Integration of functional genomics & pathway information to - PowerPoint PPT Presentation

Integration of functional genomics & pathway information to elucidate deregulation of signal transduction and drugs' mode of action Michael Schubert & Julio Saez-Rodriguez European Bioinformatics Institute Hinxton (Cambridge) UK www.ebi.ac.uk/saezrodriguez 1 EBI is an Outstation of the European Molecular Biology Laboratory

How do cells process extracellular signals? Ligand EGF Receptor EGFR Shc Grb2/Sos Ras Mediators (kinases, adaptors,...) Raf MEK ERK Transcription factors fos Gene expression Phenotype

How do cells process extracellular signals? EGF EGFR Shc Grb2/Sos Ras Raf MEK ERK fos Phenotype

How do cells process extracellular signals? il6 tnfa il1a il1a ifn tgfa igf il1r il6r tnfr il1r ifn egfr igfr a20 irs1t shc pip2 jak1 irs1s jak2 rasgap pi3k grb2 stat3 stat1 pip3 traf6 pp2a sitpec sos traf2 pdk1 ras rac akt casp9 pak stat33 stat13 stat11 cot ask1 raf1 map3k1 map3k7 nik gsk3 ikk mek12 mkk7 mkk3 mkk4 mkk6 mtor jnk12 p38 erk12 ikb prak p90rsk hsp27 p70s6 nfkb p90rskn p38n jnk12n erk12n mdm2 p53 atf2 creb ck2 msk12 nfkbn elk1 cjun stat1n stat3n atf1 histh3 cfos Phenotype

How is signal processing altered in disease? Normal Diseased                                                                                                                                                             Can we revert disease phenotype ... or target diseased cells with new therapies?

(Some) challenges in drug discovery • Identify drug targets for a certain disease • Characterization of mode of action Identifying the molecular ? pathways targeted by a compound and its off- target effects Dissecting what follows functionally the drug/substrate ? interaction

There is information available at different levels Healthy Genetic Diseased DNA alteration Both Environment mRNA Proteins Network Phenotype

Characterization of drugs at biochemical level with mechanistic models of signalling networks DNA mRNA Proteins Perturb with drugs and ligands Network Measure phosphorylation of proteins Phenotype

Comparison of primary hepatocytes and hepatocelluar carcinoma using logic models a. Specific Networks Focus Primary HepG2 Hep3B Huh7 Drug-target Analysis Disease Classifiers Comparative Network Analysis Generic Scaffold of network logical models Primary HCC Model Graph Process Train to Cell Response CNO Data high high low low 0.11 0.15 0.095 0.17 N.A. 0.46 0.48 Only HCC Both Connectivity Add links Only Primary New link Error that lower error 0.10 0.08 Saez-Rodriguez J, Alexopoulos LG, Zheng M, Morris MK, Lauffenburger DA, Sorger PK, Cancer Research 71(16), 2011

Experimental design to characterize differences between healthy and cancerous liver cells Construct map of canonical il6 il6 tnfa il1a il1a lps lps ifn ifn tgfa tgfa tnfa igf igf pathways il1r il1r il6r tnfr tlr4 tlr4 il6r ifn ifn igfr igfr tnfr egfr egfr irs1t irs1t a20 a20 shc shc pip2 pip2 jak1 irs1s irs1s jak2 jak2 jak1 rasgap rasgap pi3k pi3k grb2 grb2 stat3 stat3 Select stat1 stat1 pip3 pip3 traf6 traf6 pp2a pp2a sitpec sitpec sos sos traf2 traf2 pdk1 pdk1 ras ras rac rac - perturbations akt akt casp9 casp9 pak pak (chemical inhibitors = drugs) stat33 stat13 stat11 stat33 stat13 stat11 cot cot ask1 ask1 raf1 raf1 map3k1 map3k1 map3k7 map3k7 & nik nik GSK3 gsk3 - signals (phosphorylations ikk ikk mek12 mkk7 mek12 mkk7 mkk3 mkk3 mkk4 mkk4 mkk6 mkk6 measurable with Luminex/xMAP mtor mtor jnk12 jnk12 technology) p38 p38 erk12 erk12 ikb ikb prak prak p90rsk p90rsk as distributed in the network as hsp27 hsp27 possible p70s6 p70s6 nfkb nfkb p90rskn p38n p90rskn p38n jnk12n erk12n jnk12n erk12n mdm2 mdm2 Stimulus Perturbation p53 p53 atf2 atf2 creb creb ck2 ck2 msk12 msk12 nfkbn nfkbn Readout Perturb&Read elk1 elk1 cjun cjun stat1n stat1n stat3n stat3n atf1 atf1 histh3 histh3 cfos cfos

