Hal Dietz, MD

Victor A. McKusick Professor of Medicine and Genetics

Investigator, Howard Hughes Medical Institute

Johns Hopkins University School of Medicine

Marfan Foundation Annual Conference 2017

Research Update

“Listening to Nature’s Cues”

Marfan syndrome

Fibrillin-1

Dietz…and Francomano Nature, 1991

MFS LAP

LAP

L T

B P

TGFβ

Latent Complex

Neptune et al. Nature Gen, 2003

Judge et al. JCI, 2004

Ng et al. JCI, 2004

Habashi et al. Science, 2006

Cohn et al. Nature Med, 2007

Excessive TGF

Activation

P P

pSmad2/3

Smad4

P

TGFβ

Smad2/3

pSmad2/3

Smad4

P

TF

cytoplasm

nucleus

Excessive TGF

Signaling

Emphysema

Aortic Aneurysm

Mitral Valve Prolapse

Myopathy

Phenotypic

Consequences

Microfibrils composed of

Fibrillin-1

(All rescued by

systemic Rx of

MFS mice with

TGF-neutralizing

antibody)

Fibrillin-1 Mutations Lead to Excessive TGFβ Signaling in MFS

LAP

LAP TGFβ

L

T

B

P

AngI

AngII

AT1 AT2

­TSP-1

­TGFb signaling

­Proliferation

¯Apoptosis

­Fibrosis

­MMP2, MMP9

¯Proliferation

­Apoptosis

¯Fibrosis

¯MMP9

­TGFb ligands

­TGFb receptors

The Angiotensin II Type 1 Receptor Blocker (ARB) Losartan

Habashi…and Dietz, Science, 2006

Losartan

Aortic root growth

(mm/6 months)

Aortic wall thickness

(micrometers)

Aortic wall architecture

(score 1-4)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Gro

wth

(m

m)

p = 0.55

p < 0.0001

p < 0.0001 p < 0.001 p < 0.02

Wild-type

n = 11

Placebo

n = 10

Propranolol

n = 7

Losartan

n = 5

C1039G/+

50

60

70

80

90

100

110

120

(m

icro

me

te

rs

)

p = 0.67

p < 0.002

p < 0.0001 p = 0.17 p < 0.05

Wild-type

n = 11

Placebo

n = 10

Propranolol

n = 7

Losartan

n = 5

C1039G/+

1

1.5

2

2.5

3

3.5

4

Sc

ore

p = 0.23

p < 0.002

p < 0.0001 p = 0.47 p < 0.05

Wild-type

n = 11

Placebo

n = 10

Propranolol

n = 7

Losartan

n = 5

C1039G/+

Aort

ic R

oot

Gro

wth

pSmad2

β-Actin

Wild-type C1039G/+

Losartan

C1039G/+

Placebo

Wild-type

C1039G/+

Placebo

C1039G/+

Propranolol

C1039G/+

Losartan

Lacro et al. NEJM, 2014 (mean z=4.0)

Atenolol

(mean 2.8; up to 4.0mg/kg/day) +0.069cm/yr -0.14z/yr

Losartan

(mean 1.2; up to 1.4mg/kg/day) +0.075cm/yr -0.11z/yr

Untreated pediatric patients:

Ladoucer et al., 2007 +0.11cm/yr

Rossi-Foulkes et al., 1999 +0.18cm/yr

Tahernia et al., 1993 +0.16cm/yr

Salim et al. 1994 +0.21cm/yr

Treated with typical atenolol dosing:

Brooke et al., 2008 (mean z=3.25) +0.17cm/yr +0.24z/yr

Brooke et al., 2008 (mean z=7.21) +0.35cm/yr +0.46z/yr

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 2 3 4 5 6 7WT

n=11

Placeb

o

n=16

Low Los

5mg/kg

n=10

High Prop

40mg/kg

n=9

Med Prop

20mg/kg

n=8

High Los

50mg/kg

n=12

Med Los

25mg/kg

n=11

Ao R

oot

Gro

wth

(m

m /

6 m

onth

s)

MFS

<0.001

NS NS

<0.05

NS

<0.001

<0.001

<0.01

<0.05

<0.0001

<0.00001 <0.000001

P P

TGF

P P

Canonical TGFβ

Signaling

pSmad2/3

Smad4

P

pSmad2/3

Smad4

P

MEK P

ERK P

Noncanonical TGFβ

Signaling (MAPK)

JNK P

p38 P

TAK P

Activation of ERK MAPK in Marfan Mice

pSmad2

Smad2

pERK1/2

ERK1/2

pMEK1 MEK1

β-Actin

WT C1039G/+

Holm et al. Science, 2011; Habashi et al. Science, 2011

TGFβ

ShcA

MEK1

pERK1/2

RDEA119 Plac RDEA Plac RDEA

Wild Type C1039G/+

-0.10

0.00

0.10

0.20

0.30

1 2 3 4

p<0.05

n=6 n=9 n=6 n=12

p<0.05

p<0.0001

p=0.15

p=0.91

2m

o A

ort

ic R

oo

t G

row

th (

mm

)

Pregnant women with MFS or LDS have an elevated risk of aortic tear.

