Summary of Study ST002066

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 PR001308. The data can be accessed directly via it's Project DOI: 10.21228/M8N98X 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 IDST002066
Study TitleGlutaminase inhibition impairs CD8 T cell activation in STK11/Lkb1 deficient lung cancer
Study TypeBiomedical research
Study SummaryTo characterize the impact of KRAS co-mutations KEAP1 and STK11/Lkb1 on the metabolic and immune microenvironment of lung adenocarcinoma in immune-intact models, we generated a cohort of genetically engineered mouse models (GEMMs) to reflect the diverse tumor suppressor landscape seen in patients. Mice carrying the KrasG12D allele (K)21 were crossed with Keap1flox/flox (KK)22 and/or Lkb1flox/flox (KKL, KL)23 mice and genetic recombination induced by intranasal inhalation of Ad5-CMV-Cre adenovirus. We interrogated the metabolites present in lung tumor nodules collected from cohorts of KK, KL and KKL mice.
Institute
The Walter and Eliza Hall Institute of Medical Research
LaboratoryKate Sutherland
Last NameSarah
First NameBest
Address1G, Royal Parade, Parkville VIC 3052, Australia
Emailbest@wehi.edu.au
Phone+61-3-9345-2452
Submit Date2022-01-18
Num Groups5
Total Subjects25
Num Males17
Num Females8
Study CommentsL lobe of mice lung
PublicationsGlutaminase inhibition impairs CD8 T cell activation in STK11/Lkb1 deficient lung cancer
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-05-02
Release Version1
Best Sarah Best Sarah
https://dx.doi.org/10.21228/M8N98X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001308
Project DOI:doi: 10.21228/M8N98X
Project Title:Glutaminase inhibition impairs CD8 T cell activation in STK11/Lkb1 deficient lung cancer
Project Type:untargeted LCMS metabolomics of GM mice lung tumors upon treatment
Project Summary:The tumor microenvironment (TME) contains a rich source of nutrients that sustain cell growth and ultimately facilitate tumor progression. The availability of glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert anti-tumor function. Recently, inhibition of glutaminase, the enzyme that hydrolyzes glutamine to glutamate, has garnered interest as an approach to both decrease tumor metabolism and growth, while increasing available glutamine in the TME for effector T cells. Checkpoint blockade immunotherapy unleashes anti-tumor effector capabilities of T cells, and although there are good responses in many solid tumors, a significant proportion of patients respond poorly. In lung adenocarcinoma, response to immunotherapy is reported to be impaired in KRAS-mutant patients harboring concurrent KEAP1 and STK11/Lkb1 mutations. To investigate the metabolism and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated a series of murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape seen in patients. Here we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1 checkpoint immunotherapy. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and cytotoxic activation of tumor-infiltrating CD8 T cells. Thus, CD8 T cells exposed to checkpoint immunotherapy have a dependence on glutamine and reliance on glutaminase activity and are negatively impacted by the glutaminase inhibitor in this highly activated state. Therefore, we discern that the combination of immunotherapy and glutaminase inhibition is not efficacious for CD8 T cell activation in the tumor microenvironment.
Institute:The Walter and Eliza Hall Institute of Medical Research
Laboratory:Kate Sutherland
Last Name:Best
First Name:Sarah
Address:1G, Royal Parade, Parkville VIC 3052
Email:best@wehi.edu.au
Phone:+61-3-9345-2452
Publications:Glutaminase inhibition impairs CD8 T cell activation in STK11/Lkb1 deficient lung cancer
Contributors:Sarah A. Best, Patrick M. Gubser, Shalini Sethumadhavan, Ariena Kersbergen, Yashira L. Negrón Abril, Joshua Goldford, Katherine Sellers, Waruni Abeysekera, Alexandra L. Garnham, Jackson A. McDonald, Clare E. Weeden, Dovile Anderson, David Pirman, Thomas P. Roddy, Darren J. Creek, Axel Kallies, Gillian Kingsbury and Kate D. Sutherland

Subject:

Subject ID:SU002148
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6 GEMMs
Age Or Age Range:Mutants: K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre
Gender:Male and female

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id treatment
SA194269Blank_1Extraction blank
SA194270Blank_4Extraction blank
SA194271Blank_3Extraction blank
SA194272Blank_2Extraction blank
SA194273K_5Kras
SA194274K_4Kras
SA194275K_1Kras
SA194276K_2Kras
SA194277K_3Kras
SA194278KK_5Kras/Keap1
SA194279KK_4Kras/Keap1
SA194280KK_3Kras/Keap1
SA194281KK_2Kras/Keap1
SA194282KKL_5Kras/Keap1/Lkb1
SA194283KKL_4Kras/Keap1/Lkb1
SA194284KKL_3Kras/Keap1/Lkb1
SA194285KKL_1Kras/Keap1/Lkb1
SA194286KL_5Kras/Lkb1
SA194287KL_4Kras/Lkb1
SA194288KL_3Kras/Lkb1
SA194289KL_1Kras/Lkb1
SA194290KL_2Kras/Lkb1
SA194291KP_3Kras/p53
SA194292KP_4Kras/p53
SA194293KP_2Kras/p53
SA194294QC_4QC
SA194295QC_3QC
SA194296QC_2QC
SA194297QC_1QC
SA194299KK_1YFP(WT)/Kras/Keap1/p53(WT)
SA194298KP_1YFP(WT)/Kras/Keap1(WT)/p53
Showing results 1 to 31 of 31

