Targeting the TGF-β superfamily in Myelodysplastic Syndromes (MDS) Amit Verma Albert Einstein College of Medicine Bronx, NY
Targeting the TGF-β superfamily in Myelodysplastic Syndromes (MDS)
Amit Verma
Albert Einstein College of MedicineBronx, NY
1. Greenberg P, et al. Blood. 1997;89:2079-2088. Erratum in Blood. 1998;91:1100.2. Adebonojo SA, et al. Chest. 1999;115:1507-1513.
AJCC-UICC Stage IPSS Risk Category
Life expectancy is shorter for US patients with MDS than those with lung cancer*†[1,2]
Low IaInt-1 IIaInt-2 IIIaHigh IV
Lung Cancer
Surv
ival
(Yrs
)
01
8
109
4
23
567
MDS
8.0
5.4
2.41.2
5.7
3.5
1.10.4
Reduced Survival Is an Inherent Feature of MDS: Even for low risk subgroups
*Adjusted for age (lung cancer, median 66 yrs; MDS, median 69 yrs) and risk/stage.[1,2]
†All histological subtypes.[1,2]
Need for therapeutic approaches that increase blood counts
Ineffective Hematopoiesis is seen in MDSHypercellular marrows with decreased peripheral blood counts
Increased Cell death (Apoptosis/Cell cycle arrest)Decreased Proliferation / Differentiation
Pro Apoptotic / Myelosuppresive/ Inflammatory cytokine signaling
Tumor necrosis factor α↑ TNFα mRNA in MDS BMs↑ TNF production by MDS
Macrophages Pro-apoptotic Type 1
receptors increased in low grade MDS and decreased in
high grade cases and AMLAnti-TNF therapies
(Remicaid & Enbrel) show efficacy
Thalidomide can degrade TNF mRNA
Transforming growth factor β
↑ membrane bound TGF β on
progenitors
↑ conc. in serum
Vascular Endothelial growth factor (VEGF)
Secreted by malignant clone and ALIP cells
Expression in BM correlates with disease
severity
Higher expression of high affinity VEGFR1
Interferon γ (IFN g)
Interleukin 1β
Fibroblast Growth factor (FGF)
Hepatocyte growth factor (HGF)
Macrophage Inhibitory Protein (MIPα)
Other TGF-βfamily members
GDF11, GDF15
ALK, activin-like kinase receptor; GDF, growth differentiation factor; TGF-β, transforming growth factor β.
TGF-beta family members regulate hematopoiesis
+
P
PP
Transphosphorylated TβR complex
Phosphorylated SMAD2/3 complex
SMAD2 SMAD3
P
TIF γSMAD4
SMAD6/7
+
P
P P
P
PP
ALK4
Activin receptor ligands, GDFs
TGF-β
Altered erythroid differentiation
P
Stem cell proliferation and quiescence
ALK5
BM, bone marrow; IHC, immunohistochemistry. Zhou L, et al. Blood. 2008;112:3434-43.
Activated SMAD2/3 seen in MDS BM samples
0
0.1
0.4
0.2
0.3
0.5
MDS Controls
Phos
po-S
MA
D2
IHC
stai
ning
inte
nsity
/fiel
d
MDS Controls0
50
200
100
150
250
Num
ber o
f pho
spo-
SMA
D2+
cel
ls/h
ot fi
eldN = 20
N = 8
N = 20
N = 8
p < 0.005 p < 0.005
* *
MD
SC
ontr
ols
Phospho-SMAD2 IHC
Anti-
TBR
I sh
RN
A +
TGF
Scr s
hRN
A +
TGF
PhaseGFP+
K562
TGFβ − + − +Anti-TBRI-shRNA − − + +
Scr-shRNA + + − −
p-smad2
smad2
actin
Inhibition of SMAD2 activation can stimulate MDS hematopoiesis in vitro
0
10
20
30
40
50
60
BFU-E
CFU-GM
BFU-E
CFU-GM
BFU-E
CFU-GM
BFU-E
CFU-GM
BFU-E
CFU-GM
BFU-E
CFU-GM
Scr shRNA
anti-TBRI
MDS 1 MDS 3 MDS 4 MDS 5MDS 2 MEAN
No.