Differences between normal and transformed hepatocytes: targets for therapies? TGF α Ins TNF α All Primary HCC lines EGFR INSR TNFR some HCC TGF 5 Grb2 TRAF2/ IRS-1 MAP3K7 pERK12 0 PI3K 2 p38 ERK1/2 { pHSP27 MAP3K7 Rac 0 Prak PDK1 Fold increase of signal Insulin MAP3K1 NIK 1 { 18 HepG2 Hep3B { Primary HSP27 AKT pAKT Hu7 Focus AND 0 5 GSK3 TNF TGF ,TNF IKK pGSK3 PI3Ki PI3Ki 1 NFAT cat 0 I B I b 0 NF B Only active in HCC cell lines: Difference in NFkB activation: ! ! Insulin → .. → AKT → GSK3 TNF dependent only in HCC TNF+TGF α in primary HSP27 phosphorylation: ! ERK mediated in primary Saez-Rodriguez J, Alexopoulos LG, Zheng M, Lauffenburger DA, Sorger PK, Cancer Research 71(16) 1-12, 2011

Characterization of drug mode of action at biochemical level Identification of off-target effect of Gefitinib (EGFR inhibitor) on IL1-alpha pathway (cJun activation) Mitsos et al PLoS Comp Bio 2009

Characterization of drug mode of action at biochemical level + Precise characterization at biochemical level - Limited scope (measurement limitations) - No direct connection to phenotype Identification of off-target effect of Gefitinib (EGFR inhibitor) on IL1-alpha pathway (cJun activation) Mitsos et al PLoS Comp Bio 2009

Genome-wide, non-mechanistic characterization of drugs using gene expression DNA mRNA Proteins Perturb with drugs Network Measure changes in Phenotype gene expression

Use drug- & disease-induced transcriptional changes for drug discovery & repurposing (A) (C) + expression gene 0 GEO MSigDB - Array 1 2 3 4 n cMap _ _ _ _ _ Express e e e e e n n n n n e e e e e g g g g g Disease + drug Drug gene Experiment gene expression expression signature signature Disease signature Drug response signature (B) + expression gene 0 - 1 2 3 4 n Disease Drug _ _ _ _ _ e e e e e n n n n n signature signature e e e e e g g g g g F Iorio T Rittman H Ge Disease signature Analyse with approach (A) M Menden => disease-to-drug matching J Saez-Rodriguez Drug A response signature Drug Discovery Analyse with approach (B) Drug B response signature Today, in press => drug-to-drug matching

DvD: An R/Cytoscape pipeline for drug repurposing using public repositories of gene expression data - Compare drug & disease signatures with dynamic access to databases (Array Express, GEO), and Connectivity Map Pacini C Iorio F Gonçalves E Iskar M Klabunde T Bork P Saez-Rodriguez J, Bioinformatics, 2013

Signature matching: e.g. Topiramate (anticonvulsant) identified as treatment for IBD * Prednisolone = established compound for Crohn’s disease ** Trinitobenzene Sulfonic Acid (TNBS) Drug-disease score A 0 5186324 Topiramate Prednisolone Vehicle only TNBS + vehicle 12,13-EODE Tolbutamide Yohimbine 5213008 Tomelukast Phenanthridinone 5162773 5151277 TNBS + topiramate TNBS + prednisolone Clotrimazole 5140203 B 5 Gross pathology score Genistein Fasudil 4 5230742 3 **** 5182598 2 1 0 TNBS + veh TNBS + top TNBS + pred Vehicle Dudley et al, Sci Trans Med 2011

Guilt by association: fasudil (vessel obstructions) identified as enhancer of autophagy Iorio et al, PNAS 2010