A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE

REGULATORS OF NOD2 SIGNALING

Gabriel NuñezMarch 7th, 2013

Department of PathologyUniversity of Michigan

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

NOD2 Introduction

NOD2 and Crohn’s Disease

SNP8R702W

SNP12G908R SNP13

1007fs

Genome-wide siRNA Screening Strategy

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50

100

150

200

250

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450

Assay Baseline knockdown ofPOSITIVEregulator

knockdown ofNEGATIVEregulator

Luciferase Reporter activity

(+ MDP)

(+ MDP)

(+ MDP)

siRNA Screen Schematic

Fluorescence basedCell Viability Assay (n=3)

Luminescence basedNF-κB Assay (n=3)

IL-8 ELISAAssay (n=2)

siRNA Library (20nM, 48 hours)

Total - 68 plates - 18,110 genes

NF-κB Luciferase inhibition and Viability Data

Normal (Bell) Distribution

Normal (Bell) Distribution

(RIPK2)

(Non-targeting)

(Non-targeting)

(Lysed)

PosRegs

NegRegs

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

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

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

Comparison of Luciferase and IL-8 assay hits

Positive Regulators (906)

Negative Regulators (928)

Luciferase IL-8

Luciferase IL-8

Overlap94 genes

Overlap77 genes

Top 500Positive Regulators

Top 500Negative Regulators

Luc IL-8 Luc IL-8

Legend

Increased Signal

Not significantly changed

Decreased Signal

Gen

es

non Targe

ting contro

lRIPK2

RELA

UBE2D3

CCL13

DONSON

IL2RA

TNFS

F18

LIME1

DDX6

GRIN3BCAD

DAP3CIRBP

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0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

13 Validated LUC hits are present within loci associated with CD/IBD by GWAS

siRNA treatment

Nor

mal

ized

LUC

indu

ction

upo

n M

DP sti

mul

ation

NegRegs

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)

PosRegs

47

17

7

NOD2 genotyping

NOD2 +/+NOD2 +/-NOD2 -/-

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 genotypeGroup 1 – WT NOD2 that responded normally to MDP Group 2 – homozygous for CD associated NOD2 mutations – MDP unresponsiveGroup 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 patientsMichigan IBD patient databank(Dr. Ellen Zimmermann)

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)

non Targe

ting contro

l

IL27RA

NMUR2

CDC25CRGL3

DONSON

ACRV1

loc51252

flj23049

ADAMTS8

GRIN3B

ZNF2

95

Genome A

ve0

0.20.40.60.8

11.21.41.61.8

2

23 Luciferase hits

Rela

tive

Nor

m L

UC

indu

ction

(M

DP)

non targe

ting contro

l

DONSON

GABRA6

ACRV1M

C3RGPT

CDC25C

IL27RA

C1QTNF8

ADAMTS

8

PPAP2CPHF3

RGS12

ZNF2

95

GRIN3B

SUSD

2

ARRDC1

loc5

1252

DYX1C1

MSL

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tralp

ush

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40 IL-8 hits

Rela

tive

Nor

m IL

-8

indu

ction

(MDP

)

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 GWASconsistent with an important role for the NOD2 pathway inthe pathogenesis of Crohn’s disease.

- SNPs from 43 genes validated as NOD2 regulators are beinggenotyped in a cohort of nearly 6000 Crohn’s disease patients and healthy controls