PDF file - 45K, ALK and FN1-ALK tumors are sensitive to TAE684 treatment

PDF file - 129K, A novel fusion: FN1-ALK

XLS file - 68K, Phospho-tyrosine Peptides of Tyrosine Kinases Identified by LC-MS/MS in Ovarian Tissues

<p>PDF file - 50K, Hyperphosphorylation of signaling molecules in serous carcinomas bearing ALK</p>

XLS file - 31K, Patient Diagnostic Information

Supplementary Data from Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer

XLS file - 40K, Abundance of phospho-peptides corresponding to specific signaling molecules in selective serous carcinoma patients

PDF file - 175K, Activation and ALK inhibitor sensitivity of down stream molecules in 293T cells expressing ALK and FN1-ALK

<p>PDF file - 127K, Detection of two common ALK peptides containing phospho-Y1507 in 4 patients</p>

To understand drug combination effect, it is necessary to decipher the interactions between drug targets many of which are signaling molecules. Previously, such signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed, de novo reconstruction of signaling interactions from large-scale molecular profiling is still lagging, compared to similar efforts in transcriptional and protein-protein interaction networks. To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry -based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including "novel' substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.

Protein posttranslational modifications (PTMs) have typically been studied independently, yet many proteins are modified by more than one PTMtype, and cell signaling pathways somehow integrate this information. We coupled immunoprecipitation using PTM-specific antibodies with tandem mass tag (TMT) mass spectrometry to simultaneously examine phosphorylation, methylation, and acetylation in 45 lung cancer cell lines compared to normal lung tissue and to cell lines treated with anticancer drugs. This simultaneous, large-scale, integrative analysis of these PTMs using a cluster-filtered network (CFN) approach revealed that cell signaling pathways were outlined by clustering patterns in PTMs. Weused the t-distributed stochastic neighbor embedding (t-SNE) method to identify PTM clusters and then integrated each with known protein-protein interactions (PPIs) to elucidate functional cell signaling pathways. The CFN identified known and previously unknown cell signaling pathways in lung cancer cells that were not present in normal lung epithelial tissue. In various proteins modified by more than one type of PTM, the incidence of those PTMs exhibited inverse relationships, suggesting that molecular exclusive "OR" gates determine a large number of signal transduction events. Wealso showed that the acetyltransferase EP300 appears to be a hub in the network of pathways involving different PTMs. In addition, the data shed light on themechanismof action of geldanamycin, an HSP90 inhibitor. Together, the findings reveal that cell signaling pathways mediated by acetylation, methylation, and phosphorylation regulate the cytoskeleton, membrane traffic, and RNA binding protein-mediated control of gene expression.

Lung cancer is the most common type of cancer in the U.S., with 5 yr. survival rates of 18% and 6% for non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) patients, respectively. In spite of success with targeted therapies for 15-20% of patients with NSCLCs, the success is generally short-lived as the tumors become resistant to treatment. In contrast to NSCLC, no successful targeted therapies have been identified in SCLC. Further understanding of the regulatory cell signaling networks for SCLCs, may enable the discovery of new actionable lung cancer drivers. Posttranslational modifications (PTMs), including phosphorylation, acetylation and methylation, act alone and in combination to regulate protein function, cellular behavior and epigenetics. All three types of PTMs are deregulated in various cancers. In this study we developed a highly quantitative MS/MS approach combining TMT labeling with a number of motif and site-specific antibodies to identify deregulated signaling pathways between tumor and normal specimens. PTM-specific antibodies were used to enrich modified peptides followed by 6-plex TMT mass-spec analyses to quantitatively measure over 15,300 differentially phosphorylated, acetylated, and methylated sites on over 4,600 proteins from 17 SCLC patient tissues and 5 para-normals. Understanding cell-signaling networks at the intersection of multiple pathways and protein modification systems in SCLC lung cancers leads to the identification of new druggable disease drivers. Citation Format: Klarisa Rikova, Ben Hall, Tyler Levy, Anthony Possemato, Mike Aguiar, Sean Beausoleil, Jian Min Ren, Kimberly Lee Lee, Scott Lonning, Michael Comb. Proteomic analysis identifies multi-dimensional deregulated signaling pathways in SCLC lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-067.

