THE PYRUVATE KINASE ACTIVATOR AG-348 IMPROVES MURINE β-THALASSEMIC ANEMIA AND CORRECTS INEFFECTIVE ERYTHROPOIESIS

A Mae1, E. Beneduce1, A. Siciliano1, P. A. Kosinski2, A. Janin3, C. Lebouef3, A. Iolascon4, L. De Falco4, L. Dang2, C. Kung2, L. De Franceschi1

1Department of Medicine, University of Verona-AOUI Verona, Verona; Italy; 2Agios Pharmaceucals, Inc., Cambridge, MA, USA 3Inserm, U1165, Université Paris 7- Denis Diderot, AP-HP, Hôpital Saint- Louis, F-75010, Paris, France; 4CEINGE and Dept. of Biochemistry, University of Naples, Naples; Italy

EHA- 8-12 Jun,e 2016 β-Thalassemic Syndromes are Worlwide Distributed Hereditary Red Cell Disorder and is a Model of Pathological Erythropoiesis

• 7% of global population is a carrier for severe hemoglobinopathies and β thalassemias are one of the most common inherited red cell disorders

• β thalassemias are characterized by absent or reduced synthesis of β globin chains, resulting in accumulation of free α-chain and free pathological heme.

Weatherall DJ et al. Ann Human Biol 32: 117, 2005; Weatherall DJ Bull WHO 79: 704, 2001; Rund D et al. NEJM 353: 1135, 2005 In βthalassemic Syndromes is due to both Ineffective Erythropoiesis and Reduced RBC Survival in the Peripheral Circulation

" thalassemic RBCs PS

!![K+] K+ !![Mg2+] - KCC " Oxidative stress !O2 O2 MPs Cl-

P Heinz body P H2O2 Fe Free ! globin chain Fe Free pathological iron Loss of interactions !OH Clustered band 3 between skeleton proteins

IgG anti-band 3

In β-thalassemias, the intra-cellular accumulation of free α-globin chains, free pathological iron and free heme result in high ROS production. Ginzburg Y et al. Blood 118: 4321, 2011; De Franceschi L et al. Oxidative Medicine and Cellular Longevity 2013, Matte A ARS 23: 1284, 2015 Potentiation of Endogenous Anti-oxidant Systems as Novel Therapeutic Strategy in β-thalassemia

• In β-thalassemia, exogenous anti-oxidants have been largely studied to limit ROS cytotoxity and ineffective erythropoiesis

• Potentiation of endogenous anti-oxidant systems might be a novel interesting therapeutic strategy to face chronic and severe oxidative stress such as in β- thalassemia.

Franco SS et al. Haematologica 99: 267, 2014; Matte A ARS 23: 1284, 2015; Rund D et al. NEJM 353: 1135, 2005; Macari ER et al. Blood 117: 5987, 2011; Schmidt HHHW et al. ARS 23:1130, 2015 ATP is important for activity of anti-oxidant systems and low ATP levels have been reported in β-thal RBCs

• In RBCs, ATP is generated through glycolysis and pyruvate kinase-R is involved in the final step of ATP production

• β-thal intermedia RBCs show 3-fold faster glucose metabolism compared to controls

• Under oxygenation/deoxygenation condition, β-thal major and β-thal /HbE RBCs show reduced ATP content compared to healthy

RBCs Ting YL et al. BJH 88: 547, 1994; Chakrabarty I et al. Arch Med Res 2012

AG-348: Allosteric acvator of the red cell isoform of pyruvate kinase (PK-R)

AG-348 binds at PK-R enzyme dimer-dimer AG-348 increases ATP levels in healthy interface to promote active tetramer volunteers (NCT02149966) ) L Dosing period Placebo m P / T g 200 15 mg q12hr µ A

(

n i n

60 mg q12hr o e i t g

a 120 mg q24hr n r a t 100 h n 120 mg q12hr c e

c e t n 360 mg q12hr u o l c o 700 mg q12hr s d

b 0 o A o Mean + SD l b

0 5 10 15 20 Time (day)

AG-348 is currently in Phase 2 testing in patients with pyruvate kinase deficiency (NCT02476916)

Kung C et al. 55th ASH Annual meeting 2013, Abstract 2180 Presented at EHA, 2015, Abstract S138 6 Ineffective erythropoiesis is present in PK Deficiency

• PK deficiency is the most common hereditary abnormality in glycolytic pathway, resulting in a large clinical spectrum of hemolytic anemia

• Abnormalities in erythropoiesis, (dyserythropoiesis and ineffective erythropoiesis) have been described in few patients with PK deficiency as well as in a PK mutated mouse model, suggesting a possible role of PK in erythroid maturation events

Zanella A et al. 13: 57, 2000; Aizawa S et al. AJH 74: 68, 2003; Aizawa S et al. Exp Hematol 33: 1292, 2005; Haija MA et al Ped Blood Cancer 61: 1463, 2014 Study Aim

To evaluate the impact of AG-348 on anemia and ineffective erythropoiesis in a mouse model of β thal intermedia Study Design

• Hbb3th/+ mice were used as model of b-thalassemia intermedia and C57B6/2J as WT controls. Two month-old females were treated with either vehicle or AG-348 at 50 mg/kg bid by oral gavage.

• Hematological parameters, red cell indices and reticulocytes were determined.

• Flow cytometric analysis of erythroid precursors from bone marrow and spleen was performed using the CD44 and TER-119.

• Annexin-V positivity of orthochromatic erythroblasts was assessed.

• Histological analysis of spleen and liver was carried independently by two pathologists.