of C
olon
ies
P=0.007
P=0.049
**
Zhou et al, Blood, 2008
0
10
20
30
40
50
60
WBC(x10^3/cc)
Hgb (g/dl) Hct (%) Plt (x10^5/cc)
WT Alb/TGF+
*
*
Alb/TGF+ transgenic mice develop anemia and mimic human bone marrow failure
Megakaryocytes
Erythroid progenitors
0
20
40
60
80
100
120
140
160
Erythroid Myeloid
Col
onie
s/10
000
BM c
ells
PlaceboSD-208 30mg/kg/d
p=.03 * p=.04 *
0123456789
10
Placeb
o
10mg/k
g/d
20mg/k
g/d
30mg/k
g/d
P=.04
Incr
ease
in H
emot
ocrit
af
ter t
reat
men
t
SD-208 x 2 wks
*
Small molecule inhibitor of ALK5 (TGF-b receptor I), SD-208, can improve hematopoiesis in TGF overexpressing mice and can raise their hematocrit
Zhou et al, Blood, 2008
RA, refractory anemia; RAEB; RA with excess blasts;RARS, RA with ringed sideroblasts. Zhou L, et al. Cancer Res. 2011;71:955-63.
Why is SMAD2/3 signaling activated in MDS?
SMAD2SMAD7
**Controls
Log
2 (S
MA
D2
gene
exp
ress
ion)
Log
2 (S
MA
D7
gene
exp
ress
ion)
5
6
9
7
8
10
4 7.5
8.0
9.5
8.5
9.0
10.0
Controls(N = 17)
RA(N = 55)
RARS(N = 48)
RAEB(N = 80)
5q−(N = 16)
0 10
Controls(N = 17)
RA(N = 55)
RARS(N = 48)
RAEB(N = 80)
5q−(N = 16)
MDS
Negative regulator Smad7 is reduced
Lowest expression
ALK, activin-like kinase receptor; GDF, growth differentiation factor; TGF-β, transforming growth factor β.
SMAD7 is a negative regulator of ALK4 and ALK5
+
P
PP
Transphosphorylated TβR complex (ALK5)
Phosphorylated SMAD2/3 complex
SMAD2 SMAD3
P
TIF γSMAD4
SMAD6/7
+
P
P P
P
PP
ALK4
Activin receptor ligands, GDFs
TGF-β
Altered erythroid differentiation
P
Inhibition of proliferation
0
10
20
30
40
50
60
70
80
90
100
Zhou L, et al. Cancer Res. 2011;71:955-63.
SMAD7 is reduced in MDSControls MDS
SMA
D7
IHC
p < 0.05
Low-grade MDS
High-grade MDS
ControlsSt
aini
ng fo
r SM
AD
7(%
)
Strong staining
Weak or no staining
Fold
Cha
nge
*
*
00.5
11.5
22.5
33.5
4*
Scr shRNA + TGF
Scr shRNA + TGF + LY
SMAD7 shRNA + TGF
SMAD7 shRNA + TGF + LY
Reduced SMAD7 leads to increased sensitivity to TGF-β
0
20
40
60
80
100
120
140
DMSO 1ng/mlTGF
10ng/mlTGF
DMSO 1ng/mlTGF
10ng/mlTGF
Scr control shRNA SMAD7 shRNA
% C
ontro
l Ery
thro
id C
olon
ies
***
…….That can be reversed by inhibition of TGF-b receptor kinase
HR MDS, high-risk MDS; LR MDS, low-risk MDS;miR-21, microribonucleic acid 21; UTR, untranslated region. Bhagat TD, et al. Blood. 2013;121:2875-81.
Why is SMAD7 decreased in MDS?Human SMAD7 3’ UTR
Nor
mal
ized
miR
-21
expr
essi
on
* *
Mea
n in
tens
ity
miR
-21
(Log
2)
0
2
4
6
8
10
12
Controls MDS
*
02468
10121416
CONTROL ALL MDS LR MDS HR MDS
*
All MDSControl
0.1k 0.2k 0.3k 0.4k0.4k 0.5k 0.6k 0.7k 0.8k 0.9k 1k 1.1k 1.2k 1.3k 1.4k 1.5kGeneHuman SMAD7 SNM_005904 3’ UTR Length: 1518
Conserved sites for miRNA families conserved in human, mouse, rat, dog, and chickenmiR-216miR-15/16/195/424/497 miR-21 miR-25/32/92/363/367
miR-17-5p/20/93.mr/10
miR-181
KeySites conserved in human, mouse, rat, dog, and chicken
Less conserved sites8 mer 7 mer-m8 7 mer-1A
8 mer 7 mer-m8 7 mer-1A
miR-21 is increased in MDS and has a putative binding site on the SMAD7 3’ UTR
Parallel Transcriptional Analysis reveals miR-21 overexpression in MDS/AML Stem and Progenitor cells
Barreyro et al. Blood 2012
MDS
0
0.2
0.4
0.6
0.8
1
1.2
Fold
Cha
nge
(mir2
1/co
ntro
l)
WT Smad7 UTR Mut Smad7 UTR
**
miR-21 binds to SMAD7 3’UTR and leads to reduced levels of SMAD7
