Abstract IA11: Combinatorial screens in Drosophila cells

Our laboratory is combining a number of omics approaches (Mass Spec, RNAi, phosphoproteomics, and transcriptomics) to expand our understanding of the structure of signaling networks. Applying these approaches has allowed us to identify additional components of a number of signaling networks including the MAPK/ERK, AKT/PI3K, Hippo, Insulin and autophagy pathways (Friedman et al., 2011; Vinayagam et al., 2013; Neumuller et al., 2013; Kwon et al., 2013; Zirin et al., 2015; Vinayagam et al., 2016). An important finding from large-scale studies is that we could only functionally validate about half of the network components using single-gene loss of function studies, suggesting a high level of functional redundancy within network. For example, using Mass Spec we generated an Insulin pathway interactome of about 560 proteins and only 50% were found to affect pathway output as measured using phospho-specific antibodies (Vinayagam et al., 2016). To interrogate redundancy, we established a robust platform for performing combinatorial screens and used it to identify synthetic lethal (SL) interactions with a number of tumor suppressors such as TSC1 and TSC2 (Housden et al., 2015). Importantly, almost all the SL interactions we discovered in the fly screens turned out to be conserved in mammalian cells (collaboration with Alex Valvezan and Brendan Manning). These studies have led to identification of promising drug targets, and, as some of these are known targets of FDA approved drugs, drug candidates. To expand SL screens in Drosophila cells, we have developed two new powerful screening approaches: 1. Pooled CRISPR and CRISPRa screens, and 2. Variable Dose Analysis, a screening method that exhibits significant reduced noise compared to previous SL screening methods. These approaches allow us to not only identify evolutionary conserved SL interactions that can lead to promising drug targets, but also to build a network of SL interactions that we will be able to compare to yeast SL network in particular. Results from these studies will provide fundamental information on the extent of conservation of SL across organisms, an issue that has not been addressed rigorously yet. References Friedman AA, Tucker G, Singh R, Yan D, Vinayagam A, Hu Y, Binari R, Hong P, Sun X, Porto M, Pacifico S, Murali T, Finley RL Jr, Asara JM, Berger B, Perrimon N. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. Sci Signal. 2011 Oct 25;4(196):rs10. PMID: 22028469. Vinayagam A, Hu Y, Kulkarni M, Roesel C, Sopko R, Mohr SE, Perrimon N. Protein complex-based analysis framework for high-throughput data sets. Sci Signal. 2013 Feb 26;6(264):rs5. PMID: 23443684. Neumuller RA, Gross T, Samsonova AA, Vinayagam A, Buckner M, Founk K, Hu Y, Sharifpoor S, Rosebrock AP, Andrews B, Winston F, Perrimon N. Conserved regulators of nucleolar size revealed by global phenotypic analyses. Sci Signal. 2013 Aug 20;6(289):ra70. PMID: 23962978. Kwon Y, Vinayagam A, Sun X, Dephoure N, Gygi SP, Hong P, Perrimon N. The Hippo signaling pathway interactome. Science. 2013 Nov 8;342(6159):737-40. PMID: 24114784. Zirin J, Nieuwenhuis J, Samsonova A, Tao R, Perrimon N. Regulators of autophagosome formation in Drosophila muscles. PLoS Genet. 2015 Feb 18;11(2):e1005006. PMID: 25692684. Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902. Vinayagam A, Kulkarni MM, Sopko R, Sun X, Hu Y, Nand A, Villalta C, Moghimi A, Yang X, Mohr SE, Hong P, Asara JM, Perrimon N. An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling. Cell Rep. 2016 Sep 13;16(11):3062-74. PMID: 27626673. Citation Format: Norbert Perrimon. Combinatorial screens in Drosophila cells [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA11.