Summary of Study ST002587

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	ST002587
Study Title	Stable isotope tracer analysis (SITA) using 13C6-glucose and 13C5-glutamine measured by GC/MS in H1703 cells ± SMARCA4/A2 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 H1703 cells upon SMARCA4/A2-restoration. Conversely, the 13C5-glutamine SITA in H1703 cells revealed that SMARCA4/A2-restoration decreased glutamine uptake and utilization via glutaminolysis to fuel the TCA cycle as indicated by reduced glutamine metabolites (glutamate, α-KG, metabolites) and TCA cycle intermediates (succinate, fumarate, malate, aspartate, 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	2023-04-26
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:	SU002689
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
SA259218	43_Ala_C13_Gln_3	Alanine 18h and C13 Glutamine 60min
SA259219	41_Ala_C13_Gln_1	Alanine 18h and C13 Glutamine 60min
SA259220	42_Ala_C13_Gln_2	Alanine 18h and C13 Glutamine 60min
SA259221	44_Ala_C13_Gln_4	Alanine 18h and C13 Glutamine 60min
SA259222	45_Ala_Gln	Alanine 18h and Glutamine 60min
SA259223	2_C13_Glu_2	C13 Glucose 30min
SA259224	1_C13_Glu_1	C13 Glucose 30min
SA259225	3_C13_Glu_3	C13 Glucose 30min
SA259226	4_C13_Glu_4	C13 Glucose 30min
SA259227	22_C13_Gln_2	C13 Glutamine 60min
SA259228	24_C13_Gln_4	C13 Glutamine 60min
SA259229	21_C13_Gln_1	C13 Glutamine 60min
SA259230	23_C13_Gln_3	C13 Glutamine 60min
SA259231	5_Glu	Glucose 30min
SA259232	25_Gln	Glutamine 60min
SA259233	13_A2_C13_Glu_3	SMACA2 restoration + C13 Glucose 30min
SA259234	12_A2_C13_Glu_2	SMACA2 restoration + C13 Glucose 30min
SA259235	11_A2_C13_Glu_1	SMACA2 restoration + C13 Glucose 30min
SA259236	14_A2_C13_Glu_4	SMACA2 restoration + C13 Glucose 30min
SA259237	31_A2_C13_Gln_1	SMACA2 restoration + C13 Glutamine 60min
SA259238	34_A2_C13_Gln_4	SMACA2 restoration + C13 Glutamine 60min
SA259239	33_A2_C13_Gln_3	SMACA2 restoration + C13 Glutamine 60min
SA259240	32_A2_C13_Gln_2	SMACA2 restoration + C13 Glutamine 60min
SA259241	15_A2_Glu	SMACA2 restoration + Glucose 30min
SA259242	35_A2_Gln	SMACA2 restoration + Glutamine 60min
SA259243	16_A4A2_C13_Glu_1	SMACA4 and A2 restoration + C13 Glucose 30min
SA259244	18_A4A2_C13_Glu_3	SMACA4 and A2 restoration + C13 Glucose 30min
SA259245	17_A4A2_C13_Glu_2	SMACA4 and A2 restoration + C13 Glucose 30min
SA259246	19_A4A2_C13_Glu_4	SMACA4 and A2 restoration + C13 Glucose 30min
SA259247	37_A4A2_C13_Gln_2	SMACA4 and A2 restoration + C13 Glutamine 60min
SA259248	39_A4A2_C13_Gln_4	SMACA4 and A2 restoration + C13 Glutamine 60min
SA259249	38_A4A2_C13_Gln_3	SMACA4 and A2 restoration + C13 Glutamine 60min
SA259250	36_A4A2_C13_Gln_1	SMACA4 and A2 restoration + C13 Glutamine 60min
SA259251	20_A4A2_Glu	SMACA4 and A2 restoration + Glucose 30min
SA259252	40_A4A2_Gln	SMACA4 and A2 restoration + Glutamine 60min
SA259253	6_A4_C13_Glu_1	SMACA4 restoration + C13 Glucose 30min
SA259254	8_A4_C13_Glu_3	SMACA4 restoration + C13 Glucose 30min
SA259255	7_A4_C13_Glu_2	SMACA4 restoration + C13 Glucose 30min
SA259256	9_A4_C13_Glu_4	SMACA4 restoration + C13 Glucose 30min
SA259257	26_A4_C13_Gln_1	SMACA4 restoration + C13 Glutamine 60min
SA259258	29_A4_C13_Gln_4	SMACA4 restoration + C13 Glutamine 60min
SA259259	28_A4_C13_Gln_3	SMACA4 restoration + C13 Glutamine 60min
SA259260	27_A4_C13_Gln_2	SMACA4 restoration + C13 Glutamine 60min
SA259261	10_A4_Glu	SMACA4 restoration + Glucose 30min
SA259262	30_A4_Gln	SMACA4 restoration + Glutamine 60min

Showing results 1 to 45 of 45

Showing results 1 to 45 of 45

Collection:

Collection ID:	CO002682
Collection Summary:	H1703 with or without SMARCA4/A2 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:	TR002701
Treatment Summary:	H1703 with or without SMARCA4/A2 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:

TR002701

Treatment Summary:

H1703 with or without SMARCA4/A2 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:	SP002695
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:

SP002695

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	AN004259
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:	CH003165
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:	MS004006
Analysis ID:	AN004259
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