A
B
D
SMAD7
Actin
miR-21 Inh
mm Control
HL-60TF-1
A
0
20
40
60
80
100 Control TGF-b
TGF-b + miR-21 Inh
TGF-b + mm contro
Nor
mal
ized
mir-
21
expr
essi
on
Scr shRNASMAD7 shRNA
0
0.1
0.2
0.3
0.4
0.5
mmcontrol
Anti-miR-21LNA
**
B C
MDS1 MDS2
miR-21 Inh
mm Control
miR-21 Inh
mm Control
SMAD7
Actin
pSMAD2
Actin
MDS3
miR-21 Inh
mm Control
*
miR-21 Inh
mm Control
Inhibition of miR-21 can abrogate the effects of TGF−β on hematopoietic cells
% C
ontro
l Ery
thro
id C
olon
y Fo
rmat
ion
Bhagat et al, Blood, 2013
Mou
se 1
125
Mou
se 7
35
Mir2
1 in
hibi
tor
δRBC
1.7x
10^6
/uL
Plac
ebo
cont
rol
δRBC
-0.2
8x10
^6/u
L
Mea
n ch
ange
post
trea
tmen
t
SMAD7 IHC p-SMAD2 IHC
*
Treatment with mir21 inhibitor leads to increase in red blood cells in TGF transgenic mice:
A B
*
RBC(x10^6)
Hgb(gm/DL)
-1
0
1
2
3
4
5
6 miR-21 Inhibitor
mm control
HCT
* *
0
30
60
90
120
150
180
BFU-E CFU-GM
ControlmiR21 Inh
% C
ontro
l Col
onie
s *
Inhibition of miR-21 stimulates erythropoiesis in MDS.
LY-215 is effective in vitro and in vivo in MDS
02468
1012141618
PLACEBO LY-215
N=17
N=17*P=0.02
MEA
N IN
CR
EASE
IN
HEM
ATO
CR
IT
TGF transgenic mice treated for 2 weeks
0
200
400
600
800
1000
1200
1400
Control LY 100nM LY 200nM LY 500nM
ErythroidMyeloid
* *
MDS BM samples
LY-2157299 (Galunisertib) is an oral ALK5 inhibitor
Zhou L, et al. Cancer Res. 2011;71:955-63.
A Study of LY2157299 in Participants With Low/Int-1 Myelodysplastic Syndromes; ClinicalTrials.gov Identifier: NCT02008318
Phase 2 Study of Monotherapy Galunisertib (LY2157299 Monohydrate) in
Very Low-, Low-, and Intermediate-risk Patients with Myelodysplastic Syndromes
David Valcarcel1, Amit Verma2, Uwe Platzbecker3, Valeria Santini4, Aristoteles Giagounidis5, Maria Diez-Campelo6, Jan Janssen7, Richard F Schlenk8, Gianluca Gaidano9, Jaime Perez
de Oteyza10, Ann L Cleverly11, Alan Y Chiang12, Michael M Lahn12, Durisala Desaiah12, Susan C Guba12, Alan List13, Rami Komrokji13
1Hematology, Vall d'Hebrón University Hospital, Vall d'Hebrón, Barcelona, Spain; 2Department of Medicine, Albert Einstein Cancer Center, Bronx, NY, USA; 3Universitätsklinikum Carl Gustav Carus an der
Technischen Universität, Dresden, Germany; 4AOU Careggi, University of Florence, Florence, Italy; 5Hematology, Oncology and Palliative Care, Marien Hospital, Düsseldorf, Germany; 6Hospital Clinico De
Salamanca, Salamanca, Spain; 7Onkologische Gemeinschaftspraxis Dres Westerstede, Germany; 8Internal Medicine, University of Ulm, Ulm, Germany; 9Hematology, Department of Translational Medicine, Amedeo
Avogadro Novara, Italy; 10Hematology, Hospital HM Sanchinarro, Madrid, Spain; 11Eli Lilly and Company, ErlWood, ELCL, UK; 12Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA; 13 H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL, USA
57th American Society of Hematology Annual Meeting; December 5-8, 2015; Orlando, Fl., USA
Hematological Improvement in Patients Treated with Galunisertib, 150 mg BID
Baseline Trans-FusionNeed
Number of
Patients (N)
Median number of transfused
units at baseline
Post Treatment (N)TI During any 8
WeeksHI
(TR/TI or HGB Increase) During
any 8 weeks≥ 4 Units 24 7.5 4 9/24 (37.5%)1-3 Units 4 2.5 2 00 Units 10 0 9 1Total 38 10/38 (26.3%)
