murine tgfβ-antagonist (rap-1332) inhibits fibrosis in a...
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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