{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001429","ANALYSIS_ID":"AN002390","VERSION":"1","CREATED_ON":"July 19, 2020, 7:22 pm"},

"PROJECT":{"PROJECT_TITLE":"MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with Host Cell Factor-1","PROJECT_SUMMARY":"MYC is an oncoprotein transcription factor that is overexpressed in the majority cancers. Although MYC itself is considered undruggable, it may be possible to inhibit MYC by targeting the co-factors it uses to drive oncogenic gene expression patterns. Here, we use loss- and gain- of function approaches to interrogate how one MYC co-factor—Host Cell Factor (HCF)-1—contributes to MYC activity in a Burkitt lymphoma setting. We identify high-confidence direct targets of the MYC–HCF-1 interaction that are regulated through a recruitment-independent mechanism, including genes that control mitochondrial function and rate-limiting steps for ribosome biogenesis and translation. We describe how these gene expression events impact cell growth and metabolism, and demonstrate that the MYC–HCF-1 interaction is essential for tumor maintenance in vivo. This work highlights the MYC–HCF-1 interaction as a focal point for development of novel anti-cancer therapies.","INSTITUTE":"Vanderbilt University","LAST_NAME":"Codreanu","FIRST_NAME":"Simona","ADDRESS":"1234 Stevenson Center Lane","EMAIL":"simona.codreanu@vanderbilt.edu","PHONE":"6158758422"},

"STUDY":{"STUDY_TITLE":"MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with Host Cell Factor-1","STUDY_SUMMARY":"MYC is an oncoprotein transcription factor that is overexpressed in the majority cancers. Although MYC itself is considered undruggable, it may be possible to inhibit MYC by targeting the co-factors it uses to drive oncogenic gene expression patterns. Here, we use loss- and gain- of function approaches to interrogate how one MYC co-factor—Host Cell Factor (HCF)-1—contributes to MYC activity in a Burkitt lymphoma setting. We identify high-confidence direct targets of the MYC–HCF-1 interaction that are regulated through a recruitment-independent mechanism, including genes that control mitochondrial function and rate-limiting steps for ribosome biogenesis and translation. We describe how these gene expression events impact cell growth and metabolism, and demonstrate that the MYC–HCF-1 interaction is essential for tumor maintenance in vivo. This work highlights the MYC–HCF-1 interaction as a focal point for development of novel anti-cancer therapies.","INSTITUTE":"Vanderbilt University","LAST_NAME":"Codreanu","FIRST_NAME":"Simona","ADDRESS":"1234 Stevenson Center Lane","EMAIL":"simona.codreanu@vanderbilt.edu","PHONE":"6158758422"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENOTYPE_STRAIN":"Burkitt lymphoma cell line, wild type and two mutants, 4A and VP16","GENDER":"Not applicable"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"WT-1",
"Factors":{"genotype":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_WT1"}
},
{
"Subject ID":"-",
"Sample ID":"WT-2",
"Factors":{"genotype":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_WT2"}
},
{
"Subject ID":"-",
"Sample ID":"WT-3",
"Factors":{"genotype":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_WT3"}
},
{
"Subject ID":"-",
"Sample ID":"WT-4",
"Factors":{"genotype":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_WT4"}
},
{
"Subject ID":"-",
"Sample ID":"WT-5",
"Factors":{"genotype":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_WT5"}
},
{
"Subject ID":"-",
"Sample ID":"4A-1",
"Factors":{"genotype":"4A"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_4A1"}
},
{
"Subject ID":"-",
"Sample ID":"4A-2",
"Factors":{"genotype":"4A"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_4A2"}
},
{
"Subject ID":"-",
"Sample ID":"4A-3",
"Factors":{"genotype":"4A"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_4A3"}
},
{
"Subject ID":"-",
"Sample ID":"4A-4",
"Factors":{"genotype":"4A"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_4A4"}
},
{
"Subject ID":"-",
"Sample ID":"4A-5",
"Factors":{"genotype":"4A"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_4A5"}
},
{
