Summary of Study ST001838

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 PR001160. The data can be accessed directly via it's Project DOI: 10.21228/M8RT34 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 IDST001838
Study TitleReversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies
Study SummaryGlioblastoma is an aggressive brain malignancy with a dismal prognosis. With emerging evidence that disproves the immune privileged environment in the brain, there is much interest in examining various immunotherapy strategies to treat these incurable cancers. Unfortunately, to date, clinical studies investigating immunotherapy regimens have not provided much evidence of efficacy, leading to questions about the suitability of immunotherapy strategies for these tumors. Inadequate inherent populations of lymphocytes in tumor (TILs) and limited trafficking of systemic circulating T cells into the central nervous system (CNS) likely contribute to the poor response to immunotherapy treatment for primary CNS cancers. This paucity of TILs is in concert with the finding of epigenetic silencing of genes that promote immune cell movement (chemotaxis) to the tumor. In this study we evaluated the ability of GSK126, a blood-brain barrier permeable small molecule inhibitor of EZH2, to reverse the epigenetic silencing of chemokines like CXCL9 and CXCL10. When combined with anti-PD-1 treatment, these IFN driven chemokines promote T cell infiltration, resulting in decreased tumor growth and enhanced survival in immunocompetent murine sub-cutaneous and intracranial tumor syngeneic models of GBM. Examination of the tumor micro-environment revealed that the decrease in tumor growth in the mice treated with the drug combination was accompanied by increased tumor CD8 T cell infiltration along with higher IFN expression. Additionally, a significant increase in CXCR3+ T cells in the draining lymph nodes was also found. Taken together, our data suggests that in glioblastoma, epigenetic modulation using GSK126 could improve current immunotherapy strategies by reversing the epigenetic changes that enable immune cell evasion leading to enhanced immune cell trafficking to the tumor.
Institute
National Cancer Institute
DepartmentNeuro-Oncology Branch
LaboratoryCancer Metabolism
Last NameDowdy
First NameTyrone
Address37 convent dr, Bldg 37 rm 1142
Emailtyrone.dowdy@nih.gov
Phone2407607066
Submit Date2021-06-11
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2021-06-30
Release Version1
Tyrone Dowdy Tyrone Dowdy
https://dx.doi.org/10.21228/M8RT34
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001160
Project DOI:doi: 10.21228/M8RT34
Project Title:Reversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies
Project Summary:Glioblastoma is an aggressive brain malignancy with a dismal prognosis. With emerging evidence that disproves the immune privileged environment in the brain, there is much interest in examining various immunotherapy strategies to treat these incurable cancers. Unfortunately, to date, clinical studies investigating immunotherapy regimens have not provided much evidence of efficacy, leading to questions about the suitability of immunotherapy strategies for these tumors. Inadequate inherent populations of lymphocytes in tumor (TILs) and limited trafficking of systemic circulating T cells into the central nervous system (CNS) likely contribute to the poor response to immunotherapy treatment for primary CNS cancers. This paucity of TILs is in concert with the finding of epigenetic silencing of genes that promote immune cell movement (chemotaxis) to the tumor. In this study we evaluated the ability of GSK126, a blood-brain barrier permeable small molecule inhibitor of EZH2, to reverse the epigenetic silencing of chemokines like CXCL9 and CXCL10. When combined with anti-PD-1 treatment, these IFN driven chemokines promote T cell infiltration, resulting in decreased tumor growth and enhanced survival in immunocompetent murine sub-cutaneous and intracranial tumor syngeneic models of GBM. Examination of the tumor micro-environment revealed that the decrease in tumor growth in the mice treated with the drug combination was accompanied by increased tumor CD8 T cell infiltration along with higher IFN expression. Additionally, a significant increase in CXCR3+ T cells in the draining lymph nodes was also found. Taken together, our data suggests that in glioblastoma, epigenetic modulation using GSK126 could improve current immunotherapy strategies by reversing the epigenetic changes that enable immune cell evasion leading to enhanced immune c-ll trafficking to the tumor.
Institute:National Cancer Institute
Department:Neuro-Oncology Branch
Laboratory:Cancer Metabolism
Last Name:Dowdy
First Name:Tyrone
Address:37 convent dr, Bldg 37 rm 1142
Email:tyrone.dowdy@nih.gov
Phone:2407607066
Contributors:Nivedita M. Ratnam1, Heather M. Sonnemann1, Stephen C. Frederico1, Huanwen Chen1, Marsha-Kay N.D. Hutchinson1, Tyrone Dowdy1, Caitlin M. Reid1, Jinkyu Jung1, Wei Zhang1, Hua Song1, Meili Zhang1, Dionne Davis1, Mioara Larion1, Amber J. Giles1 and Mark R. Gilbert1*.