While this risk has historically been attributed to hemodynamic stress, the

vast majority of dissections occur within the weeks after delivery, and this

risk is not altered by Cesarean section or antihypertensive agents.

What initiates toward the

end of pregnancy, is

maintained after delivery

and might synergize with

pathogenic events

previously defined for

aneurysm and dissection?

Oxytocin

- needed to initiate uterine contraction

and milk letdown

- release peaks at the end of pregnancy

and is sustained during breast feeding

- receptor upregulated in the aorta in

response to estrogen and pregnancy

- mediates its effects on peripheral

tissues through ERK1/2 activation Habashi et al. unpublished

Day Post Partum

Pe

rce

nt

Su

rviv

al

p<0.01

From 4% to 70% survival simply by

removing pups at birth (eliminating

lactation-induced oxytocin release)

Profound Protection from Postpartum Aortic Dissection Simply

by Removing Pups at Birth (and Prevention of Lactation-

Induced Oxytocin Release)

Habashi et al. unpublished

desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT

Oxytocin antagonist

(150X greater potency for the oxytocin vs. vasopressin receptor)

Pe

rce

nt

su

rviv

al

No Rx

Oxytocin antagonist

p < 0.001

Administration of an Oxytocin-Blocking Drug in the 3rd Trimester

and After Birth Provides Full Protection from Aortic Dissection

(Despite Retained Ability to Both Deliver Pups and Breast Feed)

Habashi et al. unpublished

Perc

en

t S

urv

iva

l

Day Post Partum

Predisposition for Pregnancy-Associated Aortic Dissection

is Proportional to ERK Activation

Habashi et al. unpublished

pERK1/2

β-Actin

WT

WT

w/pups MFS

MFS

w/pups

MFS

pups

removed

MFS

w/pups

+antag

WT WT

w/pups

MFS MFS

w/pups

MFS

pups

removed

MFS

w/pups

+antag

pE

RK

1/2

/ β

-Actin

p<0.005 p<0.05

p=0.05 p<0.05

p<0.05

2.5

2.0

1.5

1.0

0.5

0.0

Trametinib – FDA approved ERKi

P<0.0001 Pe

rcen

t S

urv

iva

l

Postpartum Day

mgR-/-preg (n=45)

mgR-/-preg, ERKi (n=20)

32 genes in the critical interval

MAP3K4 (encodes a MAPK kinase –

the kind of protein that activates

ERK)

Five exceptional families with defined FBN1 mutation showing discrete

intrafamilial variation in phenotypic severity were subjected to linkage

analysis to map a protective modifier locus for Marfan syndrome.

Chromosome 6 Position (cM)

Pa

ram

etr

ic L

OD

Sco

re

LOD=4.044

Alex Doyle

(w/ Bart Loeys, Julie DeBacker and Anne De Paepe)

How does nature modify Marfan syndrome?

MA

P3K

4 /

β-A

CT

IN

0

1

2

3

1 2 3

<0.05

<0.01

<0.001

Control

Marfan

Mild Severe

Protection from Vascular Disease in MFS Associates with Reduced

Expression of MAP3K4 in Cultured Dermal Fibroblasts

Map3k4 haploinsufficiency abrogates abnormal aortic root

growth in a mouse models of MFS A

oR

Gro

wth

(m

m/8

mo

)

0

0.2

0.4

0.6

1 2 3 4

p<0.0002

NS

p<0.01

NS

WT M3K4+/− MFS MFS

M3K4+/−

Wild-Type M3K4+/− Marfan Marfan M3K4+/−

MAP3K4

pERK1/2

pp38

pJNK1/2

β-Actin

0.0

0.5

1.0

1.5

1 2 3 4 0.0

0.5

1.0

1.5

2.0

1 2 3 4

<0.05

NS

<0.05 <0.01

NS

<0.05

0.0

0.5

1.0

1.5

1 2 3 4

<0.05

NS

<0.05

Wild-Type

M3K4+/−

Marfan

Marfan

M3K4+/−

MAP3K4 pERK1/2 pp38

AT1R AT2R

PLC

DAG IP3

PKC

pERK1/2 (+/- pJNK, pp38)

TGFβ

Definition of an Axis for Vascular Disease in Marfan Syndrome

Enzastaurin

CCB

RDEA119/

MAP3K4i

Hydralazine

Losartan

NAb

Prevents Aneurysm (7)

AT2KO

Worsens Aneurysm (3)

OXTR Oxytocin

OXTR blocker

Target Genes

Angiotensin-II

Doyle & Doyle et al. unpublished

Modification of Marfan Syndrome in Mice:

The C57BL/6J (BL6) mouse background is protected

The Sv129 mouse background shows accelerated vascular disease

0.0

0.2

0.4

0.6

0.8

1.0

1 2 3 4 BL6 Sv129 BL6 Sv129 Wild-type C1039G/+

AR

Gro

wth

: 2

-6m

o (

mm

)

p=0.77

p=0.03

p<0.0001 C1039G/+; BL6 C1039G/+; Sv129

AorootAoroot

0 1 2 3 4 5 6 7 8 9 10

Months of Age

100

80

60

40

20

0

% S

urv

ivin

g

WT C57BL6/J

WT Sv129

MFS C57BL6/J

MFS Sv129

0

50

100

150

1 2

Blo

od

Pre

ssure

(m

mH

g)