• RT-PCR analysis (including hepcidin and erythroferrone) and immunoblot analysis were carried out on sorted erythroid precursors and on liver cells.

Franco SS et al. Haematologica 99: 267, 2014; Liu J et al. Blood 121: e43, 2013; Matte A et al. Antioxid Redox Signal 23: 1284, 2015 AG-348 treatment significantly ameliorates anemia in a mouse model of β-thalassemia

WT WT A0001480 WT AG-348

Hbbth3/+ Hbbth3/+ vehicle Hbbth3/+ AG-348 Hbbth3/+ A0001480 17 52 * * * Baseline AG-348 (21 d) * * 15 48

) 13 ) fL dL * 44 (g/ WT 11 * Hb MCV ( MCV 40 9

36 7

Hbbth3/+ 5 32 0 7 14 21 0 7 14 21 20 * * 280 18 *

) 230 *

16 uL * ) 180

dL *

14 cells 4 130 * ( 10 ( MCH(g/ 12 * 25 10 Retics

8 0 0 7 14 21 0 7 14 21 Time (days) Long-term (7 weeks treatment) with AG-348 showed increased Hb and reduction in circulating erythroblasts

60

16 (%) * 50

) 14 40 dL

(g/ 12 °* 30 Erythroblasts

Hb °* 10 * 20

8 10 Circulating 6 0 WT Vehicle Wt AG-348 Hbb3th/+ Hbb3th/+ WT Vehicle Wt AG-348 Hbb3th/+ Hbb3th/+ Vehicle AG-348 Vehicle AG-348

*p< 0.05 compared to WT °p<0.05 compared to vehicle (V)

In β-thalassemic mice, AG-348 treatment ameliorates the ineffective erythropoiesis and is associated with decreased Erfe expression

40 2.5 Hbbth3/+ Vehicle (%) * Hbbth3/+ A-0001480 AG-348

2 high 35 1.5 *° FSC-A + 30 1 ° ratio Ter119 + 0.5 25 CD44 0 WT WT Hbbth3/+ Hbbth3/+ spleen weight/mouse weight weight weight/mouse spleen V AG-348 V AG-348 20 Spleen

70 WT WT vehicle 60 WT A-0001480 WT AG-348 ° * Erfe

° Hbbth3/+ Hbbth3/+ vehicle 50 Hbbth3/+ A-0001480 Hbbth3/+ AG-348

40 (%)

expression °* 30

Cells °° Gapdh 20 * on

* relative 10 °

0 mRNA WT WT Hbbth3/+ Hbbth3/+ I II III IV V AG-348 V AG-348

Erythroblast Populations In β-thalassemic Mouse Erythroid Precursors, AG-348 Treatment Reduces ROS Levels and the Amount of Apoptotic Orthochromatic E.

° ) 3 WT WT vehicle WT A-0001480 WT AG-348 15 20 Hbbth3/+ Hbbth3/+ vehicle 20 * Hbbth3/+ A-0001480 Hbbth3/+ AG-348 ° 15 10 15

* cells (%) * + * cells (%) ° 10 + 10 * ROS levelsfluoresc*10ROS ( 5 ° * 5

Annexin V 5 Annexin V

0 I II III IV 0 0 bone marrow spleen Erythroblast Populations

*p< 0.05 compared to WT °p<0.05 compared to vehicle (V)

In β-thalassemic mice, AG-348 treatment reduces ROS production and alpha chain aggregates in RBCs and increased RBC survival

15 * WT ) 3 °* 100 ° ° WT AG348 10 90 Hbb3th/+ ° Hbb3th/+ AG348 80 ° 5 ° 70 * ° (%) ° ° 60 0 cells ° ROS levelsfluoresc*10ROS ( WT WT Hbbth3/+ Hbbth3/+ ° ° V AG-348 V AG-348 50 * 40 * ° ° CFSE positive positive CFSE 30 * 1 ° 20 * * ° β 0.95 *

(%) Hb °* 10 * ° 0.9 total α / * 0 Hb 0.85 0 10 20 30 40 50 Time (days) 0.8 Soluble

0.75 *p< 0.05 compared to WT WT WT Hbbth3/+ Hbbth3/+ WT WT Hbbth3/+ Hbbth3/+ °p<0.05 compared to vehicle (V) V AG-348 V AG-348 V AG-348 V AG-348 β-thalassemic AG-348 treated mice showed reduced liver iron overload and increased Hepcidin expression

WT Hbbth3/+

Vehicle

Liver- Hamp

*

°* expression °* on Gapdh relative

mRNA WT WT Hbbth3/+ Hbbth3/+ AG-348 V AG-348 V AG-348

*p< 0.05 compared to WT °p<0.05 compared to vehicle (V)

Conclusions

² In β thalassemic mice, AG-348: • Reduces ineffective erythropoiesis, extramedullar erythropoiesis, Erfe expression and ROS levels • Increases Hb levels, reduces reticulocyte count and circulating erythroblasts • Significantly increases RBC survival • Reduces liver iron overload and increases Hamp

² AG-348 might represent a novel therapeutic approach in clinical management of anemia in β thalassemic syndromes. University of Verona; Verona, Italy Alessandro Matte, Enrica Federti, Francesca Lupo, Angela Siciliano, Elisabetta Beneduce

CEINGE-University Federico II , Naples; Italy Achille Iolascon, Luigia De Falco,

Dept of Pathology, Hopital S Louis, Paris, France Anne Janin, Christophe Lebouf

Agios Lenny Dang, Penelope Kosinski, Charles Kung