*14 patients could not decrease their transfusion requirement by 4 units/8 weeks per IWG 2006 criteria.If only the ≥4 units at baseline patients are included, then the response rate is 9/24 or 37.5%
TI, transfusion independence; HI, hematological improvement; TR, transfusion reduction; HGB, hemoglobin
]14*
Heatmap of Biomarkers vs. Response Demonstrated no Correlation with mutations
Res
pond
erN
on-R
espo
nder
Stem Cell Differentiation Block (CD34+CD38-Lin-CD33-) is associated with response to TGF-β inhibitor
0
5
10
15
20
25
Responders Non-Responders
HSCs (Lin-, CD33-, CD34+, CD38+)
CD38
Lin-CD33- cells
CD
34CD38
CD
34
Lin-CD33- cells
38% 45%
1% 96%
Responder
Non-Responder%ag
e H
SCs
*
Pre-
treat
men
t
Lineage
CD
33
CD38C
D34
Viable, single cells Lin-CD33- cells
CD45Rα
CD
123
FSC
IL1R
AP
CD34+CD38+ cellsCD123-CD45Rα- cells
Lineage
CD
33
CD38
CD
34
Viable, single cells Lin-CD33- cells
CD45Rα
CD
123
FSC
IL1R
AP
CD34+CD38+ cellsCD123-CD45Rα- cells
Response to TGF-β inhibition associated with increased progenitor differentiation
Cyc
le 6
38% 45%
16% 75%
75%
63%
GMP 28%
CMP
MEP
GMP 37%
Increased %age of CD34+/CD38+
progenitors
Decreased %age of Aberrant (IL1RAP)
HSCs
IgG, immunoglobulin G. Ravi Kumar, Acceleron
Sotatercept and Luspatercept (ACE-536) act as novel ligand traps for TGF-β superfamily ligands
Modifiedextracellular
domainof ActRIIB
Fc domainof human
IgG1 antibody
LuspaterceptACE-536
Extracellulardomain
of ActRIIA
Fc domainof human
IgG1 antibody
Sotatercept(ACE-011)
Amino acid homology between ECDof sotatercept and ACE-536 is ~ 60%
The murine orthologues of these molecules are RAP-011 and RAP-536;extracellular domains are identical, but linked to murine IgG2a Fc domain
TBS, tris-buffered saline; M:E, myeloid:erythroid. Suragani RN, et al. Nat Med. 2014;20:408-14.
ACE-536 correct anemia by promoting late stage erythropoeisis
Studies using RAP-536, murine analogue of luspatercept
Inhibits SMAD2/3 signalingMDS+TBS MDS+RAP-536
Increases Hb
* p < 0.001 vs WT+TBS** p < 0.05 vs MDS+TBS
*
**
14
12
10
8
6WT+
TBS
MDS+
TBS
MDS+
RAP-536
Hb
(g/d
L)
WT+TBS MDS+TBS MDS+RAP-536Erythroid precursors Non-erythroid cells
Normalizes M:E ratio in BM
74%
26%
48%52% 68%
32%
ACE-536 increases hemoglobin and reduces transfusion burden in patients with low or
intermediate-1 risk myelodysplastic syndromes (MDS): Final results from a phase 2 study
Uwe Platzbecker, MD1, Ulrich Germing, MD*,2, Aristoteles Giagounidis, MD PhD3, Katharina Götze, MD*,4, Philipp Kiewe, MD*,5, Karin Tina Mayer, MD*,6, Oliver Ottmann, MD7, Markus Radsak, MD*,8, Thomas
Wolff, MD9, Detlef Haase, MD*,10, Monty Hankin*,11, Dawn Wilson*,11, Xiaosha Zhang*,11, Abderrahmane Laadem, MD12, Matthew L. Sherman, MD11, and Kenneth M. Attie, MD
1Universitätsklinikum Carl Gustav Carus, Dresden; 2Universitätsklinikum Düsseldorf; 3Marien Hospital Düsseldorf;4Technical University of Munich; 5Onkologischer Schwerpunktam Oskar-Helene-Heim, Berlin; 6Universitätsklinikum
Bonn; 7Klinikum der J.W. Goethe-Universität Frankfurt; 8University Medical Center - Johannes Gutenberg-Universität,Mainz; 9OncoResearch Lerchenfeld UG, Hamburg; 10Department of Hematology and Medical Oncology, UniversityMedicine of Göttingen, Germany; 11Acceleron Pharma, Cambridge, MA; 12Celgene Corporation, Summit, NJ, USA
Study supported by Acceleron and CelgenePlatzbecker U, et al. Presentation presented at ASH 2014.
