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