Lung cancer is the most common type of cancer in the U.S., with 5 yr. survival rates of 18% and 6% for non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) patients, respectively. In spite of success with targeted therapies for 15-20% of patients with NSCLCs, the success is generally short-lived as the tumors become resistant to treatment. In contrast to NSCLC, no successful targeted therapies have been identified in SCLC. Further understanding of the regulatory cell signaling networks for SCLCs, may enable the discovery of new actionable lung cancer drivers. Posttranslational modifications (PTMs), including phosphorylation, acetylation and methylation, act alone and in combination to regulate protein function, cellular behavior and epigenetics. All three types of PTMs are deregulated in various cancers. In this study we developed a highly quantitative MS/MS approach combining TMT labeling with a number of motif and site-specific antibodies to identify deregulated signaling pathways between tumor and normal specimens. PTM-specific antibodies were used to enrich modified peptides followed by 6-plex TMT mass-spec analyses to quantitatively measure over 15,300 differentially phosphorylated, acetylated, and methylated sites on over 4,600 proteins from 17 SCLC patient tissues and 5 para-normals. Understanding cell-signaling networks at the intersection of multiple pathways and protein modification systems in SCLC lung cancers leads to the identification of new druggable disease drivers. Citation Format: Klarisa Rikova, Ben Hall, Tyler Levy, Anthony Possemato, Mike Aguiar, Sean Beausoleil, Jian Min Ren, Kimberly Lee Lee, Scott Lonning, Michael Comb. Proteomic analysis identifies multi-dimensional deregulated signaling pathways in SCLC lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-067.

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

Abstract Akt isoforms exhibit different functional properties. To address the signaling differences between Akt1, Akt2 and Akt3 we examined the phosphoproteomes of a set of isogenic mouse cell lines that express different Akt isoforms. Based on this screen, we identified a total of 606 Akt phosphorylation targets, of which many are phosphorylated in an isoform specific manner. Bioinformatics analyses of these data revealed that Akt isoforms regulate differentially multiple cellular functions. One of these functions was RNA metabolism which was represented by 25 proteins phosphorylated by at least one of the Akt isoforms. One of these proteins was IWS1, which is involved in the assembly of RNA Pol II transcriptional elongation complex, and which was found to be phosphorylated at the conserved site Ser720/Thr721 by Akt3 and Akt1. Here we show that this phosphorylation event is required for the recruitment of the histone methyltransferase SETD2 to the complex and the trimethylation of histone H3 at K36 in the body of the transcribed genes. H3K36me3 provides a docking site for MRG15 and its binding partner, the splicing suppressor PTB, and regulates PTB-dependent alternative splicing. One of the targets is FGFR-2 whose alternative splicing gives rise to two isoforms, IIIb, which is expressed in epithelial cells and IIIc, which is expressed in mesenchymal cells, promotes EMT and is associated with more aggressive tumors. IWS1 phosphorylation by Akt3/Akt1 shifts splicing toward the IIIc isoform and promotes tumor growth and invasiveness both in culture and in animals. Addressing the expression of FGFR-2 in a set of lung-derived normal and tumor samples revealed that whereas the overall expression was similar in both, there was a shift toward the IIIc isoform in the tumor samples. More important, the relative expression of the IIIc and IIIb isoforms in non-small-cell-lung-carcinomas (NSCLCs) correlated with the stoichiometry of IWS1 phosphorylation and the latter correlated with Akt phosphorylation and Akt3 expression. These findings combined, underpin the importance of this pathway in the pathogenesis of lung cancer. Overall, our data suggest that Akt isoform-dependent phosphorylation events are essential for RNA processing and provide novel insights into the role of Akt in carcinogenesis. Citation Format: Ioannis Sanidas, Christos Polytarchou, Maria Hatziapostolou, Scott A. Ezell, Filippos Kottakis, Lan Hu, Ailan Guo, Jianxin Xie, Michael J. Comb, Dimitrios Iliopoulos, Philip N. Tsichlis. A phosphoproteomics analysis reveals Akt isoform-specific signals that link RNA splicing to non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2014-4777

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.