#METABOLOMICS WORKBENCH amelia_palermo_20230410_145425 DATATRACK_ID:3854 STUDY_ID:ST002577 ANALYSIS_ID:AN004246 PROJECT_ID:PR001662 VERSION 1 CREATED_ON April 25, 2023, 10:03 am #PROJECT PR:PROJECT_TITLE Hydroxyproline Modulates Adaptive PD-L1 Expression and Autophagy PR:PROJECT_SUMMARY The immune checkpoint protein PD-L1 plays critical roles in both immune system PR:PROJECT_SUMMARY homeostasis and tumor progression. Impaired PD-1/PD-L1 function promotes PR:PROJECT_SUMMARY autoimmunity and PD-L1 expression within tumors promotes immune evasion. If and PR:PROJECT_SUMMARY how changes in metabolism or defined metabolites regulate PD-L1 expression is PR:PROJECT_SUMMARY not fully understood. Here, using a metabolomics activity screening-based PR:PROJECT_SUMMARY approach, we have determined that hydroxyproline (Hyp) significantly and PR:PROJECT_SUMMARY directly enhances adaptive (i.e., IFN-γ induced) PD-L1 expression in multiple PR:PROJECT_SUMMARY relevant myeloid and cancer cell types. Mechanistic studies reveal that Hyp acts PR:PROJECT_SUMMARY as an inhibitor of IFN-γ-induced autophagic flux, which allows it to regulate PR:PROJECT_SUMMARY this negative feedback mechanism, thereby contributing to its overall effect on PR:PROJECT_SUMMARY PD-L1 expression. Due to its prevalence in fibrotic tumors, these findings PR:PROJECT_SUMMARY suggest that hydroxyproline could contribute to the establishment of an PR:PROJECT_SUMMARY immunosuppressive tumor microenvironment and that Hyp metabolism could be PR:PROJECT_SUMMARY targeted to pharmacologically control PD-L1 expression for the treatment of PR:PROJECT_SUMMARY cancer or autoimmune diseases. PR:INSTITUTE Scripps Research Institute/University of California, Los Angeles PR:LAST_NAME Palermo PR:FIRST_NAME Amelia PR:ADDRESS Los Angeles, CA, USA 90095 PR:EMAIL apalermo@mednet.ucla.edu PR:PHONE 8582811389 #STUDY ST:STUDY_TITLE Hydroxyproline Modulates Adaptive PD-L1 Expression and Autophagy ST:STUDY_SUMMARY The immune checkpoint protein PD-L1 plays critical roles in both immune system ST:STUDY_SUMMARY homeostasis and tumor progression. Impaired PD-1/PD-L1 function promotes ST:STUDY_SUMMARY autoimmunity and PD-L1 expression within tumors promotes immune evasion. If and ST:STUDY_SUMMARY how changes in metabolism or defined metabolites regulate PD-L1 expression is ST:STUDY_SUMMARY not fully understood. Here, using a metabolomics activity screening-based ST:STUDY_SUMMARY approach, we have determined that hydroxyproline (Hyp) significantly and ST:STUDY_SUMMARY directly enhances adaptive (i.e., IFN-γ induced) PD-L1 expression in multiple ST:STUDY_SUMMARY relevant myeloid and cancer cell types. Mechanistic studies reveal that Hyp acts ST:STUDY_SUMMARY as an inhibitor of IFN-γ-induced autophagic flux, which allows it to regulate ST:STUDY_SUMMARY this negative feedback mechanism, thereby contributing to its overall effect on ST:STUDY_SUMMARY PD-L1 expression. Due to its prevalence in fibrotic tumors, these findings ST:STUDY_SUMMARY suggest that hydroxyproline could contribute to the establishment of an ST:STUDY_SUMMARY immunosuppressive tumor microenvironment and that Hyp metabolism could be ST:STUDY_SUMMARY targeted to pharmacologically control PD-L1 expression for the treatment of ST:STUDY_SUMMARY cancer or autoimmune diseases. ST:INSTITUTE Scripps Research Institute/University of California, Los Angeles ST:LAST_NAME Palermo ST:FIRST_NAME Amelia ST:ADDRESS University of California, Los Angeles, CA, USA ST:EMAIL apalermo@mednet.ucla.edu ST:PHONE 8582811389 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - EXP1_S1_HILICneg Time:T0 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ab_b_3_01_3032.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S2_HILICneg Time:T0 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_cu_b_4_01_3033.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S3_HILICneg Time:T0 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_da_b_5_01_3034.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S4_HILICneg Time:T0 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ef_b_6_01_3035.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S5_HILICneg Time:T0 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_fa_b_7_01_3036.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S6_HILICneg Time:T24 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_fg_b_8_01_3038.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S7_HILICneg Time:T24 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_gh_9_01_3010.