#METABOLOMICS WORKBENCH Ofelia4m1_20170824_022855 DATATRACK_ID:1225 STUDY_ID:ST000898 ANALYSIS_ID:AN001460 PROJECT_ID:PR000624 VERSION 1 CREATED_ON November 13, 2017, 1:31 pm #PROJECT PR:PROJECT_TITLE TAp73 is a marker of glutamine addiction in medulloblastoma PR:PROJECT_TYPE siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control PR:PROJECT_TYPE siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 PR:PROJECT_TYPE according to the supplier’s protocol for 48 hours PR:PROJECT_SUMMARY Metabolically-targeted therapies hold the promise of offering an effective and PR:PROJECT_SUMMARY less toxic treatment for tumours including medulloblastoma, the most common PR:PROJECT_SUMMARY malignant brain tumour of childhood. Current treatment relies on the sensitivity PR:PROJECT_SUMMARY of these tumours to DNA damage that was discovered more than 50 years ago. PR:PROJECT_SUMMARY Finding new tumour-specific susceptibilities to complement sensitivity to DNA PR:PROJECT_SUMMARY damage is key to developing new more effective adjuvant therapies. The specific PR:PROJECT_SUMMARY metabolic program of tumours is an attractive vulnerability, as restriction diet PR:PROJECT_SUMMARY are low cost and easy to implement. Here, we present compelling pre-clinical PR:PROJECT_SUMMARY evidence that glutamine restriction diet can be used as an adjuvant treatment PR:PROJECT_SUMMARY for p73-expressing medulloblastoma. PR:INSTITUTE Queen Mary University of London PR:DEPARTMENT Blizard Institute PR:LABORATORY Centre for Genomics and Child Health PR:LAST_NAME Marino PR:FIRST_NAME Silvia PR:ADDRESS 4 Newark Street, E1 2AT, London PR:EMAIL s.marino@qmul.ac.uk PR:PHONE +44 20 7882 2360 PR:FUNDING_SOURCE Children with Cancer UK fellowship (Reference Nº2014/178) #STUDY ST:STUDY_TITLE TAp73 is a marker of glutamine addiction in medulloblastoma ST:STUDY_TYPE siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control ST:STUDY_TYPE siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 ST:STUDY_TYPE according to the supplier’s protocol for 48 hours ST:STUDY_SUMMARY Metabolically-targeted therapies hold the promise of offering an effective and ST:STUDY_SUMMARY less toxic treatment for tumours including medulloblastoma, the most common ST:STUDY_SUMMARY malignant brain tumour of childhood. Current treatment relies on the sensitivity ST:STUDY_SUMMARY of these tumours to DNA damage that was discovered more than 50 years ago. ST:STUDY_SUMMARY Finding new tumour-specific susceptibilities to complement sensitivity to DNA ST:STUDY_SUMMARY damage is key to developing new more effective adjuvant therapies. The specific ST:STUDY_SUMMARY metabolic program of tumours is an attractive vulnerability, as restriction diet ST:STUDY_SUMMARY are low cost and easy to implement. Here, we present compelling pre-clinical ST:STUDY_SUMMARY evidence that glutamine restriction diet can be used as an adjuvant treatment ST:STUDY_SUMMARY for p73-expressing medulloblastoma. ST:INSTITUTE Queen Mary University of London ST:DEPARTMENT Blizard Institute ST:LABORATORY Centre for Genomics and Child Health ST:LAST_NAME Marino ST:FIRST_NAME Silvia ST:ADDRESS 4 Newark Street, E1 2AT, London ST:EMAIL s.marino@qmul.ac.uk ST:PHONE +44 20 7882 2360 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 18 ST:STUDY_COMMENTS We include 3 biological replicate with 3 technical replicates for each ST:STUDY_COMMENTS condition. #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS Daoy (ATCC® HTB-186™) SU:SUBJECT_COMMENTS cancer cell line #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Control 1 Control_1.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 1 Control_1.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 1 Control_1.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 2 Control_2.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 2 Control_2.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 2 Control_2.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 3 Control_3.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 3 Control_3.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 3 Control_3.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 4 Control_4.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 4 Control_4.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 4 Control_4.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 5 Control_5.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 5 Control_5.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 5 Control_5.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 6 Control_6.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 6 Control_6.