"Subject ID":"-",
"Sample ID":"VP-1",
"Factors":{"genotype":"VP"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_VP1"}
},
{
"Subject ID":"-",
"Sample ID":"VP-2",
"Factors":{"genotype":"VP"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_VP2"}
},
{
"Subject ID":"-",
"Sample ID":"VP-3",
"Factors":{"genotype":"VP"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_VP3"}
},
{
"Subject ID":"-",
"Sample ID":"VP-4",
"Factors":{"genotype":"VP"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_VP4"}
},
{
"Subject ID":"-",
"Sample ID":"VP-5",
"Factors":{"genotype":"VP"},
"Additional sample data":{"RAW_FILE_NAME":"SC_20190611_RPLCp_FMS_Tansey_VP5"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"To understand the cellular consequences of modulating the MYC–HCF-1 interaction, we engineered a system that allows us to express the 4A or VP16 HBM mutant MYC proteins as the sole form of MYC in a cell. We chose Ramos cells, a Burkitt lymphoma (BL)-derived line in which a t(8;14) translocation places one c-MYC allele under regulatory control of the immunoglobulin heavy chain enhancer. The untranslocated c-MYC allele is not expressed in these cells. Because sequences encoding the MYC HBM are contained within exon 3, we used CRISPR/Cas9-triggered homologous recombination of the translocated MYC allele to integrate an exon 3 switchable cassette for wild-type (WT) MYC, 4A, or VP16 HBM mutants, and confirmed appropriate integration by Southern blotting. Thus, we successfully generated a system for inducible, selective, and bidirectional modulation of the MYC−HCF-1 interaction in the context of an archetypal MYC-driven cancer cell line.","SAMPLE_TYPE":"Lymphoma cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"There is no treatment analyzed by MS in this project."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The global, untargeted metabolomics study was performed on switchable MYC allele Ramos cell lines treated with 20 nM 4-OHT. Individual cell pellet samples were lysed using 200 µl ice cold lysis buffer (1:1:2, Acetonitrile : Methanol : Ammonium Bicarbonate 0.1 M, pH 8.0, LC-MS grade) and sonicated using a probe tip sonicator, 10 pulses at 30% power, cooling down on ice between samples. A BCA was used to determine the protein concentration for individual samples, and adjusted to 200 µg total protein in 200 µl of lysis buffer. Isotopically labeled standard molecules, Phenylalanine-D8 and Biotin-D2, were added to each sample to assess sample preparation. Samples were subjected to protein precipitation by addition of 800 µL of ice cold methanol (4X by volume), and incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 minutes to eliminate precipitated proteins and supernatant(s) were transferred to a clean Eppendorf tube and dried down in vacuo. Samples were stored at -80°C until further LC-MS analysis.","SAMPLEPREP_PROTOCOL_ID":"Global untargeted method_MYC_Project","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"For HILIC analysis metabolite extracts (10 μl injection volume) were separated on a SeQuant ZIC-HILIC 3.5-μm, 2.1 mm × 100 mm column (Millipore Corporation, Darmstadt, Germany) held at 40°C. Liquid chromatography was performed at a 200 μl min−1 using solvent A (5 mM Ammonium formate in 90% H2O, 10% acetonitrile) and solvent B (5 mM Ammonium formate in 90% acetonitrile, 10% H2O) with the following gradient: 95% B for 2 min, 95-40% B over 16 min, 40% B held 2 min, and 40-95% B over 15 min, 95% B held 10 min (gradient length 45 min).","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"EMD Millipore ZIC-HILIC (100 x 2.1 mm, 3.5 um)","FLOW_RATE":"0.2 mL/min","COLUMN_TEMPERATURE":"40","METHODS_FILENAME":"Global untargeted method_MYC_Project","SOLVENT_A":"5mM Ammonium formate in 90% water, 10% acetonitrile","SOLVENT_B":"5mM Ammonium formate in 90% acetonitrile, 10% water"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"FMS and DDA acquisition over a mass range of m/z 70-1050 data were imported, processed, normalized and reviewed using Progenesis QI","MS_RESULTS_FILE":"ST001429_AN002390_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}