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S8_HILICneg Time:T24 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_hc_b_10_01_3040.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S9_HILICneg Time:T24 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ie_11_01_3015.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S10_HILICneg Time:T24 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_iy_12_01_3016.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S11_HILICneg Time:T48 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ja_13_01_3019.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S12_HILICneg Time:T48 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_lf_14_01_3020.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S13_HILICneg Time:T48 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_mn_15_01_3021.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S14_HILICneg Time:T48 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_mo_16_01_3022.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S15_HILICneg Time:T48 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ne_17_01_3023.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S16_HILICneg Time:T72 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_pq_18_01_3026.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S17_HILICneg Time:T72 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_qj_19_01_3027.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S18_HILICneg Time:T72 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_rs_20_01_3028.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S19_HILICneg Time:T72 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_ya_21_01_3029.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S20_HILICneg Time:T72 RAW_FILE_NAME=20180420_PD-L1_HILICneg_sample_yq_22_01_3030.mzXML SUBJECT_SAMPLE_FACTORS - EXP1_S1_HILICpos Time:T0 RAW_FILE_NAME=20180228_sample_1.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S2_HILICpos Time:T0 RAW_FILE_NAME=20180228_sample_2.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S3_HILICpos Time:T0 RAW_FILE_NAME=20180228_sample_3.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S4_HILICpos Time:T0 RAW_FILE_NAME=20180228_sample_4.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S5_HILICpos Time:T0 RAW_FILE_NAME=20180228_sample_5.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S6_HILICpos Time:T24 RAW_FILE_NAME=20180228_sample_6.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S7_HILICpos Time:T24 RAW_FILE_NAME=20180228_sample_7.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S8_HILICpos Time:T24 RAW_FILE_NAME=20180228_sample_8.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S9_HILICpos Time:T24 RAW_FILE_NAME=20180228_sample_9.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S10_HILICpos Time:T24 RAW_FILE_NAME=20180228_sample_10.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S11_HILICpos Time:T48 RAW_FILE_NAME=20180228_sample_11.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S12_HILICpos Time:T48 RAW_FILE_NAME=20180228_sample_12.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S13_HILICpos Time:T48 RAW_FILE_NAME=20180228_sample_13.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S14_HILICpos Time:T48 RAW_FILE_NAME=20180228_sample_14.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S15_HILICpos Time:T48 RAW_FILE_NAME=20180228_sample_15.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S16_HILICpos Time:T72 RAW_FILE_NAME=20180228_sample_16.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S17_HILICpos Time:T72 RAW_FILE_NAME=20180228_sample_17.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S18_HILICpos Time:T72 RAW_FILE_NAME=20180228_sample_18.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S19_HILICpos Time:T72 RAW_FILE_NAME=20180228_sample_19.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S20_HILICpos Time:T72 RAW_FILE_NAME=20180228_sample_20.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S1_RPpos Time:T0 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample1.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S2_Rppos Time:T0 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample2.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S3_Rppos Time:T0 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample3.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S4_Rppos Time:T0 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample4.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S5_Rppos Time:T0 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample5.