A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE REGULATORS OF NOD2 SIGNALING

1. A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE REGULATORS OF NOD2 SIGNALING Gabriel Nuñez March 7th, 2013 Department of Pathology University of Michigan

2. Outline • Introduction : NOD2 and Crohn’s Disease • Year 1 – Genome-wide siRNA Screen to identify regulators of NOD2 signaling • Year 2 - Exome sequencing of a small cohort of Crohn’s Disease patients in an attempt to identify clinically relevant components of the NOD2 signaling pathway

3. NOD2 Introduction

4. NOD2 and Crohn’s Disease SNP8 R702W SNP12 G908R SNP13 1007fs

5. (+ MDP) (+ MDP) (+ MDP) Genome-wide siRNA Screening Strategy

6. siRNA Screen Schematic siRNA Library (20nM, 48 hours) Total - 68 plates - 18,110 genes IL-8 ELISA Assay (n=2) Fluorescence based Cell Viability Assay (n=3) Luminescence based NF-κB Assay (n=3)

7. NF-κB Luciferase inhibition and Viability Data Normal (Bell) Distribution (RIPK2) Pos Regs Neg Regs (Non-targeting) Normal (Bell) Distribution (Non-targeting) (Lysed)

8. siRNA screen identifies known components of the NOD2 signaling pathway (Literature)

9. Known Components of the NF-kB signaling pathways such as the Proteasome and Nuclear Pore were hits in the screen Ref: Warner et al., Sci. Signal. 6, rs3 (2013)‏

10. Genes that interact with core components of the NOD2 signaling pathway were hits in the screen Ref: Warner et al., Sci. Signal. 6, rs3 (2013)‏

11. Comparison of Luciferase and IL-8 assay hits Top 500 Negative Regulators Top 500 Positive Regulators Positive Regulators (906) Luc IL-8 Luc IL-8 Luciferase IL-8 Overlap 94 genes Legend Increased Signal Not significantly changed Decreased Signal Genes Negative Regulators (928) Luciferase IL-8 Overlap 77 genes

12. Pos Regs CD/IBD loci taken from Jostinset al., (2012). Nature. 491:119. 9 of these genes were also IL-8 hits, 9 more IL-8 specific hits are present in CD/IBD risk loci (not shown)

13. Are any of these putative NOD2 regulators involved in Crohn’s Disease? n = 71 U of M CD patients Michigan IBD patient databank (Dr. Ellen Zimmermann) Strategy: select CD patients whose monocytes are impaired in their response to MDP despite WT NOD2 Patient stratification based on MDP response and NOD2 genotype Group 1 – WT NOD2 that responded normally to MDP Group 2 – homozygous for CD associated NOD2 mutations – MDP unresponsive Group 3* – WT NOD2 but unresponsive to MDP (8 patients – Exome sequencing) * HYPOTHESIS: some other component(s) of the NOD2 signaling pathway may be disrupted

14. Ongoing follow up of Crohn’s disease SNPs identified as validated NOD2 regulators • 1021 coding SNPs were identified in genes that hits in our siRNA screen • 135 coding SNPs were identified in genes that were validated hit from our secondary siRNA screen • mass array genotyping of 72 prioritized SNPs from 43 genes is being carried out in a larger cohort of • nearly 6000 CD patients and healthy controls to identify risk alleles (Judy Cho, NIDDK IBD consortium)

15. Overall Conclusions from the siRNA screen • - we identified and validated 211 genes that either positively or • negativelyregulate NOD2 signaling • - 82 of the validated hits affected both NF-κB induction • and IL-8 secretion • - 22 genes identified as NOD2 regulators in our screen are • found in loci known to be associated with CD risk by GWAS • consistent with an important role for the NOD2 pathway in • the pathogenesis of Crohn’s disease. • - SNPs from 43 genes validated as NOD2 regulators are being • genotyped in a cohort of nearly 6000 Crohn’s disease patients • and healthy controls