1. A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE REGULATORS OF NOD2 SIGNALING Gabriel Nuñez March 7 th, 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. Genome-wide siRNA Screening Strategy (+ MDP)

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

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

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

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

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

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

12. CD/IBD loci taken from Jostins et 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) Pos Regs

13. Are any of these putative NOD2 regulators involved in Crohn’s Disease? 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 n = 71 U of M CD patients Michigan IBD patient databank (Dr. Ellen Zimmermann)

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 negatively regulate 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