BL6 Sv129

NS

Systolic

Diastolic

0

250

500

750

1 2

BL6 Sv129

NS

Pu

lse

Ra

te

(bp

m)

Doyle & Doyle et al. unpublished

The predisposition imposed by the Sv129 background remains

responsive to both losartan and ERK1/2 inhibition (RDEA-119)

-0.2

0.2

0.6

1.0

1 2 3 4

Plac Los Plac Los

Wild-type C1039G/+

<0.0001

<0.00001 <0.00001

AR

Gro

wth

: 2

-6m

o (

mm

)

-0.2

0.0

0.2

0.4

0.6

1 2 3 4

<0.001

<0.001 <0.01

AR

Gro

wth

: 2

-4m

o (

mm

)

Plac RDEA Plac RDEA

Wild-type C1039G/+

Wild-Type C1039G/+

C57BL6/J Sv129 C57BL6/J Sv129

pSmad2

pSmad3

pPKCα/β

pPKCδ

pPKCθ

pRAF

pMEK

pERK1/2

β-Actin

Smad/PKC/ERK Activation is Increased in the Aortic Root

of Marfan Mice on a Sv129 Background

Doyle & Doyle et al. unpublished

0

20

40

60

80

1 2 3 4

Me

an

Lin

ear

Inte

rcept (µ

m)

NS

<0.001

<0.01

BL6 129 BL6 129

Wild-Type

Marfan

WT BL6 WT 129 Marfan BL6 Marfan 129

<0.0001

50um

0

5

10

15

20

1 2 3 4

Kyphosis

Index

(mm

) NS

<0.0001 <0.05

BL6 129 BL6 129

Wild Type

Marfan

WT BL6 WT 129 Marfan BL6 Marfan 129

<0.00001

Single-point QTL genome scan

0

1

2

3

4

Chromosome

lod

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X

3.82

LOD – 4.76

p=0.008 (genome adjusted)

LOD – 4.78

p=0.008 (genome adjusted)

Chromosome

4

3

2

1

0

LO

D

5 11

Pronounced epistasis – LOD=12.8 when 2 loci are considered in combination

Genes of interest – Mouse Chromosome 5:

Rabgef1 Integrator of TGFβ & RAS-ERK signaling

Rasa4 Ca+2-dependent Ras GTPase-activator that inhibits ERK

Mmp17 Regulates ERK activation; p.X579W

Genes of interest – Mouse Chromosome 11

Bptf Regulates TGFβ-dependent embryonic events and ERK

Prkca Ca+2-activated protein kinase that directly activates ERK

Map2k6 Activates MAPKs & activated by Map3k4 (human modifier!);

p.G76E

Modifier Candidate Genes:

Theme – integration of Ca+2 / TGFβ / MAPK signaling

Map2k6

Mmp17

129 +/- +/- -/- BL6 +/+ +/+

129 +/+ +/- -/- BL6 +/- +/+

Dosage of knockout alleles

Total 0 0 1 1 2 4 4

1.2

1.5

1.8

2.1

2.4

1 2 3 4 5 6 7

<0.01

MFS

Pure 129

Mixed

Pure BL6

NS

Aort

ic R

oot

at 2m

o (

mm

)

1.2

1.5

1.8

2.1

2.4

1 2 3 4 5 6 7

NS

Taken together, these data suggest that natural

genetic variation that attenuates TGFβ and/or

ERK1/2 activation in both people and mice with

Marfan syndrome has the ability to protect from

vascular disease.

Drugs that mimic nature’s strategies represent

novel and potentially potent therapeutic options

for the care of people with Marfan syndrome.

TGFβ antagonists

AT1 antagonists (ARBs)

ERK antagonists

Oxytocin antagonists

PLC/IP3/PKC antagonists

MAP3K4 antagonists

MAP2K6 antagonists

p300 inhibitors

EGFR antagonists

Optimistic prospects for disease treatment

Weak tissues Obilgate Tissue Failure

Dietz lab: Hamza Aziz

James Beckett

Ben Brooke

Juan Calderon

Sara Cooke

Alex Doyle

Jef Doyle

Elena Gallo

Russell Gould

Jennifer Habashi

Tammy Holm

Adam Johnson

Dan Judge

Ben Kang

Bart Loeys

Checco Ramirez

Jason Cooke

Dan Rifkin

Anne De Paepe

Julie De Backer

GenTAC Investigators

MIBAVA Investigators

Mark Lindsay

David Loch

Bart Loeys

Javid Moslehi

Sarah Parker

Rosanne Rouf

Joseph Shin

Robert Wardlow

Nicole Wilson

Jennifer van Eyk

Dave Huso

William S. Smilow Center

For Marfan Syndrome Research