#METABOLOMICS WORKBENCH Codreags00_20200715_095049 DATATRACK_ID:2098 STUDY_ID:ST001429 ANALYSIS_ID:AN002390 PROJECT_ID:PR000981 VERSION 1 CREATED_ON July 19, 2020, 7:22 pm #PROJECT PR:PROJECT_TITLE MYC regulates ribosome biogenesis and mitochondrial gene expression programs PR:PROJECT_TITLE through its interaction with Host Cell Factor-1 PR:PROJECT_SUMMARY MYC is an oncoprotein transcription factor that is overexpressed in the majority PR:PROJECT_SUMMARY cancers. Although MYC itself is considered undruggable, it may be possible to PR:PROJECT_SUMMARY inhibit MYC by targeting the co-factors it uses to drive oncogenic gene PR:PROJECT_SUMMARY expression patterns. Here, we use loss- and gain- of function approaches to PR:PROJECT_SUMMARY interrogate how one MYC co-factor—Host Cell Factor (HCF)-1—contributes to PR:PROJECT_SUMMARY MYC activity in a Burkitt lymphoma setting. We identify high-confidence direct PR:PROJECT_SUMMARY targets of the MYC–HCF-1 interaction that are regulated through a PR:PROJECT_SUMMARY recruitment-independent mechanism, including genes that control mitochondrial PR:PROJECT_SUMMARY function and rate-limiting steps for ribosome biogenesis and translation. We PR:PROJECT_SUMMARY describe how these gene expression events impact cell growth and metabolism, and PR:PROJECT_SUMMARY demonstrate that the MYC–HCF-1 interaction is essential for tumor maintenance PR:PROJECT_SUMMARY in vivo. This work highlights the MYC–HCF-1 interaction as a focal point for PR:PROJECT_SUMMARY development of novel anti-cancer therapies. PR:INSTITUTE Vanderbilt University PR:LAST_NAME Codreanu PR:FIRST_NAME Simona PR:ADDRESS 1234 Stevenson Center Lane PR:EMAIL simona.codreanu@vanderbilt.edu PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE MYC regulates ribosome biogenesis and mitochondrial gene expression programs ST:STUDY_TITLE through its interaction with Host Cell Factor-1 ST:STUDY_SUMMARY MYC is an oncoprotein transcription factor that is overexpressed in the majority ST:STUDY_SUMMARY cancers. Although MYC itself is considered undruggable, it may be possible to ST:STUDY_SUMMARY inhibit MYC by targeting the co-factors it uses to drive oncogenic gene ST:STUDY_SUMMARY expression patterns. Here, we use loss- and gain- of function approaches to ST:STUDY_SUMMARY interrogate how one MYC co-factor—Host Cell Factor (HCF)-1—contributes to ST:STUDY_SUMMARY MYC activity in a Burkitt lymphoma setting. We identify high-confidence direct ST:STUDY_SUMMARY targets of the MYC–HCF-1 interaction that are regulated through a ST:STUDY_SUMMARY recruitment-independent mechanism, including genes that control mitochondrial ST:STUDY_SUMMARY function and rate-limiting steps for ribosome biogenesis and translation. We ST:STUDY_SUMMARY describe how these gene expression events impact cell growth and metabolism, and ST:STUDY_SUMMARY demonstrate that the MYC–HCF-1 interaction is essential for tumor maintenance ST:STUDY_SUMMARY in vivo. This work highlights the MYC–HCF-1 interaction as a focal point for ST:STUDY_SUMMARY development of novel anti-cancer therapies. ST:INSTITUTE Vanderbilt University ST:LAST_NAME Codreanu ST:FIRST_NAME Simona ST:ADDRESS 1234 Stevenson Center Lane ST:EMAIL simona.codreanu@vanderbilt.edu ST:PHONE 6158758422 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN Burkitt lymphoma cell line, wild type and two mutants, 4A and VP16 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 - WT-1 genotype:WT RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_WT1 SUBJECT_SAMPLE_FACTORS - WT-2 genotype:WT RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_WT2 SUBJECT_SAMPLE_FACTORS - WT-3 genotype:WT RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_WT3 SUBJECT_SAMPLE_FACTORS - WT-4 genotype:WT RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_WT4 SUBJECT_SAMPLE_FACTORS - WT-5 genotype:WT RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_WT5 SUBJECT_SAMPLE_FACTORS - 4A-1 genotype:4A RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_4A1 SUBJECT_SAMPLE_FACTORS - 4A-2 genotype:4A RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_4A2 SUBJECT_SAMPLE_FACTORS - 4A-3 genotype:4A RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_4A3 SUBJECT_SAMPLE_FACTORS - 4A-4 genotype:4A RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_4A4 SUBJECT_SAMPLE_FACTORS - 4A-5 genotype:4A RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_4A5 SUBJECT_SAMPLE_FACTORS - VP-1 genotype:VP RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_VP1 SUBJECT_SAMPLE_FACTORS - VP-2 genotype:VP RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_VP2 SUBJECT_SAMPLE_FACTORS - VP-3 genotype:VP RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_VP3 SUBJECT_SAMPLE_FACTORS - VP-4 genotype:VP RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_VP4 SUBJECT_SAMPLE_FACTORS - VP-5 genotype:VP RAW_FILE_NAME=SC_20190611_RPLCp_FMS_Tansey_VP5 #COLLECTION CO:COLLECTION_SUMMARY To understand the cellular consequences of modulating the MYC–HCF-1 CO:COLLECTION_SUMMARY interaction, we engineered a system that allows us to express the 4A or VP16 HBM CO:COLLECTION_SUMMARY mutant MYC proteins as the sole form of MYC in a cell. We chose Ramos cells, a CO:COLLECTION_SUMMARY Burkitt lymphoma (BL)-derived line in which a t(8;14) translocation places one CO:COLLECTION_SUMMARY c-MYC allele under regulatory control of the immunoglobulin heavy chain CO:COLLECTION_SUMMARY enhancer. The untranslocated c-MYC allele is not expressed in these cells. CO:COLLECTION_SUMMARY Because sequences encoding the MYC HBM are contained within exon 3, we used CO:COLLECTION_SUMMARY CRISPR/Cas9-triggered homologous recombination of the translocated MYC allele to CO:COLLECTION_SUMMARY integrate an exon 3 switchable cassette for wild-type (WT) MYC, 4A, or VP16 HBM CO:COLLECTION_SUMMARY mutants, and confirmed appropriate integration by Southern blotting. Thus, we CO:COLLECTION_SUMMARY successfully generated a system for inducible, selective, and bidirectional CO:COLLECTION_SUMMARY modulation of the MYC−HCF-1 interaction in the context of an archetypal CO:COLLECTION_SUMMARY MYC-driven cancer cell line. CO:SAMPLE_TYPE Lymphoma cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY There is no treatment analyzed by MS in this project. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The global, untargeted metabolomics study was performed on switchable MYC allele SP:SAMPLEPREP_SUMMARY Ramos cell lines treated with 20 nM 4-OHT. Individual cell pellet samples were SP:SAMPLEPREP_SUMMARY lysed using 200 µl ice cold lysis buffer (1:1:2, Acetonitrile : Methanol : SP:SAMPLEPREP_SUMMARY Ammonium Bicarbonate 0.1 M, pH 8.0, LC-MS grade) and sonicated using a probe tip SP:SAMPLEPREP_SUMMARY sonicator, 10 pulses at 30% power, cooling down on ice between samples. A BCA SP:SAMPLEPREP_SUMMARY was used to determine the protein concentration for individual samples, and SP:SAMPLEPREP_SUMMARY adjusted to 200 µg total protein in 200 µl of lysis buffer. Isotopically SP:SAMPLEPREP_SUMMARY labeled standard molecules, Phenylalanine-D8 and Biotin-D2, were added to each SP:SAMPLEPREP_SUMMARY sample to assess sample preparation. Samples were subjected to protein SP:SAMPLEPREP_SUMMARY precipitation by addition of 800 µL of ice cold methanol (4X by volume), and SP:SAMPLEPREP_SUMMARY incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 SP:SAMPLEPREP_SUMMARY minutes to eliminate precipitated proteins and supernatant(s) were transferred SP:SAMPLEPREP_SUMMARY to a clean Eppendorf tube and dried down in vacuo. Samples were stored at -80°C SP:SAMPLEPREP_SUMMARY until further LC-MS analysis. SP:SAMPLEPREP_PROTOCOL_ID Global untargeted method_MYC_Project SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For HILIC analysis metabolite extracts (10 μl injection volume) were separated CH:CHROMATOGRAPHY_SUMMARY on a SeQuant ZIC-HILIC 3.5-μm, 2.1 mm × 100 mm column (Millipore CH:CHROMATOGRAPHY_SUMMARY Corporation, Darmstadt, Germany) held at 40°C. Liquid chromatography was CH:CHROMATOGRAPHY_SUMMARY performed at a 200 μl min−1 using solvent A (5 mM Ammonium formate in 90% CH:CHROMATOGRAPHY_SUMMARY H2O, 10% acetonitrile) and solvent B (5 mM Ammonium formate in 90% acetonitrile, CH:CHROMATOGRAPHY_SUMMARY 10% H2O) with the following gradient: 95% B for 2 min, 95-40% B over 16 min, 40% CH:CHROMATOGRAPHY_SUMMARY B held 2 min, and 40-95% B over 15 min, 95% B held 10 min (gradient length 45 CH:CHROMATOGRAPHY_SUMMARY min). CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME EMD Millipore ZIC-HILIC (100 x 2.1 mm, 3.5 um) CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 40 CH:METHODS_FILENAME Global untargeted method_MYC_Project CH:SOLVENT_A 5mM Ammonium formate in 90% water, 10% acetonitrile CH:SOLVENT_B 5mM Ammonium formate in 90% acetonitrile, 10% water #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 POSITIVE MS:MS_COMMENTS FMS and DDA acquisition over a mass range of m/z 70-1050 data were imported, MS:MS_COMMENTS processed, normalized and reviewed using Progenesis QI MS:MS_RESULTS_FILE ST001429_AN002390_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END