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Ingenuity Pathways Analysis v5.5 Adam Corner, PhD ([email protected]) Agenda • Introduction to Ingenuity • Sample Studies: – – – – IPA in Biomarker Discovery Research IPA in Copy Number/Disease Mechanism Research IPA in Metabolic Disorders IPA in Metabolomics & Molecular Toxicology Ingenuity Systems • Partial Customer List Ingenuity Systems is the leading provider of products and services that enable analysis and exploration of biological data – There is more data to analyze and explore then there is time or resources – Need to understand biological systems at molecular, cellular and organismal level – Essential to have the right content and tools for the job • User base reflects strong adoption in research community – Top pharmaceutical companies – Large biotechs – Academic and government research labs Discover the Biology 3 The Challenge Data Analysis Across Multiple Dimensions of Biology Systematic Generation of Novel Biological and Therapeutic Insights Disease Processes Cellular Processes Find genes implicated in disease Cancer Identify related cellular processes, pathways Apoptosis Angiogenesis Molecules Fas Vegf Experimental Platforms Generate hypothesis of molecular mechanism Informed in vivo, in vitro assays Discover the Biology 4 The Challenge Data Analysis Across Multiple Dimensions of Biology Systematic Generation of Novel Biological and Therapeutic Insights Drug Induced Injury/Pathology Disease Processes Cellular Processes Cancer Toxicological Responses Apoptosis Angiogenesis Xenobiotic Insult Molecules Fas Vegf Experimental Platforms Drug Treatment Discover the Biology 5 The Solution: Ingenuity Pathways Analysis Value Solution IPA is a software application that enables researchers to analyse & understand the complex biological and chemical systems at the core of life science research Discover the Biology 6 Discovery Providing Value Throughout Drug Discovery & Development Process Understand disease processes, identify and validate targets Biomarkers Identify novel biomarkers, understand their role in disease pathways Toxicology Identify potential mechanism of toxicity Pharmacogenomics Gain insight into differential response to therapeutics Discover the Biology 7 Ingenuity Publications Peer-Reviewed Research Articles Citing IPA 600 528 Total # of Citations 500 400 300 200 100 0 2003 2004 2005 2006 through September 2007 Full bibliography available at www.ingenuity.com The Ingenuity Platform Ingenuity Pathways Knowledge Base Ontology and Knowledge Infrastructure Client Solutions Ingenuity Pathways Analysis Portal & Enterprise Search Enablement • ~ 1.8 million findings manually extracted from full text • ~160 curated metabolic and cell signaling pathways • Chemical and drug info • Signatures • Scalable best-in-class Content Acquisition processes Specialist Analytics • Ingenuity Ontology of ca. 600,000 biological objects and processes in 12 major branches • Robust, up-to-date synonym library • Knowledge Infrastructure tools and processes for structuring biological and chemical knowledge Enterprise KM Infrastructure 9 Ingenuity Pathways Knowledge Base Database that is the core of Ingenuity’s Solutions • The IPKB is a database that houses the most context-rich, high quality content available • Expert Extraction from full text of journals • • Current coverage of ~300 journals, plus review articles and textbooks Manually extracted by trained Ph.D. scientists • Biological Database Import: • OMIM, GO, EntrezGene • Internally curated knowledge: • • Signaling & Metabolic Pathways Drug/Target/Disease relationships • All findings structured for computation • Ingenuity Ontology covers protein, gene, protein complex, cell, cellular component, tissue, organ, small molecule & disease concepts and their interrelationships Simple Data Upload Process • Flexible upload process eliminates time consuming file formatting steps • Supports analysis of id lists with mixed identifiers •Array identifiers •Gene identifiers •Protein identifiers •Metabolomic identifiers • Stream lined workflows through 3rd Party vendors • API capabilities Discover the Biology 14 Agenda • Introduction to Ingenuity • Sample Studies: – – – – IPA in Biomarker Discovery Research IPA in Copy Number/Disease Mechanism Research IPA in Metabolic Disorders Research IPA in Metabolomics & Mol. Toxicology Research IPA in Biomarker Discovery Research • Goal of study: Identify diagnostic biomarkers for RA from rat transcriptomic and human proteomic data • Results: multiple inflammatory pathways are perturbed across all data sets (Toll Like receptor signaling and IL6 & IL10 signaling). Multiple genes associated with chemotaxis of leukocytes upregulated in both mRNA and protein levels. Putative biomarkers supported by previous experimental data findings e.g CCL5 • Specific bottleneck addressed by IPA: Anchoring of molecular profiles to phenotypes associated with RA. Integration of data from multiple platforms (transcriptomic and proteomic) and disease models (CIA in rat & Erosive RA in humans). Confirmation that putative biomarkers identified are available in suitable biofluids. Genomics data Species: rat Several pathways are commonly perturbed across multiple datasets – including IL-6 signaling, IL-10 signaling and Toll-like receptor signaling. Do known phenotypes e.g leukocyte counts in Erosive RA patients correlate with cellular processes? Proteomics data Species: human Many genes that regulate “chemotaxis of leukocytes” were elevated, giving confidence that profiles generated from both approaches can be anchored to biological processes central to rheumatoid arthritis. CCL5 expression has already been implicated in rodent models of collagen induced arthritis. In particular, Gene View’s for CCL4, CCL5, S100A8 and S100A9 confirmed that assays are available for detection of human proteins from peripheral blood samples. IPA in Copy Number/Disease Mechanism Research • Goal of study: Identify genes relevant to malignancy in melanoma samples using SNP copy number analysis • Results: IPA analysis of Chr.3 amplified region showed multiple genes associated with cancer and controlled by known cancer regulating genes. Multiple molecular relationships identified between