Murine TGFβ-antagonist (RAP-1332) Inhibits Fibrosis in a Murine Model of Myelofibrosis

Rajasekhar NVS Suragani, PhD

Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF)

Janus Kinase2 (JAK2) V617F mutation: 95% in PV, ~50-60% in ET and PMF

• Calreticulin (CALR): 20-25% in ET and PMF

• Myeloproliferative leukemia virus (MPL) oncogene: less than 10% for ET and PMF

Central role of TGFβ signaling pathway in fibrosis1

PV ET PMF

Myelofibrosis

Post-PV Post-ET

Murine model of MPN: JAK2(V617F) transgenic expression2

Elevated RBC (PV), elevated platelets (ET), elevated WBC with progression into Myelofibrosis with age

Splenomegaly; mean survival age of ~17 months

Mutations leading to constitutively active signaling and uncontrolled proliferation of BM myeloid lineage cells and ultimately cause fibrosis

1Rosemary JA et al., Nature Reviews (2012) 2Shu Xing et al., Blood (2008)

TGFβ expression and initiation of fibrosis in the JAK2(V617F) murine model

Treatment of TGFβ antagonist (ACE-1332) in JAK2(V617F) transgenic mice

Combination treatments of RAP-1332 and ruxolitinib (JAK2 inhibitor) in JAK2(V617F) transgenic mice

Outline

JAK2(V617F) transgenic mice display elevated RBC, Platelets, WBC and splenomegaly

6

8

10

12

14

16

18

2 4 6 8 2 3 4 5 8 10 12

RB

C (

10

6 c

ells

/μL)

0

10

20

30

40

50

60

2 4 6 8 2 3 4 5 8 10 12

WB

C (

10

3 c

ells

/μL)

Wild type JAK2(V617F) Age (months)

0

400

800

1200

1600

2 4 6 8 2 3 5 8 10 12

Pla

tele

ts(1

03/μ

L)

Wild type JAK2(V617F)

Age (months)

2 4 6 8 2 3 4 5 8 10 12

wt 2

B

wt 4

B

wt 6

B

wt 7

B

JAK 2

-2M

JAK2-

3M

JAK2-

4B

JAK2-

5M

JAK2-

8M

JAK2-

10M

JAK2-

12M

0

500

1000

1500

2000

Sple

en w

eigh

t (m

g)RBC Spleen weight

WBC Platelets

Wild type JAK2(V617F) Wild type JAK2(V617F)

BM Fibrosis starts in JAK2(V617F) transgenic mice at 5-months and worsens with age

Fibrosis (Reticulin, 20x)

2 5 8 Age (months)

Serum levels of TGFβ1 and 3 but not TGFβ2 ligands are elevated in JAK2(V617F) transgenic mice

TGF1

WT 2

mos

WT 6

mos

JAK2 2

mos

JAK2 5

mos

JAK2 8

mos

JAK2 1

0mos

JAK2 1

2mos

0

2000

4000

6000

8000

10000

p

g/m

L

TGF2

WT 2

mos

WT 6

mos

JAK2

2mos

JAK2

5mos

JAK2

8mos

JAK2

10m

os

JAK2

12m

os

0

20

40

60

80

pg/m

L

TGF3

WT 2

mos

WT 6

mos

JAK2 2

mos

JAK2 5

mos

JAK2 8

mos

JAK2 1

0mos

JAK2 1

2mos

0

200

400

600

800

pg/

mL

TNFalpha

WT 2

mos

WT 6

mos

JAK2 2

mos

JAK2 5

mos

JAK2 8

mos

JAK2 1

0mos

JAK2 1

2mos

0

2000

4000

6000

pg/

ml

IL-6

WT 2

mos

WT 6

mos

JAK2 2

mos

JAK2 5

mos

JAK2 8

mos

JAK2 1

0mos

JAK2 1

2mos

0

100

200

300

400

pg/

ml

Wild type JAK2(V617F)

Age (months)

2 6 2 5 8 10 12

Wild type JAK2(V617F)

Age (months)

2 6 2 5 8 10 12

Wild type JAK2(V617F)

Age (months)

2 6 2 5 8 10 12

Wild type JAK2(V617F) Age (months)

2 6 2 5 8 10 12 Wild type JAK2(V617F)

Age (months)

2 6 2 5 8 10 12

TGFβ1 TGFβ2 TGFβ3

TNFα IL-6

ACE-1332 binds tightly to TGFβ1 and TGFβ3 ligands but not to TGFβ2

ACE-1332 is a protein biologic engineered to bind and inhibit signaling by TGFβ1 and TGFβ3 but not TGFβ2

Does not bind other ligands in the TGFβ-superfamily RAP-1332 is murine ortholog of ACE-1332

RAP-1332

TGFβ1 TGFβ2 TGFβ3

Ligands KD (pM) @ 37oC IC50 (ng/ml)

A549 RGA

TGFβ1 5.5 3.8

TGFβ2 400 No Inhibition

TGFβ3 2.2 3.1

Biacore Cell Based Assay

Genotype Sex Age

(Mos) N Treatment Dosage Frequency

Duration (months)

