Pedro A. Martinez, PhD December 7 th, 2015

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RAP-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA-1 Function in Murine β-thalassemia Pedro A. Martinez, PhD December 7 th, 2015

Outline Background Mechanism of ACE-536 Ligand binding profile Molecular Targets RNA-seq analysis GATA1 negative regulation via GDF11 Conclusions

ACE-536 (Luspatercept) 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 Robust stimulation of RBC production in mice, rat and cynomolgus monkeys ACE-536 Modified Extracellular Domain of ActRIIB Fc Domain of human IgG 1 Antibody Co-developed with Celgene RAP-536 is murine ortholog of ACE-536

Effect of ACE-536 (Luspatercept) on Erythropoiesis 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

RAP-536 decreases elevated psmad2/3 and attenuates anemia in a murine model of β-thalassemia (Hbb -/- ) RBC EPO Reticulocytes * 5 WT th1/th1 th1/th1 + RAP-536 WT th1/th1 th1/th1 + RAP-536 th1/th1 th1/th1 + RAP-536 # # # p< 0.001 vs wt; ** p< 0.01, *p<0.05 vs th1/th1 Suragani et al., Blood 2014

RAP-536 corrects Ineffective Erythropoiesis and associated co-morbidities in a β-thalassemia mouse model (Hbb -/- ) Improved Bone Mineral Density Decreased Liver Iron WT β-thal+tbs β-thal+rap-536 Reduced Spleen Size WT β-thal+tbs β-thal+rap-536 Improved RBC Morphology 6 WT βthal+tbs βthal+rap-536 βthal+tbs βthal+rap-536

RAP-536 decreases elevated psmad2/3, attenuates anemia and decreased erythroid hyperplasia in a mouse model of MDS psmad2/3 Hgb 7 Suragani et al., Nature Medicine 2014 *p< 0.05 vs MDS+TBS ### p< 0.001 vs WT+TBS

ACE-536(Luspatercept) inhibits Smad2/3 signaling ligands Smad2/3 signaling Ligands Biacore K D (nm) @ 37 o C ACE-536 Cell Based Assay IC 50 (ng/ml) A204 RGA GDF-8 3.00 88 GDF-11 0.71 7.1 Activin B 0.27 >14,400 Activin A No binding >33,000 TGF-b1 No binding >33,000 8

GDF11 (pg/ml) GDF11 (pg/ml) Elevated levels of serum GDF11 detected in patients with MDS and β-thalassemia * Normal sera MDS sera Normal: 6 MDS patients: 8 Normal sera β-thalassemia sera Normal: 5 Thalassemia patients: 19 9 * p< 0.05

RBC (10 6 cells/ml) Hgb (gm/dl) GDF11 and other Smad2/3 ligands contribute to the activity of Luspatercept Red Blood Cells Hemoglobin 12 11.5 11 10.5 *** ** 17 16.5 16 15.5 15 *** *** 10 14.5 9.5 14 9 13.5 8.5 VEH RAP-536 Anti-activin B Anti-GDF8/11 Anti-actB + mab Anti-GDF8/11 13 VEH RAP-536 Anti-activin B Anti-GDF8/11 Anti-actB + mab Anti-GDF8/11 10 Dosage: 10mg/kg, twice/week for 2 weeks, N= 5/group ** P<0.01, *** P< 0.001 vs VEH

Flow cytometric cell sorting of β-thalassemic erythroid precursors (basophilic) for RNA sequencing analysis CD71 + Ter119 + Fsc high (Basophilic erythroblasts) 11 β-thalassemic mice were treated with VEH or RAP-536 Splenic basophilic erythroblasts were sorted via flow post 16hrs treatment RNA was isolated from these cells for RNA-seq

Differentially regulated genes by RAP-536 treatment in β- thalassemic erythroid precursors VEH RAP-536 74 genes were differentially expressed by RAP-536 (absolute fold change >1.5, FDR P-value <0.05) Upregulated pathways: Respiratory Electron Transport Oxidative phosphorylation Porphyrin Metabolism Proteasomal Pathway Downregulated IL-12 12

Gene Set Enrichment Analysis (GSEA)-Transcription Factors Transcription Factors Enrichment Score HSF_Q6 2.1767392 GATA_C 1.8235506 NEF2_01 1.7020864 Unbiased enrichment analysis - GATA1, NEF2, and HSF transcription factors are activated while NfκB, etc., are repressed 158 GATA1 downstream targets were differentially upregulated by RAP-536 treatment 13

GATA-1 transcription factor downstream target genes are differentially regulated by RAP-536 treatment RAP-536 VEH Pathways Expression Genes Erythroid differentiation Heme biosynthetic pathway Protein quality control Up Up Up Gata1, Fog1, p21, Klf1, Hri, Atf4, Bcl-xl, Alas2, Abcb6, Ppox, Mthfr, Bach1, Fech Nqo1, Prdx2, Sod2, Hsp s, Psmc3 Proliferation and cell death Down Fos, Jun, Igf1r These data indicate that psmad2/3 negatively regulates erythropoiesis 14

Control GDF-11 GDF-11 + ACE-536 Mouse erythroid leukemic (MEL) and murine primary fetal liver erythroid cells as a model Differentiating erythroid cells (MEL cells treated with DMSO) or Fetal liver cells Fetal liver erythroid cells GDF11 Phosphorylation of Smad2/3 GATA1 availability MEL Reactive Oxygen Species What is the role of ACE-536 psmad2 60kDa GAPDH 37kDA 15 GDF11(100 ng/ml -1 ), 30 mins

GATA1 levels are decreased in the nucleus with GDF11 treatment ACE-536 reverses this effect in MEL cells 16 Control GDF11 GDF11 ACE-536 GATA1

MFI - GATA1 GATA1 levels are decreased in the nucleus with GDF11 treatment ACE-536 reverses this effect in MEL cells Control GDF11 GDF11 ACE-536 * DAPI GATA1 HSP70 17 Control GDF11 GDF11 + ACE-536

GATA1 levels are decreased in the nucleus with GDF11 treatment ACE-536 reverses this effect in MEL cells DAPI GATA1 HSP70 Control GDF11 GDF11 ACE-536 Nuclear extract 18

GATA1 levels are decreased in the nucleus with GDF11 treatment ACE-536 reverses this effect in murine fetal liver erythroid precursors Control GDF11 GDF11 ACE-536 19 GATA1

β-thalassemia + ACE-536 β-thalassemia GATA1 levels are restored in the nucleus of β-thalassemic murine BM cells GATA1 DAPI Merge 20

ROS is induced with GDF11 treatment and reduced upon cotreatment with ACE-536 in MEL cells GDF11 GDF11 + ACE536 21

Model - β-thalassemia GDF11 = Erythroid differentiation is inhibited ROS psmad2/3 C-Cas-3 Nucleus GA TA1 22

Model - β-thalassemia + ACE536 GDF11 ACE-536 ROS = Block removed Proper erythroid maturation psmad2/3 C-Cas-3 Nucleus GATA1 23

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 decreases ROS and restores GATA1 availability Luspatercept is currently being tested in patients with β-thalassemia 24 and MDS

25 Acknowledgements Dr. Suragani Dr. Kumar Dr. Bhasin Collaborator at Beth Israel Medical Center at Harvard Dr. Attie Dr. Sherman Steve Ertel Acceleron Team Celgene Team

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