Subject:

Subject ID:SU001915
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:albino C57BL/6
Age Or Age Range:6-8 weeks
Gender:Female
Animal Animal Supplier:Jackson Laboratories (Bar Harbor, ME)
Animal Housing:NCI-Bethesda Animal Facility

Factors:

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

mb_sample_id local_sample_id Treatment Gsk Exposure Time (h)
SA171094blank_S22.dblank -
SA171095Blank_S14.dblank -
SA171096blank_Sr62.dblank -
SA170972SubCu7_Tum_g1_10GSK 10
SA170973SubCu7_DLN_g1_10GSK 10
SA170974SubCu7_NLN_g1_10GSK 10
SA170975SubCu8_NLN_g1_10GSK 10
SA170976SubCu8_DLN_g1_10GSK 10
SA170977SubCu8_Tum_g1_10GSK 10
SA170978LLN_g1 _Intra8_10hGSK 10.0
SA170979LLN_g1 _Intra7_10hGSK 10.0
SA170980SER_g1 _Intra8_10hGSK 10.0
SA170981SER_g1 _Intra7_10hGSK 10.0
SA170982SER_g1 _Subcutaneous8_10hGSK 10.0
SA170983SER_g1 _Subcutaneous7_10hGSK 10.0
SA170984BRN_g1 _Intra8_10hGSK 10.0
SA170985BRN_g1 _Intra7_10hGSK 10.0
SA170986SER_g1 _Subcutaneous3_2hGSK 2.0
SA170987SER_g1 _Subcutaneous2_2hGSK 2.0
SA170988SER_g1 _Subcutaneous1_2hGSK 2.0
SA170989SER_g1 _Intra3_2hGSK 2.0
SA170990LLN_g1 _Intra1_2hGSK 2.0
SA170991LLN_g1 _Intra3_2hGSK 2.0
SA170992SER_g1 _Intra1_2hGSK 2.0
SA170993SER_g1 _Intra2_2hGSK 2.0
SA170994LLN_g1 _Intra2_2hGSK 2.0
SA170995BRN_g1 _Intra2_2hGSK 2.0
SA170996BRN_g1 _Intra3_2hGSK 2.0
SA170997BRN_g1 _Intra1_2hGSK 2.0
SA170998SubCu1_Tum_g1_2hGSK 2.00
SA170999SubCu1_NLN_g1_2hGSK 2.00
SA171000SubCu3_Tum_g1_2hGSK 2.00
SA171001SubCu2_NLN_g1_2hGSK 2.00
SA171002SubCu2_Tum_g1_2hGSK 2.00
SA171003SubCu2_DLN_g1_2hGSK 2.00
SA171004SubCu3_NLN_g1_2hGSK 2.00
SA171005SubCu1_DLN_g1_2hGSK 2.00
SA171006SubCu3_DLN_g1_2hGSK 2.00
SA171007SER_g1 _Subcutaneous5_6hGSK 6.0
SA171008BRN_g1 _Intra4_6hGSK 6.0
SA171009SER_g1 _Subcutaneous4_6hGSK 6.0
SA171010SER_g1 _Subcutaneous6_6hGSK 6.0
SA171011BRN_g1 _Intra5_6hGSK 6.0
SA171012LLN_g1 _Intra4_6hGSK 6.0
SA171013LLN_g1 _Intra6_6hGSK 6.0
SA171014SER_g1 _Intra5_6hGSK 6.0
SA171015SER_g1 _Intra4_6hGSK 6.0
SA171016SER_g1 _Intra6_6hGSK 6.0
SA171017SubCu6_NLN_g1_6hGSK 6.00
