CAN EMERGING GENOMIC DATA BE LEVERAGED FOR NASH THERAPEUTICS

Arun J. Sanyal MBBS, MDZ Reno Vlahcevic Professor of Medicine, Physiology and Molecular Pathology

Virginia Commonwealth University School of Medicine

Richmond, VA

+ =

Conflicts of Interest

• Dr. Sanyal is President of Sanyal Biotechnologies

• Stock options for Genfit, Tiziana, Indalo, Durect, Exhalenz, Galmed

• Consultant- Gilead, Intercept*, Allergan*, Lilly, Novo Nordisk, Astra Zeneca-Medimmune*, Novartis, Pfizer, Genentech*, Merck, Bristol Myers*, Boehringer Ingelhiem*, Immuron*, Echosense, GE, OWL*, Birdrock, Tern, Sundise, RedX*, IFMO, Lipocine*, Innovate*, Zydus*, AMRA, Hemoshear,

• Grant support: Bristol Myers, Intercept, Gilead, Allergan, Merck, Echosense, Novartis, Boehringer Ingelhiem

* no financial remuneration in last 24 months

Genetics influences the development and progression of NASH

Biochimica et Biophysica Acta 1812 (2011) 1557–1566

PNPLA3 is associated with steatohepatitis

Sookoian and Pirola, Hepatology 2011 Jun;53(6):1883-94.

The impact of genetic risk variants on liver damage is proportional to that on hepatic fat accumulation

Standardized coefficients are adjusted for age,

sex, BMI, enrolment criteria or ethnicity

Linear relationship between liver steatosis and fibrosis

PNPLA3

TM6SF2

GCKR

MBOAT7

VARIANT

Dongiovanni, Mancina, Stender J Int Med 2017

• HSD17B13 rs72613567:TA is a common splice variant (AF ~26%).

• The rs72613567:TA variant is associated with:

• Decreased Liver Transaminases

• Reduced risk of alcoholic and non-alcoholic liver disease and cirrhosis.

• Protection from NASH in individuals with fatty liver.

N. S. Abul-Husn et al. NEJM 2018; 378(12)

HSD17B13: A Novel Genomics-Driven Drug Target

HSD17B13

SERPINA1

TM6SF2

PNPLA3

SAMM50

GPT

ERLIN1

PNPLA3

SERPINA1GOT1

HSD17B13

SAMM50

SLC39A12

Alanine aminotransferase Aspartate aminotransferase

Silencing PNPLA3 reduces lipid accumulation

Linden et al, Mol Metab. 2019 Apr; 22: 49–61.

So how does PNPLA3 cause steatohepatitis?

Study design for overexpression of PNPLA3 wildtype (WT) or PNPLA3 I148M in DIAMOND Model

Chow diet

Western diet

Chow diet

Western diet

Chow diet

Western diet

8 weeks on diet

Endpoints measured:• Weight, Insulin resistance, markers of liver injury• Gross appearance of liver• Histology: H&E, Sirius red• Assessment of pathways known to be relevant in human NASH:

• Lipid metabolism (FAS, ACC); Oxidative stress (nrf2); ER stress (eif2a, CHOP, GRP78); Apoptosis (C3, PARP cleavage); Inflammation (JNK, NFkB, p38 MAPkinase, ERK); Fibrosis (Collagen 1 and III, MMP13)

Confirmation of liver-specific PNPLA3 expression

Mouse

GAPDH

Human

PNPLA3

PNPLA3 protein expression in liver

Liver-specific human PNPLA3 expression

I148M mutant PNPLA3 induced hepatomegaly but did not worsen insulin resistance

0.00

0.02

0.04

0.06

CDNW

AAV luc WT

pnpla3

MT

pnpla3

AAV luc WT

pnpla3

MT

pnpla3

WDSW

Liv

er/

bod

y w

eigh

t ra

tio (

g)

*

*

Blo

od

glu

cose

(m

g/d

l)

Time (min)

0 15 30 45 60 75 90 105 1200

50

100

150

Insulin-tolerance test

Mutant PNPLA3 increased steatohepatitc activityC

DN

WW

DS

W

AAV luc PNPLA3 WT PNPLA3 I148MA Figure 2

0

1

2

3

His

tolo

gic

al sco

re

CDNW WDSW

B

0.0

0.5

1.0

Steatosis

CDNW WDSW

C

0

1

2

CDNW WDSW

#D

0

1

2

3

4

5

6

CDNW WDSW

##E

AAV lucPNPLA3 WT

PNPLA3 I148M

Hepatocellular ballooning score

Lobular inflammation

NAFLDActivity score

0

25

50

75

100

Normal

NAFL

NASH

CDNW

AAV luc WT

pnpla3

MT

pnpla3

AAV luc WT

pnpla3

MT

pnpla3

WDSW

Mutant PNPLA3 accelerated fibrosis

AAV lucPNPLA3 WT

PNPLA3 I148M

CD

NW

WD

SW

0.0

0.5

1.0

1.5

2.0

2.5

% p

er a

rea

CDNW WDSW

#H

Fibrosis CollagenG

0.0

0.5

1.0

1.5

2.0

CDNW

AAV luc WT

pnpla3

MT

pnpla3

AAV luc WT

pnpla3

MT

pnpla3

WDSW

His

tolo

gy s

core

**

Defining the impact of mutant PNPLA3 on metabolome and transcriptome in high fat-refined sugar diet (AAV-luc vs mt PNPLA3)

