688
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
SA259193	23_Ala_C13_Glutamine_3	Alanine 18h and C13 Glutamine 60min
SA259194	22_Ala_C13_Glutamine_2	Alanine 18h and C13 Glutamine 60min
SA259195	21_Ala_C13_Glutamine_1	Alanine 18h and C13 Glutamine 60min
SA259196	24_Ala_C13_Glutamine_4	Alanine 18h and C13 Glutamine 60min
SA259197	25_Ala_Glutamine	Alanine 18h and Glutamine 60min
SA259198	2_C13_Glucose_2	C13 Glucose 30min
SA259199	1_C13_Glucose_1	C13 Glucose 30min
SA259200	3_C13_Glucose_3	C13 Glucose 30min
SA259201	4_C13_Glucose_4	C13 Glucose 30min
SA259202	14_C13_Glutamine_4	C13 Glutamine 60min
SA259203	13_C13_Glutamine_3	C13 Glutamine 60min
SA259204	11_C13_Glutamine_1	C13 Glutamine 60min
SA259205	12_C13_Glutamine_2	C13 Glutamine 60min
SA259206	5_Glucose	Glucose 30min
SA259207	15_Glutamine	Glutamine 60min
SA259208	8_A4_C13_Glucose_3	SMACA4 repstration + C13 Glucose 30min
SA259209	7_A4_C13_Glucose_2	SMACA4 repstration + C13 Glucose 30min
SA259210	6_A4_C13_Glucose_1	SMACA4 repstration + C13 Glucose 30min
SA259211	9_A4_C13_Glucose_4	SMACA4 repstration + C13 Glucose 30min
SA259212	19_A4_C13_Glutamine_4	SMACA4 repstration + C13 Glutamine 60min
SA259213	16_A4_C13_Glutamine_1	SMACA4 repstration + C13 Glutamine 60min
SA259214	17_A4_C13_Glutamine_2	SMACA4 repstration + C13 Glutamine 60min
SA259215	18_A4_C13_Glutamine_3	SMACA4 repstration + C13 Glutamine 60min
SA259216	10_A4_Glucose	SMACA4 repstration + Glucose 30min
SA259217	20_A4_Glutamine	SMACA4 repstration + Glutamine 60min

Showing results 1 to 25 of 25

Showing results 1 to 25 of 25

Collection:

Collection ID:	CO002681
Collection Summary:	BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).
Sample Type:	Ovarian cancer cells

Treatment:

Treatment ID:	TR002700
Treatment Summary:	BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).

Treatment ID:

TR002700

Treatment Summary:

BIN-67 with or without SMARCA4 restoration cells were plated in RPMI supplemented with 6% dialyzed FBS (Wisent Bio, Cat# 080-950) at ~80% confluency the day before experiments. For isotope metabolic tracing, media was replaced with glutamine- or glucose-free RPMI supplemented with 6% dialyzed FBS and 13C5-glutamine or 13C6-glucose (Cambridge Isotopes Laboratories, Tewksbury, MA) for 1 hr or 0.5 hr, respectively. In addition, dishes were kept in unlabeled media as control. Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade).

Sample Preparation:

Sampleprep ID:	SP002694
Sampleprep Summary:	Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Sampleprep ID:

SP002694

Sampleprep Summary:

Cells were washed twice in cold saline solution (NaCl, 0.9 g/l) and metabolites were extracted with 1 ml 80% ice-cold methanol (GC/MS grade). After 2 rounds 10 min sets of sonication (30 seconds on/30 seconds off at high intensity) on slurry ice using a Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with internal control (800 ng myristic acid-D27) and dried in a cold vacuum centrifuge (Labconco) overnight. The dried pellets were reconstituted with 30 μL of 10 mg/mL methoxyamine-HCl in pyridine, and incubated for 30 min at room temperature. Samples were then derivatized with MTBSTFA for 30 min at 70 °C. A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument.

Combined analysis:

Analysis ID	AN004258
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975C
Ion Mode	POSITIVE
Units	intensity

Chromatography:

Chromatography ID:	CH003164
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:	60-320
Flow Gradient:	None
Flow Rate:	0.69 ml/min
Solvent A:	None
Solvent B:	None
Chromatography Type:	GC

MS:

MS ID:	MS004005
Analysis ID:	AN004258
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	A volume of 1 μL of sample was injected splitless with an inlet temperature of 280 oC into the GC (7890, Agilent)/MS (5975C, Agilent) instrument. Metabolites were resolved by separation on DB-5MS+DG (30 m x 250 µm x 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Helium was used as the carrier gas with a flow rate such that myristic-d27 acid eluted at approximately 18 min. The quadrupole was set at 150 ˚C, the source was at 230 °C and the GC/MS interface was at 320 ˚C. The oven program started at 60 ˚C held for 1 min, then increased at a rate of 10 ˚C/min until 320 ˚C. Bake-out was at 320 ˚C for 9 min. Metabolites were ionized by electron impact at 70 eV. All samples were injected three times: twice using scan (50-1000 m/z) mode (1x and 25x dilution for steady-state samples or 1x and 24x dilution for tracer samples) and once using selected ion monitoring (SIM) mode. All of the metabolites described in this study were validated against authentic standards confirming mass spectra and retention times. Integration of ion intensities was accomplished using Mass Hunter Quant (Agilent). Generally, M-57+. Ions (and isotopes) were analyzed. Mass isotopomer distribution analysis was carried out using in-house software using an in-house algorithm adapted from Nanchen et al as previously described.
Ion Mode:	POSITIVE