SA171018SubCu6_Tum_g1_6hGSK 6.00
SA171019SubCu5_NLN_g1_6hGSK 6.00
SA171020SubCu4_DLN_g1_6hGSK 6.00
SA171021SubCu5_DLN_g1_6hGSK 6.00
SA171022SubCu4_NLN_g1_6hGSK 6.00
SA171023SubCu4_Tum_g1_6hGSK 6.00
SA171024SubCu5_Tum_g1_6hGSK 6.00
SA171025SubCu6_DLN_g1_6hGSK 6.00
SA171026QCpool g1 D_NLN_subcu_028.dQC -
SA171027QCpool g1 D_NLN_subcu_037.dQC -
SA171028QCpool g1 D_NLN_subcu_081.dQC -
SA171029QCpool g1 D_NLN_subcu_012.dQC -
SA171030QCpool g1 SubTum_038.dQC -
SA171031QCpool g1 D_NLN_subcu_135.dQC -
SA171032QCpool g1 SubTum_136.dQC -
SA171033QCpool g1 D_NLN_subcu_055.dQC -
SA171034QcSer_S44.dQC -
SA171035QcSer_S24.dQC -
SA171036QCpool g1 SubTum_082.dQC -
SA171037QCpool g1 SubTum_056.dQC -
SA171038QcBRN_S45.dQC -
SA171039QcLLN_L1002.dQC -
SA171040QcBRN_Sr111.dQC -
SA171041QcBRN_Sr91.dQC -
SA171042QcBRN_Sr65.dQC -
SA171043SER_ctrl_g2_Intra11_2hVehicle 0.0
SA171044SER_ctrl_g2_Intra12_6hVehicle 0.0
SA171045SER_ctrl_g2_Intra13_6hVehicle 0.0
SA171046SER_ctrl_g2_Intra10_2hVehicle 0.0
SA171047SER_ctrl_g2_Subcutaneous14_6hVehicle 0.0
SA171048SER_ctrl_g2_Subcutaneous13_6hVehicle 0.0
SA171049SER_ctrl_g2_Intra14_6hVehicle 0.0
SA171050SER_ctrl_g2_Subcutaneous15_10hVehicle 0.0
SA171051SER_ctrl_g2_Subcutaneous16_10hVehicle 0.0
SA171052SER_ctrl_g2 _Intra9_2hVehicle 0.0
SA171053BRN_ctrl_g2_Intra13_6hVehicle 0.0
SA171054BRN_ctrl_g2_Intra12_6hVehicle 0.0
SA171055BRN_ctrl_g2_Intra11_2hVehicle 0.0
SA171056BRN_ctrl_g2_Intra10_2hVehicle 0.0
SA171057BRN_ctrl_g2_Intra14_6hVehicle 0.0
SA171058BRN_ctrl_g2_Intra15_10hVehicle 0.0
SA171059SER_ctrl_g2_Intra16_10hVehicle 0.0
SA171060SER_ctrl_g2_Subcutaneous10_2hVehicle 0.0
SA171061BRN_ctrl_g2_Intra16_10hVehicle 0.0
SA171062SER_ctrl_g2_Intra15_10hVehicle 0.0
SA171063SER_ctrl_g2_Subcutaneous12_6hVehicle 0.0
SA171064LLN_ctrl_g2_Intra13_6hVehicle 0.0
SA171065LLN_ctrl_g2_Intra12_6hVehicle 0.0
SA171066LLN_ctrl_g2_Intra15_10hVehicle 0.0
SA171067LLN_ctrl_g2_Intra16_10hVehicle 0.0
SA171068LLN_ctrl_g2 _Intra9_2hVehicle 0.0
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Collection:

Collection ID:CO001908
Collection Summary:All animal experiments were performed following the guidelines stipulated by the NCI-Bethesda Animal Care and Use Committee. All murine studies were performed using female albino C57BL/6 mice, 6-8 weeks of age, procured from Jackson Laboratories (Bar Harbor, ME). For sub-cutaneous tumor studies, 6x10^6 cells of stably transduced CT2A glioma cells with mCherry-firefly luciferase were injected in 100 μL PBS. For intracranial tumor studies, 1x10^2 CT2A cells with mCherry- firefly luciferase were injected in 2uL PBS. GSK126 for in vivo studies was obtained from the NCI- Drug Synthesis and Chemistry Branch and dissolved in 20% SBE-Cyclodextrin (MedChemExpress, HY-17031) pH 4-4.5 with 1N acetic acid. Vehicle was 20% SBE-Cyclodextrin pH 4-4.5 with 1N acetic acid. Water-soluble dexamethasone (Sigma Aldrich; D2915) was administered at 1mg/kg/day also by intraperitoneal injection. Anti PD-1(InVivoMAb; BE0146) or isotype control, rat IgG2a (InVivoMAb; BE0089) were also injected intraperitoneally. Subcutaneous tumor growth was measured using calipers and thereafter tumor volume was calculated using the formula for the volume of an ellipsoid given below. In the case of intracranial tumors, tumor growth was measured using the luminescence reader IVIS Ilumina and analyzed using LivingImage Software. Samples were collected from mice with three biological replicates. Serum (50 µL) was transferred to 200 μL ice-chilled (4°C) MilliQ H2O. Tissue (~16 mg) was measured from subcutaneous and intracranial samples followed by addition of 250 μL MilliQ H2O. Then, samples were sonicated at 40 amps (~30 s) until homogeneous. 80 μL of at 0.150 µg/mL debrisoquine in 60% methanol (MeOH)/40% water(aq) reagent was added. 500 μL chilled (-20°C) MeOH was added, vortexed (med) and incubated 15 min on ice. 250 μL chilled (-20°C) Chloroform was added, vortexed (high) and incubated 20 min in ice on rotating mixer. Mixture was centrifuged (13,000x g) for 18 min at 4°C. 705 μL of hydrophilic upper layer was aspirated and transferred to separate 1.5 mL microtubes, dried to completion under N2 gas sample concentrator, and stored at -80°C until LC/MS quantification of GSK126.
Sample Type:Tumor cells
Collection Method:Tumor Tissue/Serum Extract
Collection Location:Intracranial/Subcutaneous/Serum from Mice
Collection Frequency:Timepoints
Collection Duration:0, 2h, 6h and 10h
Volumeoramount Collected:Serum (50 µL)/Tumor (~16 mg)
Storage Conditions:-80℃
Collection Vials:15 mL
Storage Vials:1.7 mL
Collection Tube Temp:On Ice
Additives:Extraction reagent

Treatment:

Treatment ID:TR001928
Treatment Summary:Extracts of Gracilaria edulis were prepared through two different approaches, namely, sequential and direct, following the procedure of Subermaniam et al. (2020). For the sequential process, the solvents were used in the order of increasing polarity viz. ethyl acetate < acetone. For the direct extracts, ethyl acetate and acetone were used separately.