Wt M 4 15 TBS Iso

volume Twice

weekly 3, 6

JAK2(V617F) M 4 15 TBS Iso

volume Twice

weekly 3, 6

JAK2(V617F) M 4 15 RAP-1332 10 mg/kg Twice

weekly 3, 6

Study design: Treatment of JAK2(V617F) transgenic mice with TGFβ antagonist (RAP-1332)

Treatment initiated before the onset of fibrosis

9

RAP-1332 treatment for 3 months reduced fibrosis in BM and Spleen (JAK2 V617F mice)

JAK2 + VEH JAK2 + RAP-1332

BM

Spleen

Age of mice at study termination: 7 months

RAP-1332 treatment for 6 months reduced fibrosis in BM and Spleen in JAK2 (V617F) transgenic mice

Age of mice at study termination: 10 months

JAK2 + VEH JAK2 + RAP-1332

BM

Spleen

RAP-1332 treatment for 6 months reduced splenomegaly in JAK2(V617F) mice

* *

-29.0%

Wt JAK2(V617F) + VEH

JAK2(V617F) + RAP-1332

0

400

800

1200

1600

0 6 12 24

Plt

(1

03/m

L)

Time (weeks)

0

4

8

12

16

20

0 6 12 24

RB

C (

106

cel

ls/m

L)

Time (weeks)

Wt

JAK2+VEH

JAK2+RAP-1332

0

10

20

30

40

50

0 6 12 24

WB

C (

10

3 c

ells

/mL)

Time (weeks)

** p< 0.01 vs VEH

RBC

Spleen weight WBC

Platelets

TER-119+

Wt JAK2+VEH

JAK2+RAP-1332

RAP-1332 treatment reduced erythroid hyperplasia in Spleen and serum IL-6 levels in JAK2(V617F) mice after 6 months treatment

Wt JAK2+VEH JAK2+ RAP-1332

# # #

*

Spleen

# # # p< 0.001 vs Wt; **p< 0.01, *p< 0.05 vs JAK2+VEH

# # #

**

-48.9%

Wt JAK2+VEH JAK2+ RAP-1332

Genotype Sex Age

(Mos) N Treatment Dosage Frequency

Duration (weeks)

JAK2(V617F) M 12 9 veh Iso

volume Twice

weekly 3

JAK2(V617F) M 12 10 RAP-1332 10 mg/kg Twice

weekly 3

JAK2(V617F) M 12 10 Ruxolitinib 60 mg/kg Twice daily 3

JAK2(V617F) M 12 10 Ruxolitinib+ RAP-332

60/10 mg/kg

Twice daily/Twice

weekly 3

Combination treatment of RAP-1332 and Ruxolitinib in JAK2(V617F) transgenic mice(12 months old)

Treatment initiated at late stage of disease

14

VEH Ruxolitinib

Ruxolitinib + RAP-1332 RAP-1332

RAP-1332 or Combination treatment with Ruxolitinib reduced BM fibrosis in JAK2(V617F) transgenic mice

3-week treatment

Ruxolitinib or combination treatment with RAP-1332 reduced RBC and splenomegaly in JAK2(V617F) transgenic mice

6

8

10

12

14

RB

C (

10

6 c

ells

/μL)

** *

***

RBC

-26.4% -34.2%

Spleen weight

0

5

10

15

20

25

30

WB

C (

10

3 c

ells

/μL)

WBC

0

400

800

1200

1600

Plt

(1

03 c

ells

/μL)

Platelets

3-week treatment

* P<0.05; ** P<0.01; *** P<0.001 vs VEH

In the JAK2(V617F) transgenic mouse model of myelofibrosis, bone marrow fibrosis starts at 5 months and worsens with age

Expression of TGFβ1 and TGFβ3 ligands were elevated in JAK2(V617F) transgenic mice consistent with the initiation of fibrosis

ACE-1332, a protein biologic ligand trap inhibits specific signaling by TGFβ1 and TGFβ3 ligands but not by TGFβ2 or the other members of the family

Treatment with RAP-1332 before the onset of fibrosis reduced splenomegaly and erythroid hyperplasia inhibited fibrosis in BM and spleen decreased inflammatory cytokine levels in serum

Combination treatment of RAP-1332 and ruxolitinib decreases fibrosis compared

to ruxolitinib monotherapy while retaining the beneficial effects of decreasing splenomegaly

Summary

17

Acknowledgements

Pedro Martinez, PhD

Robert Li

Scott Pearsall, PhD

Ken Attie, MD

Matt Sherman, MD

Steve Ertel, MBA

Ravi Kumar, PhD

Acceleron Team!

RAP-1332 treatment did not effect RBC, WBC and Platelets or splenomegaly in JAK2(V617F) mice after 3 months of dosing

0

4

8

12

16

20R

BC

(1

06 c

ells

/μL)

0

400

800

1200

Plt

(1

03/μ

L)

0

10

20

30

40

WB

C (

10

3 c

ells

/μL)

0

200

400

600

800

Sple

en w

t (m

g)

N=5/group

RBC

Spleen wt WBC

Platelets