Blood. 2014;124:abstract 411.
D·MDS Deutsche MDS-Studiengruppe
Data as of 03 October 2014.
HI-E response rate by ring-sideroblast morphology, SF3B1 mutationResponse rate at higher dose levels (0.75–1.75 mg/kg)
Patient Subgroup IWG HI-E Response Rate(0.75-1.75 mg/kg)
RS positive1 19/35 (54%)
EPO < 200 U/L 14/23 (61%)
EPO ≥ 200 U/L 5/12 (42%)
RS negative1 0/4 (0%)
SF mutation2 present 18/30 (60%)
SF mutation2 absent 1/9 (11%)
An open-label, phase 2, dose-finding study of sotatercept(ACE-011) in patients with low or intermediate-1 (Int-1)-risk
myelodysplastic syndromes (MDS) or non-proliferative chronic myelomonocytic leukemia (CMML) and anemia
requiring transfusionRami Komrokji,1 Guillermo Garcia-Manero,2 Lionel Ades,3 Abderrahmane Laadem,4 Bond Vo,4 Thomas
Prebet,5 Aspasia Stamatoullas,6 Thomas Boyd, MD,7 Jacques Delaunay,8 David P. Steensma,9 Mikkael A. Sekeres,10 Odile Beyne-Rauzy11,
Jun Zou4, Kenneth Attie12, Matthew L. Sherman12, Pierre Fenaux13, and Alan F. List14
1Moffitt Cancer Center, Tampa, FL; 2University of Texas M.D. Anderson Cancer Center, Houston, TX; 3Hôpital St Louis, Paris, France; 4Celgene Corporation, Summit, NJ; 5Institut Paoli Calmettes, Marseille, France; 6Centre Henri Becquerel, Rouen,
France; 7North Star Lodge Cancer Center, Yakima, WA; 8CHU de Nantes – Hôtel Dieu, Nantes, France; 9Dana Farber Cancer Institute, Boston, MA; 10Leukemia Program, Cleveland Clinic, Cleveland, OH; 11Centre Hospitalier Universitaire Purpan
Pavillion de Médecines, Toulouse, France; 12Acceleron Pharma, Cambridge, MA; 13Service d’Hématologie Séniors, Hôpital St Louis, Université Paris 7, Paris, France; 14Malignant Hematology, Moffitt Cancer Center, Tampa, FL
Komrokji R, et al. Blood. 2014;124:abstract 3251.
Komrokji R, et al. Blood. 2014;124:abstract 3251.
Results: efficacy in HTB patients
Sotatercept dose cohortOverall(N = 45)0.1 mg/kg
(n = 7)0.3 mg/kg
(n = 6)0.5 mg/kg (n = 17)
1.0 mg/kg (n = 14)
Transfusion burden reduction≥ 4 RBC units/8 weeks, n (%) 0 4 (66.7) 7 (41.2) 8 (57.1) 19 (42)
Duration of longest response,median (range), days NA
67.5 (62–144)
150 (83–345)
87.5 (62–154)
106.0 (62–345+)
RBC-TI ≥ 56 days, n (%) 0 1 (16.7) 2 (11.8) 2 (14.3) 5 (11)
PP
P
PP
R III R II R I
Transphosphorylated TβR complex
TGF β / Activin Ligands
Smad2 Smad3
P
Smad 7
P
Conclusions: Pathogenesis of ineffective hematopoiesis
miR-21
Ineffective hematopoiesisLow Blood Counts
LY-2157299
Lustanercept, Sotatercept
SMAD2/3 pathway is overactivated in MDS HSPCs SMAD7 is a negative regulator of ALK4/5 and is
decreased in MDS Luspatercept and Sotatercept show promising
evidence of clinical activity in a cohort of lower-risk MDS patients who were anaemic and refractory to ESAs
Lustanercept is being evaluated in a multicenter Phase III trial in RARS
Conclusions
AcknowledgementsLi ZhouTushar Bhagat
Yiting YuSanchari Bhattacharyya
Orsolya GiriczMatthias Bartenstein
Marianna
Stem CellsUli SteidlBritta Will
Gene Expression dataJackie BoultwoodAndrea Pellagatti
EpigeneticsJohn Greally
Red cell studiesAmittha Wickrema
Luspatercept studyU. Platzbecker et al.
Sotatercept studyRami Komrokji, Guillermo Garcia-Manero, Alan List et al.
AcceleronRavi Kumar
Eli LillySusan Guba