RAP-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and - - PowerPoint PPT Presentation

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RAP-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and - - PowerPoint PPT Presentation

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RAP-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA1 Function in Murine -thalassemia Pedro A. Martinez, PhD June 10 th , 2016 ACE-536 is a Modified ActRIIB Receptor Fusion

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SLIDE 1

RAP-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA1 Function in Murine β-thalassemia

Pedro A. Martinez, PhD June 10th, 2016

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SLIDE 2

ACE-536 is a Modified ActRIIB Receptor Fusion Protein

  • ACE-536 is a fusion protein that consists of

a modified activin receptor (ActRIIB) - a member of the TGFβ superfamily - and the Fc of human IgG1

  • Inhibits Smad2/3 signaling and acts as a

ligand trap – GDF8, GDF11, ActB

  • Robust stimulation of RBC production in

mice, rats, cynomolgus monkeys and humans

  • Co-developed with Celgene

RAP-536 is murine ortholog of ACE-536

Modified Extracellular Domain

  • f ActRIIB

Fc Domain of human IgG1 Antibody

ACE-536

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SLIDE 3
  • ACE-536 treatment increases RBCs – however activity is distinct from EPO
  • Effect is focused on the differentiation of erythroid precursors while EPO affects

proliferation of BFU-E and CFU-E

  • ACE-536 promotes differentiation of Baso, Poly, and Ortho Erythroblasts
  • ACE-536 does not affect other cell lineages
  • ACE-536 has corrected anemia in various murine preclinical models of ineffective

erythropoiesis such as MDS and β-thalassemia

Suragani et al., Nature Medicine 2014

Effect of ACE-536 on Erythropoiesis

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SLIDE 4

RAP-536 Decreases Elevated pSmad2/3, Corrects co-morbidities and Attenuates Anemia in a Murine Model of β-thalassemia (Hbb-/-)

4 WT βthal βthal RAP-536

# # # p< 0.001 vs wt; ** p< 0.01, *p<0.05 vs th1/th1

*

WT βthal βthal RAP-536 βthal βthal RAP-536

Suragani et al., Blood 2014

RBC EPO Reticulocytes

Reduced Spleen Size

WT βthal βthal RAP-536

Decreased Liver Iron

WT βthal βthal RAP-536

pSmad2/3

WT βthal βthal RAP-536

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SLIDE 5

RNA-seq àGene Set Enrichment Analysis (GSEA) - Transcription Factors

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§ β-thalassemic mice were treated with VEH or RAP-536, splenic basophilic erythroblasts were sorted post 16hrs, and RNA was isolated for RNA-seq § Unbiased enrichment analysis - GATA1, NEF2, and HSF transcription factors are activated while NfκB, etc., are repressed

CD71+Ter119+Fschigh (Basophilic erythroblasts)

GSEA

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SLIDE 6

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158 GATA1 Downstream Target Genes are Differentially Regulated by RAP-536 Treatment in Basophilic Erythroblasts

VEH RAP-536

  • These data indicate that pSmad2/3 negatively regulates

erythropoiesis as RAP-536 binds strongly to GDF11 – preventing downstream phosphorylation of Smad2/3

Pathways Expression Genes

Erythroid differentiation

Up

Gata1, Fog1, p21, Klf1, Hri, Atf4, Bcl-xl,

Heme biosynthetic pathway

Up

Alas2, Abcb6, Ppox, Mthfr, Bach1, Fech

Protein quality control

Up

Nqo1, Prdx2, Sod2, Hsp’s, Psmc3

Proliferation and cell death

Down

Fos, Jun, Igf1r

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SLIDE 7

RAP-536 Administration to β-thal Mice Increased GATA1 and Erythroid Specific Genes in late Basophilic Erythroblasts

7

β-thalassemia VEH β-thalassemia RAP-536 β-thalassemia VEH β-thalassemia RAP-536 β-thalassemia VEH β-thalassemia RAP-536

BCL2l1 GATA-1 Fech

Normalized Expression Normalized Expression Normalized Expression

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SLIDE 8

Mouse Erythroid Leukemic (MEL) and Murine Primary Fetal Liver Erythroid Cells as a Model System

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Differentiating erythroid cells (MEL cells treated with DMSO) or Fetal liver cells GDF11 § Phosphorylation of Smad2/3 § GATA1 § Reactive Oxygen Species (ROS)

MEL

Control GDF11 GDF11 ACE536

pSmad2 – 60kDa GAPDH – 37kDA

Fetal liver erythroid cells

GDF11(100 ng/mL-1), 30 mins

+/- ACE-536

Normal: 5 Thalassemia patients: 19

Normal sera β-thalassemia sera

GDF11 (pg/ml)