Collection:

Collection ID:CO002141
Collection Summary:At defined timepoints (K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre), mice were sacrificed by cardiac puncture and lungs and plasma harvested for downstream analysis.
Sample Type:Lung
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002160
Treatment Summary:Mice were genotyped using primers outlined in the Key Resources Table. Seven-to-eight-week old conditional mice were intranasally (i.n) infected with 20 ul of 1x1010 PFU/ml Ad5-CMV-Cre virus (University of Iowa Gene Transfer Core Facility) according to standard procedures62. For anti-PD1 survival study, 20 days following Ad5-CMV-Cre, KL and KKL mice were randomly assigned to anti-PD1 or isotype control antibody (200 g RMP1-14 or Rat IgG2a; BioXCell) treatment arms (n = 6/arm). Each cycle (every 20 days) consisted of intra-peritoneal injection (200 l into left and right flank) three times over 6 days (day 0, 3, 6). Mice were collected at ethical endpoint for Kaplan Meier survival analysis. For combination anti-PD1/CB-839 study, 20 days following Ad5-CMV-Cre, KL mice were randomly assigned to vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms (study 1: n = 6/arm; study 2: n = 4/arm). On day 20, mice received one cycle of anti-PD1 or isotype control (as above) and 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29, when mice were collected 12 hours following the final dose. At harvest, mice were sacrificed by cardiac bleed and the left lung lobe was weighed and processed for flow cytometry with spleens, while right lung lobes were inflated with 4 % paraformaldehyde and processed for histology.
Treatment Compound:200 ug RMP1-14 or Rat IgG2a; Combination treatments: vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms
Treatment Route:by oral gavage
Treatment Dose:multiple dose 200 ug. Combination treatments: 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29,

Sample Preparation:

Sampleprep ID:SP002154
Sampleprep Summary:Tumor tissue was flash frozen and crushed using a mortar and pestle in liquid nitrogen. To the Eppendorf tubes 150 µL of cold 80 % acetonitrile was added and vortexed quickly. The suspension was transferred to a fresh Eppendorf tube. Another 150 µL portion of extraction solvent was added to the tubes, vortexed and the suspension combined with the first portion. To the combined extraction mixture 75 µL of CHCl3 was added and samples were mixed for 1 h in a cool room to ensure complete extraction. The samples were centrifuged at 4 °C at 14.8 g for 10 min, supernatant transferred to new tubes and evaporated at 20 °C under a stream of nitrogen. BSA assay was performed on the protein pellet by dissolving all protein in equal volume of detergent solution (4 % v/v SDS in water). Dried samples were resolubilized in appropriate volume of CHCl3 : MeOH : H2O (1 : 3 :1, v/v) mixture based on measured protein content, shaken for 15 min at room temperature, centrifuged at 4 °C at 14.8 g for 10 min. 100 µL of the supernatant was transferred to LCMS vials. 10 µL injected for metabolomics analysis.
Processing Storage Conditions:-80℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003365 AN003366
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um) SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units

Chromatography:

Chromatography ID:CH002489
Chromatography Summary:LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ultimate® 3000 RS (Thermo Fisher). Chromatographic separation was performed on a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate and (B) acetonitrile. The gradient program started at 80% B and was reduced to 50% B over 15 min, then reduced from 50% B to 5% B over 3 min, followed by wash with 5% B for another 3 min, and finally 8 min re-equilibration with 80% B. The flow rate was 0.3 mL/min and column compartment temperature was 40ºC. The total run time was 32 min with an injection sample volume of 10 µL.
Methods Filename:Metabolomics_pHILIC_Parkville_v1.meth
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um)
Column Pressure:60 bar at starting conditions
Column Temperature:25 C
Flow Gradient:0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B
Flow Rate:0.3 ml/min
Injection Temperature:4 C
Internal Standard:CAPS, CHAPS, PIPES
Sample Injection:10 uL
Solvent A:20 mM ammonium formate
Solvent B:acetonitrile
Analytical Time:32 min
Capillary Voltage:3.5 kV
Oven Temperature:25 C
Washing Buffer:syringe wash 50% IPA
Weak Wash Solvent Name:10% MeOH
Strong Wash Solvent Name:10% MeOH
Sample Loop Size:25 uL
Sample Syringe Size:25 uL
Chromatography Type:HILIC

MS:

MS ID:MS003132
Analysis ID:AN003365
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C.
Ion Mode:POSITIVE
Capillary Temperature:300 C
Capillary Voltage:4 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501 LCMS data acquisition for untargeted metabolomics analysis.pdf
  
MS ID:MS003133
Analysis ID:AN003366
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C.
Ion Mode:NEGATIVE
Capillary Temperature:300 C
Capillary Voltage:3.5 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501 LCMS data acquisition for untargeted metabolomics analysis.pdf
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