#METABOLOMICS WORKBENCH jer608_20230906_190550 DATATRACK_ID:4291 STUDY_ID:ST002853 ANALYSIS_ID:AN004674 PROJECT_ID:PR001782 VERSION 1 CREATED_ON September 12, 2023, 10:04 am #PROJECT PR:PROJECT_TITLE Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism PR:PROJECT_TITLE (Lipidomics-Human) PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) cells utilize glutamine (Gln) to support PR:PROJECT_SUMMARY proliferation and redox balance. Earlier attempts to inhibit Gln metabolism PR:PROJECT_SUMMARY using glutaminase inhibitors resulted in rapid metabolic reprogramming and PR:PROJECT_SUMMARY therapeutic resistance. We demonstrated that treating PDAC cells with a Gln PR:PROJECT_SUMMARY antagonist, 6-Diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in PR:PROJECT_SUMMARY vitro. In addition, we observed a profound decrease in tumor growth in various PR:PROJECT_SUMMARY in vivo models using DRP-104 (sirpiglenastat), a pro-drug version of DON PR:PROJECT_SUMMARY designed to circumvent DON-associated toxicity. PR:INSTITUTE New York University PR:DEPARTMENT Radiation Oncology PR:LABORATORY Alec C Kimmelman PR:LAST_NAME Encarnacion Rosado PR:FIRST_NAME Joel PR:ADDRESS Smilow Research Building Room 907G New York, NY 10016 PR:EMAIL joel.encarnacion-rosado@nyulangone.org PR:PHONE 646-501-8984 #STUDY ST:STUDY_TITLE Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism ST:STUDY_TITLE (Lipidomics-human) ST:STUDY_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) cells utilize glutamine (Gln) to support ST:STUDY_SUMMARY proliferation and redox balance. Earlier attempts to inhibit Gln metabolism ST:STUDY_SUMMARY using glutaminase inhibitors resulted in rapid metabolic reprogramming and ST:STUDY_SUMMARY therapeutic resistance. Here, we demonstrated that treating PDAC cells with a ST:STUDY_SUMMARY Gln antagonist, 6-Diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in ST:STUDY_SUMMARY vitro. In addition, we observed a profound decrease in tumor growth in various ST:STUDY_SUMMARY in vivo models using DRP-104 (sirpiglenastat), a pro-drug version of DON that ST:STUDY_SUMMARY was designed to circumvent DON associated toxicity. We found that ERK signaling ST:STUDY_SUMMARY is increased as a compensatory mechanism. Combinatorial treatment of DRP-104 and ST:STUDY_SUMMARY Trametinib led to a significant increase in survival in a syngeneic model PDAC. ST:STUDY_SUMMARY These proof-of-concept studies suggested that broadly targeting Gln metabolism ST:STUDY_SUMMARY could provide a therapeutic avenue for PDAC. The combination with an ERK ST:STUDY_SUMMARY signaling pathway inhibitor could further improve the therapeutic outcome. ST:INSTITUTE New York University ST:LAST_NAME Encarnacion Rosado ST:FIRST_NAME Joel ST:ADDRESS Smilow Research Building Room 907G New York, NY 10016 ST:EMAIL joel.encarnacion-rosado@nyulangone.org ST:PHONE 646-501-8984 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #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 - 8T-CNT1 Treatment:Vehicle RAW_FILE_NAME=S19034.mzML SUBJECT_SAMPLE_FACTORS - 8T-CNT2 Treatment:Vehicle RAW_FILE_NAME=S19035.mzML SUBJECT_SAMPLE_FACTORS - 8T-CNT3 Treatment:Vehicle RAW_FILE_NAME=S19036.mzML SUBJECT_SAMPLE_FACTORS - 8T-DON1 Treatment:DON RAW_FILE_NAME=S19037.mzML SUBJECT_SAMPLE_FACTORS - 8T-DON2 Treatment:DON RAW_FILE_NAME=S19038.mzML SUBJECT_SAMPLE_FACTORS - 8T-DON3 Treatment:DON RAW_FILE_NAME=S19039.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples were subjected to an LCMS analysis to detect and identify phospholipid CO:COLLECTION_SUMMARY molecules and quantify the relative levels of identified lipids. A lipid CO:COLLECTION_SUMMARY extraction was carried out on each sample based on the method by Vorkas et. al., CO:COLLECTION_SUMMARY cite#1and#2. The dried samples were resolubilized in 10 μL of a 4:3:1 mixture CO:COLLECTION_SUMMARY (isopropanol:acetonitrile:water) and analyzed by UPLC-MS/MS with a polarity CO:COLLECTION_SUMMARY switching method modified from Vorkas et. al., cite #1. The LC column was a CO:COLLECTION_SUMMARY WatersTM CSH-C18 (2.1 x100 mm, 1.7 μm) coupled to a Dionex Ultimate 3000TM CO:COLLECTION_SUMMARY