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S6_Rppos Time:T24 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample6.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S7_Rppos Time:T24 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample7.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S8_Rppos Time:T24 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample8.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S9_Rppos Time:T24 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample9.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S10_Rppos Time:T24 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample10.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S11_Rppos Time:T48 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample11.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S12_Rppos Time:T48 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample12.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S13_Rppos Time:T48 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample13.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S14_Rppos Time:T48 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample14.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S15_Rppos Time:T48 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample15.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S16_Rppos Time:T72 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample16.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S17_Rppos Time:T72 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample17.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S18_Rppos Time:T72 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample18.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S19_Rppos Time:T72 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample19.mzData SUBJECT_SAMPLE_FACTORS - EXP1_S20_Rppos Time:T72 RAW_FILE_NAME=022218_Exp17_PDL1_RPpos_Sample20.mzData #COLLECTION CO:COLLECTION_SUMMARY Cell pellets were washed with PBS and stored at -80 C until sample preparation CO:SAMPLE_TYPE AML cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY THP-1 cells stimulated with IFN-gamma #SAMPLEPREP SP:SAMPLEPREP_SUMMARY THP-1 cells were centrifuged and washed with PBS 3 times. The supernatant was SP:SAMPLEPREP_SUMMARY discarded, and 1 mL cold MeOH/ACN/H2O (2:2:1) was added to the cell pellets, SP:SAMPLEPREP_SUMMARY vortexed, and sonicated in an ice cold bath for 15 min, 3 times. Samples were SP:SAMPLEPREP_SUMMARY kept at – 20 ˚C for 1h, centrifuged (13000 rpm for 15 min, 4˚C). The SP:SAMPLEPREP_SUMMARY supernatant was evaporated and reconstituted in ACN/H2O (1:1) (volume normalized SP:SAMPLEPREP_SUMMARY to viable cell count). Samples were transferred to glass vials and stored at - SP:SAMPLEPREP_SUMMARY 80 ˚C until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For RP separation, an ACQUITY BEH C18 column (1.0 × 100 mm, 1.7-μm particle CH:CHROMATOGRAPHY_SUMMARY size; Waters Corporation, Milford, MA) was used. Flow was 150 μl/min, and a CH:CHROMATOGRAPHY_SUMMARY binary buffer system consisting of buffer A (0.1% FA) and buffer B (0.1% FA in CH:CHROMATOGRAPHY_SUMMARY acetonitrile) was used. The gradient for RP was: 99% A for 1 min, 1% A over 9 CH:CHROMATOGRAPHY_SUMMARY min, 35% A over 13 min, 60% A over 3 min, and held at 60% A for an additional 1 CH:CHROMATOGRAPHY_SUMMARY min. The sample injection volume was 2 μl. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker Elute CH:COLUMN_NAME ACQUITY BEH C18 (1.0 × 100 mm, 1.7-μm) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% aetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 99% A for 1 min, 1% A over 9 min, 35% A over 13 min, 60% A over 3 min, and held CH:FLOW_GRADIENT at 60% A for an additional 1 min CH:FLOW_RATE 150 μl/min CH:COLUMN_TEMPERATURE 27 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME qTOF, Impact II, Bruker, Bremen, Germany MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was calibrated using Sodium Formate peaks, before and MS:MS_COMMENTS post-run (internal calibration). Data were acquired over an m/z range of 50 to MS:MS_COMMENTS 1000 Da in positive and negative ionization mode. Electrospray source conditions MS:MS_COMMENTS were set as follows: end plate offset, 500 V; dry gas temperature, 200°C; MS:MS_COMMENTS drying gas, 6 liters/min; nebulizer, 1.6 bar; and capillary voltage, 3500 V. MS:MS_RESULTS_FILE ST002577_AN004246_Results.txt UNITS:counts Has m/z:Yes Has RT:No RT units:No RT data #END