*

* p< 0.05

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SLIDE 9

GATA1 levels were Decreased in the Nucleus with GDF11 Treatment – ACE-536 Reverses this Effect in MEL Cells

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Control GDF11 GDF11 ACE-536

DAPI GATA1 HSP70

*

Control GDF11 GDF11 + ACE-536

MFI - GATA1

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SLIDE 10

GATA1 levels were Decreased and Active Caspase 3 is Elevated Post GDF11 Treatment – ACE-536 Reverses this Effect in MEL Cells

10

MFI – Caspase 3/7 P < 0.05*

Caspase 3/7 Flow

GDF11

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SLIDE 11

11

GATA1 DAPI Merge

β-thalassemia β-thalassemia + ACE-536

GATA1 Levels are Restored in the Nucleus of β-thalassemic Murine BM Cells

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SLIDE 12

Key Points

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  • GATA1 is decreased in the nucleus with GDF11 treatment
  • Caspase 3/7 is elevated post GDF11 treatment – indicating the decrease
  • f GATA1 is possibly due to cleavage by active Caspase 3
  • Furthermore, an increase in GATA1 and erythroid genes was shown post

ACE/RAP-536

  • Because ROS is elevated in β-thal, we decided to investigate its role
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SLIDE 13

Overview of NOX Enzymes

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NOX Enzymes: NOX1, NOX2/Cybb, NOX3, NOX4, NOX5, DUOX1, DUOX2

Block et al., Nature Reviews 2012 George et al., Blood 2013

AA – Normal RBC SS – Sickle RBC

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SLIDE 14

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Effect of RAP-536 in a Murine Model of β-thalassemia (Hbbth1/th1)

  • n ROS

Suragani et al., Blood 2014

BM β-thal Spleen BM Spleen WT β-thal RAP-536

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SLIDE 15

ACE-536 Decreases NOX4 (responsible for ROS production) in MEL Cells

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SLIDE 16

RAP-536 Decreases NOX1 (responsible for ROS production) in β- thalassemic Mice to WT levels

16

*

Normalized Expression

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SLIDE 17

RAP-536 also Decreases NOX2 and NOX4 (Responsible for ROS Production) in β-thalassemic Mice

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β-thal β-thal RAP-536 β-thal β-thal RAP-536

NOX2 NOX4

Normalized Expression Normalized Expression

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SLIDE 18

GDF11 Treated Mice Show a Block in Differentiation and an Increase in NOX4

18

Suragani et al., Nature Medicine 2014

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SLIDE 19

19

GDF11

pSmad2/3 ROS C-Cas-3 TA1 GA Nucleus

= Erythroid differentiation is inhibited

Model - β-thalassemia

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SLIDE 20

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GDF11 pSmad2/3 ROS ACE-536 Nucleus

GATA1

C-Cas-3

Block removed Proper erythroid maturation

Model - β-thalassemia + ACE-536

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SLIDE 21

Conclusions

  • Luspatercept (ACE-536) binds and inhibits signaling by certain Smad 2/3

signaling ligands

  • RNA seq analysis revealed that ACE-536 upregulates genes involved in

erythroid differentiation through GATA1

  • GDF11 increases ROS and limits the availability of GATA1 in the nucleus
  • ACE-536 inhibits ROS via a decrease in NOX enzymes and restores

GATA1 availability

  • Luspatercept is currently being tested in patients with β-thalassemia and

MDS

21

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SLIDE 22

Acknowledgements

  • Dr. Suragani
  • Dr. Pearsall
  • Dr. Bhasin – Collaborator at Beth Israel Medical Center at Harvard
  • Dr. Kumar
  • Acceleron Team
  • Celgene Team

22

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SLIDE 23

GDF11 and other Smad2/3 Ligands Contribute to the Activity of Luspatercept

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8.5 9 9.5 10 10.5 11 11.5 12

VEH RAP-536 An6-ac6vin B mAb An6-GDF8/11 An6-actB + An6-GDF8/11 RBC (106 cells/µL)

*** **

13 13.5 14 14.5 15 15.5 16 16.5 17

VEH RAP-536 An6-ac6vin B mAb An6-GDF8/11 An6-actB + An6-GDF8/11 Hgb (gm/dL)

*** *** Red Blood Cells Hemoglobin

Dosage: 10mg/kg, twice/week for 2 weeks, N= 5/group ** P<0.01, *** P< 0.001 vs VEH