system and the column oven temperature was set to 55oC for the gradient elution. CO:COLLECTION_SUMMARY The flow rate of 0.3 mL/min was used with the following buffers; A) 60:40 CO:COLLECTION_SUMMARY acetonitrile:water, 10 mM ammonium formate, 0.1% formic acid and B) 90:10 CO:COLLECTION_SUMMARY isopropanol:acetonitrile, 10 mM ammonium formate, 0.1% formic acid. The gradient CO:COLLECTION_SUMMARY profile was as follows; 40-43%B (0-1.25 min), 43-50%B (1.25-2 min), 50-54%B CO:COLLECTION_SUMMARY (2-11 min), 54-70%B (11-12 min), 70-99%B (12-18 min), 70-99%B (18-32min), CO:COLLECTION_SUMMARY 99-40%B (23-24 min), hold 40%B (1 min). Injection volume was set to 1 μL for CO:COLLECTION_SUMMARY all analyses (25 min total run time per injection). MS analyses were carried out CO:COLLECTION_SUMMARY by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer CO:COLLECTION_SUMMARY operating in heated electrospray ionization mode (HESI). Method duration was 20 CO:COLLECTION_SUMMARY min with a polarity switching data-dependent Top 10 method for both positive and CO:COLLECTION_SUMMARY negative modes. Spray voltage for both positive and negative modes was 3.5kV and CO:COLLECTION_SUMMARY capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of CO:COLLECTION_SUMMARY 10, and max spray current of 100 μA. The full MS scan for both polarities CO:COLLECTION_SUMMARY utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 CO:COLLECTION_SUMMARY ms, and the scan range was from 350-2000 m/z. Tandem MS spectra for both CO:COLLECTION_SUMMARY positive and negative mode used a resolution of 15,000, AGC target of 1e5, CO:COLLECTION_SUMMARY maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, CO:COLLECTION_SUMMARY fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies CO:COLLECTION_SUMMARY (nCE) of 10, 35, 80. The minimum AGC target was 5e4 with an intensity threshold CO:COLLECTION_SUMMARY of 1e6. All data were acquired in profile mode. The resulting lipids were CO:COLLECTION_SUMMARY identified by searching the LipidBlast tandem mass spectral library of lipids CO:COLLECTION_SUMMARY cite #3. The top scoring structure match for each data-dependent spectrum was CO:COLLECTION_SUMMARY returned using an in-house script for MSPepSearch_x64. Putative lipids were CO:COLLECTION_SUMMARY sorted from high to low by their reverse dot scores, and duplicate structures CO:COLLECTION_SUMMARY were discarded, retaining only the top-scoring MS2 spectrum and the neutral CO:COLLECTION_SUMMARY chemical formula, detected m/z, and detected polarity (+ or -) of the putative CO:COLLECTION_SUMMARY lipid was recorded. References 1. Vorkas, P. A. et al. Untargeted UPLC-MS CO:COLLECTION_SUMMARY Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to CO:COLLECTION_SUMMARY Cardiovascular Disease. Anal. Chem. 87, 4184–4193 (2015). 2. Vorkas, P. A. et CO:COLLECTION_SUMMARY al. Metabolic phenotyping of atherosclerotic plaques reveals latent associations CO:COLLECTION_SUMMARY between free cholesterol and ceramide metabolism in atherogenesis. J. Proteome CO:COLLECTION_SUMMARY Res. 14, 1389–1399 (2015). 3. https://www.ncbi.nlm.nih.gov/pubmed/23817071 CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY PaTu-8988T cells were plated in a six-well plate at 1.5x10^6 cells/well and TR:TREATMENT_SUMMARY allowed to attach overnight in DMEM. Next, cells were washed with PBS twice and TR:TREATMENT_SUMMARY cultured for 24 hours in DMEM supplemented with 10% dialyzed serum was added. TR:TREATMENT_SUMMARY Cells were pre-treated with DON (25µM) overnight, media was removed and washed TR:TREATMENT_SUMMARY with PBS. Then, cells were frozen in -80C and until metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were analyzed with the global and untargeted lipidomics LCMS assays SP:SAMPLEPREP_SUMMARY after scaling the lipid extraction to a measured aliquot (~5e6/mL) for each of SP:SAMPLEPREP_SUMMARY the 6 samples. SPLASH® LIPIDOMIX® Mass Spec Standard was used as an extraction SP:SAMPLEPREP_SUMMARY standard. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME WatersTM CSH-C18 (2.1 x100 mm, 1.7 μm) CH:SOLVENT_A The flow rate of 0.3 mL/min was used with the following buffers; A) 60:40 CH:SOLVENT_A acetonitrile:water, 10 mM ammonium formate, 0.1% formic acid and B) 90:10 CH:SOLVENT_A isopropanol:acetonitrile, 10 mM ammonium formate, 0.1% formic acid. The gradient CH:SOLVENT_A profile was as follows; 40-43%B (0-1.25 min), 43-50%B (1.25-2 min), 50-54%B CH:SOLVENT_A (2-11 min), 54-70%B (11-12 min), 70-99%B (12-18 min), 70-99%B (18-32min), CH:SOLVENT_A 99-40%B (23-24 min), hold 40%B (1 min) CH:SOLVENT_B acetonitrile CH:FLOW_GRADIENT The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B (30-31 min), CH:FLOW_GRADIENT 80-80%B (31-42 min) CH:FLOW_RATE 1 μL for all analyses (25 min total run time per injection) CH:COLUMN_TEMPERATURE 55C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS MS analyses were carried out by coupling the LC system to a Thermo Q Exactive MS:MS_COMMENTS HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). MS:MS_COMMENTS Method duration was 20 min with a polarity switching data-dependent Top 10 MS:MS_COMMENTS method for both positive and negative modes. Spray voltage for both positive and MS:MS_COMMENTS negative modes was 3.5kV and capillary temperature was set to 320C with a sheath MS:MS_COMMENTS gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS MS:MS_COMMENTS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 MS:MS_COMMENTS and a maximum IT of 100 ms, and the scan range was from 350-2000 m/z. Tandem MS MS:MS_COMMENTS spectra for both positive and negative mode used a resolution of 15,000, AGC MS:MS_COMMENTS target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation MS:MS_COMMENTS offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized MS:MS_COMMENTS collision energies (nCE) of 10, 35, 80. The minimum AGC target was 5e4 with an MS:MS_COMMENTS intensity threshold of 1e6. All data were acquired in profile mode. The MS:MS_COMMENTS resulting lipids were identified by searching the LipidBlast tandem mass MS:MS_COMMENTS spectral library of lipids. The top scoring structure match for each MS:MS_COMMENTS data-dependent spectrum was returned using an in-house script for MS:MS_COMMENTS MSPepSearch_x64. Putative lipids were sorted from high to low by their reverse MS:MS_COMMENTS dot scores, and duplicate structures were discarded, retaining only the MS:MS_COMMENTS top-scoring MS2 spectrum and the neutral chemical formula, detected m/z, and MS:MS_COMMENTS detected polarity (+ or -) of the putative lipid was recorded. For feature-based MS:MS_COMMENTS analysis, an in-house python script (Ungrid) was used to detect MS1 peaks across MS:MS_COMMENTS all samples using the following parameters: a m/z discrimination threshold of 20 MS:MS_COMMENTS ppm, a minimum peak intensity of 1e5, a minimum signal-to-noise ratio of 10, and MS:MS_COMMENTS a retention time threshold of 2 min. Metabolite and feature peaks extracted in MS:MS_COMMENTS this manner were defined by either the detected feature m/z or the theoretical MS:MS_COMMENTS m/z of the expected ion type for the standard in the library (e.g., [M+H]+). The MS:MS_COMMENTS following parameters were applied: a ±5 part-per-million (ppm) tolerance, an MS:MS_COMMENTS initial retention time search window of ±0.5 min across all samples, and a MS:MS_COMMENTS ±7.5 s peak apex retention time tolerance within individual samples. An MS:MS_COMMENTS in-house statistical pipeline, Metabolize (version 1.0), was used to process the MS:MS_COMMENTS resulting data matrix of metabolite intensities for all samples and blank MS:MS_COMMENTS controls. A final peak detection was calculated based on a signal-to-noise ratio MS:MS_COMMENTS (S/N) or 3× blank controls with a floor of 1e5 (arb. units). The threshold MS:MS_COMMENTS value was input for any sample where the calculated peak intensity was lower MS:MS_COMMENTS than the blank threshold for any statistical comparisons. The “-” is for MS:MS_COMMENTS not-detected peaks. After the correction, zero values were input for MS:MS_COMMENTS non-detected values instead of the blank threshold to avoid false positive. MS:MS_RESULTS_FILE ST002853_AN004674_Results.txt UNITS:ion counts Has m/z:Yes Has RT:Yes RT units:Minutes #END