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 6 Control_6.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 7 Control_7.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 7 Control_7.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 7 Control_7.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 8 Control_8.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 8 Control_8.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 8 Control_8.3 treatment:Control SUBJECT_SAMPLE_FACTORS Control 9 Control_9.1 treatment:Control SUBJECT_SAMPLE_FACTORS Control 9 Control_9.2 treatment:Control SUBJECT_SAMPLE_FACTORS Control 9 Control_9.3 treatment:Control SUBJECT_SAMPLE_FACTORS Si p73 1 Si p73_1.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 1 Si p73_1.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 1 Si p73_1.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 2 Si p73_2.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 2 Si p73_2.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 2 Si p73_2.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 3 Si p73_3.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 3 Si p73_3.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 3 Si p73_3.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 4 Si p73_4.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 4 Si p73_4.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 4 Si p73_4.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 5 Si p73_5.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 5 Si p73_5.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 5 Si p73_5.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 6 Si p73_6.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 6 Si p73_6.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 6 Si p73_6.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 7 Si p73_7.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 7 Si p73_7.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 7 Si p73_7.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 8 Si p73_8.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 8 Si p73_8.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 8 Si p73_8.3 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 9 Si p73_9.1 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 9 Si p73_9.2 treatment:Si p73 SUBJECT_SAMPLE_FACTORS Si p73 9 Si p73_9.3 treatment:Si p73 #COLLECTION CO:COLLECTION_SUMMARY After 48 hours of transfection the cells were washed three times with ice-cold CO:COLLECTION_SUMMARY PBS (3X 5mL) and the cells were collected using a cell scraper. CO:SAMPLE_TYPE Cell CO:COLLECTION_METHOD Scraper CO:TISSUE_CELL_IDENTIFICATION Daoy cell CO:TISSUE_CELL_QUANTITY_TAKEN protein normalization #TREATMENT TR:TREATMENT_SUMMARY siRNA constructs targeting p73 (ID: 2671-AMBION) and a non-targeting control TR:TREATMENT_SUMMARY siRNA (scramble) were transfected with 10 pM siRNA with lipofectamine 3000 TR:TREATMENT_SUMMARY according to the supplier’s protocol for 48 hours TR:CELL_GROWTH_CONTAINER plate TR:CELL_GROWTH_CONFIG attached TR:CELL_GROWTH_RATE 36 hours TR:CELL_INOC_PROC lipofectamine 3000 TR:CELL_MEDIA DMEM + GlutaMAX medium (Gibco), supplemented with 10% v/v foetal bovine serum TR:CELL_MEDIA (FBS, Gibco) and penicillin/streptomycin (1 U/ml, Gibco), TR:CELL_ENVIR_COND 37°C in humidified 5% CO2 TR:CELL_HARVESTING After 48 h of transfection the cells were washed three times with ice-cold PBS TR:CELL_HARVESTING (3x5 mL) and the celles were collected using a cell scraper. Ice-cold water (3 TR:CELL_HARVESTING mL,sterile, MilliQ) was added to the samples. The suspension was transferred to TR:CELL_HARVESTING a 15 mL polypropylene tube and then snap-frozen in liquid N2 and stored on ice TR:CELL_HARVESTING for 5 min. The cells were lysed by two freeze-thaw cycles where the cells were TR:CELL_HARVESTING thawed at 37°C in a water bath and frozen using liquid N2. Subsequently, the TR:CELL_HARVESTING cells were sonicated in a TR:CELL_PCT_CONFLUENCE 80-90% TR:CELL_MEDIA_LASTCHANGED 48 hours #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples were thawed at room temperature and subjected to centrifugation for SP:SAMPLEPREP_SUMMARY 17 min at 3000 rpm and 4°C. A quality control (QC) sample was created by SP:SAMPLEPREP_SUMMARY pooling an equal volume from all samples. The aqueous supernatants were SP:SAMPLEPREP_SUMMARY transferred to clean extraction tubes followed by addition of chloroform and SP:SAMPLEPREP_SUMMARY methanol for the final proportion 2.85:4:4 water:methanol:chloroform. The SP:SAMPLEPREP_SUMMARY extraction tubes were gently vortexed and then stored at 