Dataset Information

The EML4-ALK Interactome in Lung Cancer

ABSTRACT: We experimentally generated an ALK interactome combining quantitative tyrosine phosphoproteomics and protein complex annotation using affinity purification coupled with mass spectrometry and further investigated downstream ALK signaling using quantitative tyrosine phosphoproteomics.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Epithelial Cell

DISEASE(S): Lung Cancer

SUBMITTER: John Koomen

LAB HEAD: Eric B. Haura, MD

PROVIDER: PXD004935 | Pride | 2018-10-26

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	12072011_EML4-ALK_1-1.RAW	Raw
	12072011_EML4-ALK_1-1.RAW (F002788).pride.mztab.gz	Mztab
	12072011_EML4-ALK_1-1.RAWF002788.mzid.gz	Mzid
	12072011_EML4-ALK_1-1.RAWF002788.mzid_12072011_EML4-ALK_1-1.RAW_F002788.MGF	Mzid
	12072011_EML4-ALK_1-1.RAW_F002788.dta	Raw

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Publications

Coupling an EML4-ALK-centric interactome with RNA interference identifies sensitizers to ALK inhibitors.

Zhang Guolin G Scarborough Hannah H Kim Jihye J Rozhok Andrii I AI Chen Yian Ann YA Zhang Xiaohui X Song Lanxi L Bai Yun Y Fang Bin B Liu Richard Z RZ Koomen John J Tan Aik Choon AC Degregori James J Haura Eric B EB

Science signaling 20161018 450

Patients with lung cancers harboring anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors, but acquired resistance inevitably arises. A better understanding of proximal ALK signaling mechanisms may identify sensitizers to ALK inhibitors that disrupt the balance between prosurvival and proapoptotic effector signals. Using affinity purification coupled with mass spectrometry in an ALK fusion lung cancer cell line (H3122), we generated an ALK signaling network an ...[more]

PMID: 27811184

Similar Datasets

Project description:Anaplastic Large Cell Lymphomas (ALCL) represent a subset of lymphomas in which the Anaplastic Lymphoma Kinase (ALK) gene is frequently fused to the NPM gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo, and that ALK activity is strictly required for the survival of ALK positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK positive ALCL cell lines abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPb and the anti-apoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions. Experiment Overall Design: This series of microarray experiments contains the gene expression profiles of Anaplastic Large Cell Lymphoma (ALCL) cell lines (TS [a subclone of Sup-M2] and Su-DHL1) engineered to express ALK-A5 shRNA under a doxycycline-inducible promoter or treated with cell permeable pyrrolocarbazole-derived ALK inhibitors. A mutated ALK-A5M shRNA was used as control. Briefly, cells were transduced with pLV-DsRed-tTRKRAB, expanded, and used for transduction with pLVTH-GFP-shRNA lentiviral particles. Cells were induced with doxycycline (1 microg/ml) for 12 hours, double GFP+ DsRed+ cells selected by fluorescence-activated cell sorting. Cells expressing GFP in the absence of the inducer were removed by a second flow cytometry sorting, and expanded. shRNA expression was induced by doxycycline treatment for 72 or 84 hours. Drug treatments (300 nM), were performed in TS cells with ALK inhibitors (A2 or A3), mock compound (A1), or control diluent for 6 hours. 5 micrograms of total RNA was processed and hybridized to the Affymetrix HG-U133A chip following the manufacturer's instructions.

Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK interactome, phosphoproteome, phosphotyrosine interactome and proteome as outlined below. ALK Interactome and phosphoproteome: For each experiment, three SILAC conditions (‘Light’: Lys0,Arg0, ‘Medium’: Lys4,Arg6, ‘Heavy’: Lys8,Arg10) of NB1 cells were treated for 30 minutes with each of the following ALK-targeting small-molecule inhibitors: TAE684 (100 nM), crizotinib (500 nM), and LDK378 (250 nM). The experiments were performed as specified below: Experiment 1: Light: 500 nM crizotinib, Medium: 100 nM TAE684, Heavy: 0.1% DMSO Experiment 2: Light: 100 nM TAE684, Medium: 250 nM LDK378, Heavy: 0.1% DMSO Experiment 3: Light: 250 nM LDK378, Medium: 500 nM crizotinib, Heavy: 0.1% DMSO These three triple-SILAC experiments allowed the effect of each inhibitor to be measured in duplicates. ALK phosphotyrosine interactome: To identify phosphotyrosine-specific interaction partners of ALK we used a label-free mass spectrometry-based proteomics approach. Interacting proteins to the peptide sequences (phosphorylated and non-phosphorylated peptide) indicated in the table below were identified by a peptide pull-down assay and mass spectrometry. Peptide pull-downs were performed on NB1 cell lysate prepared from cells treated for 30 minutes with LDK378 (250 nM) or DMSO. Each peptide pull-down was performed on lysates from two biological replicates. Sample name (raw file) Gene Uniprot Site Peptide sequence A1 ALK Q9UM73 pY1078 ELQSPEpYKLSKLR A2 ALK Q9UM73 pY1092 RTIMTDpYNPNYSF A3 ALK Q9UM73 pY1096 TDYNPNpYSFAGKT A4 ALK Q9UM73 pY1131 GAFGEVpYEGQVSG A5 ALK Q9UM73 pY1278 GMARDIpYRASYYR A6 ALK Q9UM73 pY1282 DIYRASpYYRKGGS A7 ALK Q9UM73 pY1283 IYRASYpYRKGGSA A8 ALK Q9UM73 pY1359 RSPGPVpYRIMTQS A9 ALK Q9UM73 pY1507 RLWNPTpYGSWFTE A10 ALK Q9UM73 pY1584 RSGNVNpYGYQQQG A11 ALK Q9UM73 pY1604 APGAGHpYEDTILK A12 ALK Q9UM73 Y1078 ELQSPEYKLSKLR B1 ALK Q9UM73 Y1092 RTIMTDYNPNYSF B2 ALK Q9UM73 Y1096 TDYNPNYSFAGKT B3 ALK Q9UM73 Y1131 GAFGEVYEGQVSG B4 ALK Q9UM73 Y1278 GMARDIYRASYYR B5 ALK Q9UM73 Y1282 DIYRASYYRKGGS B6 ALK Q9UM73 Y1283 IYRASYYRKGGSA B7 ALK Q9UM73 Y1359 RSPGPVYRIMTQS B8 ALK Q9UM73 Y1507 RLWNPTYGSWFTE B9 ALK Q9UM73 Y1584 RSGNVNYGYQQQG B10 ALK Q9UM73 Y1604 APGAGHYEDTILK C3 IRS2 Q9Y4H2 pY675 SSRSDDpYMPMSPA C4 IRS2 Q9Y4H2 pY742 SPEDSGpYMRMWSG C5 IRS2 Q9Y4H2 pY978 RSPLSDpYMNLDFS C6 IRS2 Q9Y4H2 Y675 SSRSDDYMPMSPA C7 IRS2 Q9Y4H2 Y742 SPEDSGYMRMWSG C8 IRS2 Q9Y4H2 Y978 RSPLSDYMNLDFS Additional explanations to MS raw files: -1 extension indicates Control (DMSO lysate) replicate 1. -2 extension indicates LDK378 (treated lysate) replicate 1. -3 extension indicates Control (DMSO lysate) replicate 2. -4 extension indicates LDK378 (treated lysate) replicate 2. Proteome: The NB1 cell line proteome was measured by a label-free mass spectrometry-based approach. The analysis was performed in two biological replicates.