Sample Preparation:

Sampleprep ID:SP001921
Sampleprep Summary:Serum (50 µL) was transferred to 200 μL ice-chilled (4°C) MilliQ H2O. Tissue (~16 mg) was measured from subcutaneous and intracranial samples followed by addition of 250 μL MilliQ H2O. Then, samples were sonicated at 40 amps (~30 s) until homogeneous. 80 μL of at 0.150 µg/mL debrisoquine in 60% methanol (MeOH)/40% water(aq) reagent was added. 500 μL chilled (-20°C) MeOH was added, vortexed (med) and incubated 15 min on ice. 250 μL chilled (-20°C) Chloroform was added, vortexed (high) and incubated 20 min in ice on rotating mixer. Mixture was centrifuged (13,000x g) for 18 min at 4°C. 705 μL of hydrophilic upper layer was aspirated and transferred to separate 1.5 mL microtubes, dried to completion under N2 gas sample concentrator, and stored at -80°C
Sampleprep Protocol ID:Extraction
Processing Storage Conditions:-80℃
Extraction Method:LCMS Extraction
Extract Cleanup:Bligh-Dyer biphasic separation, discard protein disk and dried to completion under N2 gas
Sample Resuspension:80 uL 60% MeOH (aq)
Sample Spiking:Internal standard 0.150 µg/mL debrisoquine(IS) was added to each

Combined analysis:

Analysis ID AN002980
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 1290 Infinity II
Column Agilent AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6545
Ion Mode POSITIVE
Units ng

Chromatography:

Chromatography ID:CH002209
Chromatography Summary:Samples were injected at 8 µL over an 8.3 min gradient on the AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column at 35°C with a flow rate of 0.220 mL/min. The LC gradient only utilized LC/MS grade reagents when preparing mobile phases, A (88:12 H2O/acetonitrile (ACN) and B 90% ACN (aq). Both mobile phases were composed with 10 mM ammonium acetate and titrated to pH 6.85 using formic acid and ammonium hydroxide. The LC gradient was initially 100% B for 0.25 min and then ramped to 55% B at 2.5 min; 49% B at 4.5 min; 35% B at 5.5 min; 20% B at 6 min; held for 0.5 min; 15% B at 7 min; 100%B at 8.3 min followed by equilibration for 1.2 min.
Instrument Name:Agilent 1290 Infinity II
Column Name:Agilent AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column
Column Pressure:600 bar
Column Temperature:35°C
Flow Gradient:The LC gradient was initially 100% B for 0.25 min and then ramped to 55% B at 2.5 min; 49% B at 4.5 min; 35% B at 5.5 min; 20% B at 6 min; held for 0.5 min; 15% B at 7 min; 100%B at 8.3 min followed by equilibration for 1.2 min.
Flow Rate:0.220 mL/min
Injection Temperature:4°C
Internal Standard:Continuous accurate mass correction was achieved by infusing proprietary Agilent Technologies API-TOF reference mass standard solution
Retention Time:2.5-2.7 min
Sample Injection:8 uL
Sampling Cone:2kV
Solvent A:88% water/12% acetonitrile; 10 mM ammonium acetate, pH 6.85
Solvent B:90% acetonitrile/10% water; 10 mM ammonium acetate, pH 6.85
Analytical Time:8.3 min
Capillary Voltage:3 kV
Sheath Liquid:N2
Chromatography Type:HILIC

MS:

MS ID:MS002770
Analysis ID:AN002980
Instrument Name:Agilent 6545
Instrument Type:QTOF
MS Type:ESI
MS Comments:MS/MS Using Masshunter Qtof Quant-My-Way 10.0 software, GSK126 was detected at elution time 2.6 min using precursor ion m/z 527.3129 and transition m/z 375.2183 generated via N2 gas collision-induced fragmentation (CID) at a collision energy (CE) of 12 V. Internal standard (IS) debrisoquine detected at elution time of 2.5 mins with precursor m/z 176.1182 and transition m/z 134.0964 generated at CE 12V.
Ion Mode:POSITIVE
Capillary Temperature:325°C
Capillary Voltage:3kV
Collision Energy:12V
Collision Gas:N2
Dry Gas Flow:9 L/min
Dry Gas Temp:250°C
Fragment Voltage:100 V
Fragmentation Method:MS/MS
Ion Source Temperature:325°C
Mass Accuracy:2 kDa
Source Temperature:325°C
Cdl Temperature:RT
Desolvation Temperature:325°C
Nebulizer:45 psig
Octpole Voltage:750V
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