8°C for 20 min prior SP:SAMPLEPREP_SUMMARY to centrifugation for 20 min at 3000 rpm and 4°C. The aqueous phases were SP:SAMPLEPREP_SUMMARY recovered and evaporated to dryness at 40°C under N2. All samples were stored SP:SAMPLEPREP_SUMMARY at -80°C after evaporation. Prior to analysis the samples were reconstituted in SP:SAMPLEPREP_SUMMARY acetonitrile:Milli-Q water 90:10 SP:PROCESSING_METHOD lysis, freeze thaw cycles and sonication (see cell harvesting) SP:PROCESSING_STORAGE_CONDITIONS Room temperature and 8°C during extraction, -80°C before and after extraction SP:EXTRACTION_METHOD Liquid Liquid Extraction using CHCl3:MeOH:H2O 4:4:2.85 SP:EXTRACT_STORAGE Extracts were stored in -80°C after evaporation SP:SAMPLE_RESUSPENSION Acetonitrile:Milli-Q water 90:10 SP:CELL_TYPE Daoy cells #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The samples were analyzed on a Acquity UPLC I-class system from Waters. A CH:CHROMATOGRAPHY_SUMMARY HILIC-Amide column (1.7 μm, i.d. 2.1x50 mm) from Waters was used for sample CH:CHROMATOGRAPHY_SUMMARY separation and a non-linear gradient elution profile from 100 % A to 100 % B was CH:CHROMATOGRAPHY_SUMMARY used. Mobile phase A consisted of 90:10 acetonitrile/water with 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate and 0.1% FA while mobile phase B consisted of 50:50 acetonitrile/ water CH:CHROMATOGRAPHY_SUMMARY with 10 mM ammonium formate and 0.1% FA. The flow rate was 0.3 ml/min, the CH:CHROMATOGRAPHY_SUMMARY column temperature 40°C and the injection volume 5 µL. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity BEH Amide (50 x 2.1mm, 1.7um) CH:FLOW_GRADIENT Non-linear gradient elution profile from 100 % A to 100 % B was used. In detail; CH:FLOW_GRADIENT 100% A was kept for 0.5 min then decreased non-linearly (slope-factor 8 in CH:FLOW_GRADIENT MassLynx) over 12.5 min to 100% B, 100% B was held for 3 min followed by 7 min CH:FLOW_GRADIENT at 100 % A to re-equilibrate the column for a total run-time of 23 min. CH:FLOW_RATE 0.3 ml/min CH:COLUMN_TEMPERATURE 40°C CH:SOLVENT_A 90:10 acetonitrile/water with 10 mM ammonium formate and 0.1% FA CH:SOLVENT_B 50:50 acetonitrile/ water with 10 mM ammonium formate and 0.1% FA CH:INJECTION_TEMPERATURE 4°C CH:SAMPLE_INJECTION 5 μl CH:ANALYTICAL_TIME 23 min total sample run-time CH:RANDOMIZATION_ORDER All samples were analyzed in a randomized order with three QC injections CH:RANDOMIZATION_ORDER interspaced every eleventh injection #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Analytical Pharmaceutical Chemistry, Department of Medicinal chemistry, Uppsala AN:LABORATORY_NAME University AN:OPERATOR_NAME Ida Erngren #MS MS:INSTRUMENT_NAME Waters Synapt G2 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The raw data was converted to NetCDF files using the software DataBridge MS:MS_COMMENTS (Masslynx version 4.1, Waters). Peak detection and retention time alignment was MS:MS_COMMENTS performed using the R based software XCMS (Smith et al. 2006). The centWave MS:MS_COMMENTS function was used for peak detection and the function parameters were set as MS:MS_COMMENTS follows; the maximal deviation in m/z between scans was set to 8 ppm, the MS:MS_COMMENTS maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal MS:MS_COMMENTS to noise ratio cutoff was set to 10. Retention time correction was performed MS:MS_COMMENTS using the “obiwarp” function. MS:CAPILLARY_TEMPERATURE 500°C MS:CAPILLARY_VOLTAGE 1 kV MS:COLLISION_ENERGY 20- 45 eV collision ramp, MSE acquistion MS:SOURCE_TEMPERATURE 120°C MS:DATAFORMAT The raw data was converted to NetCDF files using the software DataBridge MS:DATAFORMAT (Masslynx version 4.1, Waters). Peak detection and retention time alignment was MS:DATAFORMAT performed using the R based software XCMS (Smith et al. 2006). The centWave MS:DATAFORMAT function was used for peak detection and the function parameters were set as MS:DATAFORMAT follows; the maximal deviation in m/z between scans was set to 8 ppm, the MS:DATAFORMAT maximal and minimal peakwidth was set to 5 and 25 s respectively and the signal MS:DATAFORMAT to noise ratio cutoff was set to 10. Retention time correction was performed MS:DATAFORMAT using the “obiwarp” function. The resulting dataset was exported to MS:DATAFORMAT Microsoft Excel and all features with a retention time less than 45 s were MS:DATAFORMAT removed. The data was normalized using median fold change. After normalization, MS:DATAFORMAT all features with coefficient of variance (CV) >30 % in the QC samples were MS:DATAFORMAT excluded MS:SCAN_RANGE_MOVERZ m/z 50-800 MS:MS_RESULTS_FILE ST000898_AN001460_Results.txt UNITS:Peak Area #END