Dataset Information

Proteomics analysis of synergistic drug combination of midostaurin and BMS-754807 in Ewing sarcoma ASP14 cell line

ABSTRACT: We used quantitative mass spectrometry-based proteomics to unravel global changes in the phosphoproteome upon treatment of ASP14 cells (a Ewing Sarcoma cell line) according to the setup below. The combination of drugs were experimentally shown to infer synergy in terms of cell killing. Three experiments were performed using a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10): Experiment 1: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: 0.1% DMSO Experiment 2: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: BMS-754807 (20 nM) Experiment 3: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: BMS-754807 (20 nM) Heavy SILAC: 0.1% DMSO ASP14 cells were starved by replacing complete SILAC medium with SILAC minimal medium without serum over night. Cells were treated with drugs as stated above for 2h; then they were stimulated with serum for 20 minutes and harvested thereafter.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Muscle Cell

DISEASE(S): Peripheral Primitive Neuroectodermal Tumor

SUBMITTER: Kristina Bennet Emdal

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD002765 | Pride | 2017-04-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20150617_QE3_UPLC11_AKP_SA_COLLAB_ASSET_WP8_Asp14_Mix1_TiO2_1st_01.raw	Raw
	20150617_QE3_UPLC11_AKP_SA_COLLAB_ASSET_WP8_Asp14_Mix1_TiO2_2nd_02.raw	Raw
	20150617_QE3_UPLC11_AKP_SA_COLLAB_ASSET_WP8_Asp14_Mix1_TiO2_HpH_01.raw	Raw
	20150617_QE3_UPLC11_AKP_SA_COLLAB_ASSET_WP8_Asp14_Mix1_TiO2_HpH_02.raw	Raw
	20150617_QE3_UPLC11_AKP_SA_COLLAB_ASSET_WP8_Asp14_Mix1_TiO2_HpH_03.raw	Raw

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Publications

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

Radic-Sarikas Branka B Tsafou Kalliopi P KP Emdal Kristina B KB Papamarkou Theodore T Huber Kilian V M KV Mutz Cornelia C Toretsky Jeffrey A JA Bennett Keiryn L KL Olsen Jesper V JV Brunak Søren S Kovar Heinrich H Superti-Furga Giulio G

Molecular cancer therapeutics 20161115 1

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pedi ...[more]

PMID: 28062706

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Project description:We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra

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