genes amplified in Chr.3 region and genes previously associated with melanoma. • Specific bottleneck addressed by IPA: Association found between Chr3 genes amplified in copy number and cancer. Specifically between MITF and melanoma. IPA Analysis Highlights Networks of Amplified Genes Related to Cancer Genes amplified in Chr. 3 (outlined in red) directly are implicated in cancerrelated processes. They also directly regulate or are regulate by other genes known to impact cancer pathways (outlined in blue). For example: My Pathways Identifies Regulatory Events Connecting Sets of Genes Chr.3 amplified genes IPA Melanoma Genes Genes shaded gray are from the initial amplification region in Chr. 3 of melanoma cell lines. Genes with a white background are implicated in melanoma development, based on evidence from the Ingenuity Knowledge Base. Use the Connect feature to see if the Chr.3 genes are particularly enriched for connections to melanoma genes. My Pathways Reveals Strong Connections Between Genes in Amplified Region of Chr. 3 and Melanoma-Related Genes Connecting amplified region genes to melanoma implicated genes shows a connectivity dominated by MITF Many of the melanoma genes lie outside of the original amplified region of Chromosome 3 identified by Affymetrix analysis. Understanding whether those genes undergo copy number changes in the melanoma cell lines will lend further evidence that these melanoma pathways are perturbed. IPA in Metabolic Disorders Research •Goal of study: Analyse and compare gene expression profiles in skeletal muscle tissue from 8 patients with morbid obesity (MO) to identify molecular mechanisms associated to abnormalities in energy expenditure. •Results: Perturbed energy pathways such as Cardiac ß -adrenergic signaling and biological functions associated to dysregulated genes. Genes associated with synthesis of protein identified in skeletal muscle tissue from morbid obesity patients •Specific bottleneck addressed by IPA: The IPA CoreTM and Comparison analysis identified pathways and biological functions associated to dysregulated genes in skeletal muscle tissue from a subset of morbidly obese patients. Patients subgrouped based on relevant biological functions and canonical pathways Canonical Pathways across patients Cardiac ß -adrenergic Signaling:MO4 Impaired Cardiac ß adrenergic signaling in skeletal muscle tissue is associated with defects in diet-induced thermogenesis that play a role in the development of morbid obesity. Multiple Canonical Pathways cluster across patients 4-7 Comparison Functional Analysis groups patients and shows role for protein biosysnthesis in obesity Protein synthesis scores significantly in a subset of MO patients (MO4MO7). Protein synthesis capabilities arein thus Declines the rate of impaired in subset ofproteins MO biosynthesis of patients that includes in skeletal muscle tissue individuals MO4-MO7. are correlated with the onset of insulin resistance and obesity. Also, biosynthesis of protein is an important "futile cycle" that may function in adaptive thermogenesis and protection from dietinduced obesity through the consumption of ATP. Networks show clustering of dysregulated genes around a single controlling node Protein Synthesis featured heavily for a single network in patients 4-7 MYCN forms a common link between most of the downregulated genes related to the synthesis of protein E It seems likely that MYCN activity is down-regulated in skeletal muscle tissue of patient MO4, and that reduced activity of MYCN protein is responsible for the lowered expression levels of many of the genes related to synthesis of protein IPA in Metabolomics & Molecular Toxicology Research • Goal of study: Monitor global changes in metabolite levels in the liver and serum of AAP treated mice to obtain insights into the disruption of metabolic pathways related to hepatotoxicity and to identify potential biomarkers of acute liver injury. Compare and contrast with a similar study investigating the gene expression changes associated with AAP treated mice. • Results: Glutathione and Taurine Metabolic pathways implicated in cellular response to AAP exposure at both metabolite and mRNA level. Established toxicological pathologies associated with perturbed metabolites and genes. • Specific bottleneck addressed by IPA: overlaying and analysis of metabolites and genes together onto established metabolic pathways. Proprietary and Confidential 28 Glutathione Metabolism in Context of Metabolite and Transcriptome Data Significant downregulation of glutathione (GSH) as well as glutathione disulfide (GSSG) and glycine. Overlay Expression Values from gene expression study of APAP treated mice (same dose. Proprietary and Confidential 29 Functional Analysis Maps Metabolite Changes to Known APAP Induced Hepatotoxic Phenotypes Increases and decreases in metabolite levels fits well with normal role of metabolites in liver damage. Proprietary and Confidential 30 Effects on Function Analysis Highlights Potential Impact of Metabolite Changes Understand metabolite level changes in the context of their effect on cellular, organismal phenotypes. Decreased levels of glutathione suggest a net effect of increased liver damage. Proprietary and Confidential 31 Networks Highlight Interplay Between Metabolites, Genes, Pathways Networks integrate metabolic reactions with signaling and regulatory relationships. Dynamic nature of networks highlights relevance to Taurine metabolism. Proprietary and Confidential 32 Computational Pathways Analysis Accelerates Biological Understanding • Ingenuity Pathways Analysis allows scientists to – Simultaneously analyse and compare multiple datasets from different patient populations, different platforms, etc. – Gain a deeper biological understanding of the molecular and cellular mechanisms that distinguish one biological condition from another – Graphically visualise biological interactions, processes, and pathways as they change over time, dose or disease progression – Substantially decrease analysis time of microarray and other high-throughput datasets Ingenuity Systems • Free, 2 week, fully functional trial: • www.ingenuity.com • Contact details: • Adam Corner – [email protected] • Brian Dron – [email protected]