I148M PNPLA3 induces multiple metabolomics changes under Western diet conditions (vs AAV-luc)

DAGs

Oxidized AA-metabolites

nucleotides

ceramides

Plasmenyl-PC/PE

Plasmanyl PC/PEc h o w d i e t w e s t e r n d i e t

0

5 0

1 0 0

1 5 0

g r o u p s

inte

ns

ity

un

its

A A V l u c

A A V - W T P N P L A 3

A A V - M T P N P L A 3

*

triglycerides

(ADP, AMP, GMP)

• Depletion of FFA• Increased oxidized FFA• Increased AMP• Increased acylcarnitines• Decreased glutathione• Increased ceramides

WDSWPNPLA3I148M

PNPLA3WT

PNPLA3I148M

vrs AAV-Luc

PNPLA3WT

vrs AAV-Luc

C

Figure 3

Student’s t-

test (p)

log2(fold

change)

Metabolic class

Oxidized FA

Arachidonic acid-derived

oxidized FA4.98E-02 0.638

Glycerolipids

Diacylglycerols 1.29E-02 0.864

Saturated Diacylglycerols 1.10E-02 0.919

Ratio

PUFA/SFA 1.93E-02 -0.377

SM/PC 6.57E-03 -0.389

Cer/SM 2.16E-02 0.459

TG/DG 1.26E-02 -0.391

PNPLA3 I148M vs. AAV-LUC on WDSW

Gene-ontology analysis reveals an immune-inflammatory signature induced by mutant PNPLA3

Activation of STAT3 and the acute phase response by I148M PNPLA3

β actin42 kD

P-Stat3

(T705)Stat386 kD

CD WD

EV WT MT EV WT MT

STAT1 activation signature induced by mutant PNPLA3

P= 5.64e-54

Z score= 91.5

Activation of class II MHC with mutant PNPLA3

Immune reponse:

MHC Class II network activation

P= ?

Z score= ?

Cell-signaling pathway activation with disease acceleration with mtPNPLA3

Mutant PNPLA3 increases lipogenic drive, cell stress, inflammatory signals

LUC WT I148M LUC WT I148M

CDNW WDSW

PNPLA3 (I148M) activates fibrogenic signaling

AA

V l

uc

CD

AA

V l

uc

WD

AA

V- W

T C

D

AA

V- W

T W

D

AA

V- M

T C

D

AA

V- M

T W

D

0

1 0

2 0

3 0

4 0

5 0

6 0

mR

NA

(c

or

re

cte

d f

or

GA

PD

H)

*

*

0

1

2

3

4

CDNW WDSW CDNW WDSW CDNW WDSW

Vector wPNPLA3 mPNPLA3

SMA

SM

A m

RN

A

(fo

ld c

han

ge)

*

Coll 1A

42 kD

CD WD

EV WT MT EV WT MT

42 kD

46-48 kD

β actin

α-SMA

TGF-β1

CD WD

EV WT MT EV WT MT

Establishing a rational basis for using suppression of mtPNPLA3 expression as a therapeutic option

Can silencing PNPLA3 rescue the phenotype?

Mt-PNPLA3introduced

Scrambledsequences

siRNA(mt-PNPLA3)

LUC si-Scr si-PNP si-Scr si-PNP

B WDSW

GAPDH

Stat3

p-Stat3

PNPLA3

PNPLA3WT PNPLA3I148M

53

86

86

37

MW

(KD)

Silencing PNPLA3 rescues disease phenotype

0.00

0.02

0.04

0.06

His

tolo

gy

sco

re

Steatosis

*

#

PNPLA3 WT PNPLA3 I148M

His

tolo

gy

sco

re

0.0

0.5

1.0

Hepatocyte

Ballooning*

##

PNPLA3 WT PNPLA3 I148M0

1

2

Lobular Inflammation

*

#

His

tolo

gy

sco

re

PNPLA3 WT PNPLA3 I148M

0.00

0.25

0.50

0.75Fibrosis

His

tolo

gy

sco

re

PNPLA3 WT

H

Effect of mtPNPLA3 on ceramide pathways

CERAMIDE

SERINE + PALMITATE

3-KETO DIHYDRO SPHINGOSINE

SPHINGOMYELIN SPHINGOSINE

SPTLC

KDSR

SMPD CERS

DIHYDRO SPHINGOSINE

CERS

C E R S 2 C E R S 4 C E R S 5 C E R S 6

0

1

2

3

4

Re

lati

ve

mR

NA

e

xp

res

sio

n

W D -W T

W D -M T

W D -s iM T

**

* ***

*

Summary: How mutant PNPLA3 leads to NASH and fibrosis

Oxidized FA

DAG

Ceramides

α-SMA

Col1 and 3

Immune activation

p-JNK

p-STAT3

Fibrosis

Oxidative stressGSH/GSSG

TGF-β1

I148MHigh

fat diet

Sphingomyelin

Sphingosine

KDSR

Inflammation