© 2014 Nature America, Inc. All rights reserved. 13 New Jersey, USA. Malades, Paris, France. Fontenay-aux-Roses, France. Center for Research on Inflammation, Paris, France. Sorbonne Paris Cité University, Imagine Institute, Paris, France. 1 TGF- best-studied the of expansion genitor pro erythroid inhibiting and differentiation erythroid accelerating TGF- progenitors. erythroid of tiation differen and proliferation the in participate to proposed been have understood. fully not are process this in involved of a hallmark ropoiesis), stage, resulting in a suboptimal production of RBCs (ineffective eryth early precursors are liver) susceptible to and apoptosis at spleen the polychromatophilicthe in (particularly sites ullary marrow bone and in both extramed pool erythroblast immature the of expansion and compensatory in anemia chronic resulting (RBCs), cells blood red circulating of lifespan shortened a to leads also tates at stage the polychromatophilic oxygen species (ROS), which induce the death of erythroid precursors reactive of production the with associated are precipitates these and free unbound unpaired of accumulation subsequent the absent or impaired by characterized β oxidative stress and effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine loop amplification involving observations suggest that ActRIIA ligand traps may have therapeutic relevance in by erythroblasts inducing apoptosis of immature through erythroblasts the Fas–Fas ligand pathway. Taken together, these an autocrine loop amplification in erythroid Abnormal differentiation. GDF11 expression was dependent on reactive oxygen species, suggesting the existence of of GDF11 decreased oxidative stress and the amount of in splenic from erythroblasts thalassemic mice and in and erythroblasts sera from subjects with model of receptor IIA (ActRIIA) ligand trap, improved corrected ineffective erythropoiesis, anemia and limited iron overload in a mouse The of pathophysiology ineffective in erythropoiesis Thomas O Daniel Jean-Antoine Ribeil Joel Veiga Michael Dussiot in erythropoiesis An receptoractivin IIA ligand trap corrects ineffective nature medicine nature Received 12 July 2013; accepted 10 January 2014; published online 23 March 2014; INSERM UMR 1163, Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutic Implications, Paris, France. -thalassemia is a common inherited hemoglobinopathy that is is that hemoglobinopathy inherited common a is -thalassemia These authors contributed equally to this work. Correspondence should be addressed to I.C.M. ( Members of the transforming growth factor- b -thalassemia -thalassemia intermedia. Expression of growth factor differentiation 11 (GDF11), an ActRIIA ligand, was increased 4 , , Damien Grapton α 11 -globin chains precipitate in erythroid precursors precursors erythroid in precipitate chains -globin Celgene, San Francisco, California, USA.

7 . Bone morphogenetic protein-4 (BMP4), one one (BMP4), protein-4 morphogenetic Bone . 9 1 advance online publication online advance Erythropoiesis Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA. – a 10 5 -globin -globin precipitation. , 13 , , Rajesh Chopra 7 β 1 UMR 962 (Inserm-CEA-University of Paris-Sud), Fontenay-aux-Roses, France. β – -thalassemia , , Thiago T Maciel 4 superfamily members in hematopoietic hematopoietic in members superfamily , 8 , , Jean-Benoit Arlet 2– 4 . . The of presence 1 – β b 5 -globin chain production and and production chain -globin -thalassemia. GDF11 -thalassemia. inactivation also corrected the abnormal ratio of immature/mature 6 , , Etienne Paubelle β . The molecular mechanisms mechanisms . The molecular limits RBC production by production RBC limits β α 10 6 Commissariat à l’Energie Atomique (CEA)–Institut des Maladies Emergentes et des Thérapies Innovantes (iMETI), -globin subunits -globin -thalassemia β , , Victoria Sung (TGF- 1 – 5 . These expanded expanded These . 5 α , 12 13

-globin precipi -globin 1 Service d’Hématologie Clinique, Assistance Publique–Hôpitaux de Paris, Hôpital Necker, Paris, France. 3 β – , , Aurélie Fricot CNRS ERL 8254, Paris, France. 4 ) ) superfamily b , , Francine Coté -thalassemia -thalassemia is poorly understood. We report that RAP-011, an activin a 1 -globin -globin membrane resulting precipitates, in increased terminal 1 – . The The . 5 11 , , Emmanuel Payen , Olivier , HermineOlivier - - - - - roid maturation through an autocrine amplification loop involving involving loop amplification autocrine an through eryth maturation roid terminal blocks cytokine this that showed also We GDF11. of effects this ligand trap are due erythropoietic to inactivation of the the ActRIIA ligand that and mice thalassemic in erythropoiesis tive ment with a mouse version of sotatercept (RAP-011) reverses ineffec the erythropoietic effects of sotatercept is unclear. We found that treat including drug, the increased hematocrit and hemoglobin levels of effects unexpected revealed studies clinical but to disorders, treat domain. bone-loss developed was originally Sotatercept Fc IgG1 human the to linked ActRIIA of domain cellular well-defined less are and indirect be to appear effects its progenitors erythroid primary and cells leukemia myeloid sial controver somewhat is role its but noted, been have erythropoiesis in erythropoiesis ineffective with associated is and anemias sideroblastic acquired some and esis samples from with subjects blood in levels increased at found is GDF15, member, superfamily of in the spleen mice adult of tion responses stress both ­differentiation 1 doi:10.1038/nm.346 Sotatercept (ACE-011) is a ligand trap that consists of the extra the of consists that trap ligand a is (ACE-011) Sotatercept – 12– 5 1 , Céline , Chartier Céline – 4 1 5 , Geneviève Courtois , Geneviève . . Although activin induces the differentiation of K562 chronic [email protected] 4 Laboratory of Excellence GR-Ex, Paris, France. b 6 1 , -thalassemia -thalassemia by suppressing the deleterious 7 – , , Yves Beuzard 8 4 8 Département de Biothérapie, Hôpital Necker–Enfants in vitro in , 12 & Ivan C Moura 1 – β 5 8 ) or O.H. ( -thalassemia, congenital dyserythropoi congenital -thalassemia, and and , Olivier , NegreOlivier β -thalassemia b 1 -thalassemia. Inactivation -thalassemia. in vivo in – 4 , , Yelena Z Ginzburg [email protected] 6 , 7 , , Philippe Leboulch 9 , is involved in the regula the in involved is , 1 1 1 7 1 . . The molecular basis of – . Effects of activin on on activin of Effects . 5 10 6 , Celgene, Summit, 7 1 , s e l c i t r a 2 0 5 Paris Descartes–

. Another TGF- . Another INSERM U1149, in vivo in

in in vitro ). 9

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© 2014 Nature America, Inc. All rights reserved. levels measured by ELISA from serum of thalassemic mice treated for 5, 10 or 30 d with RAP-011 or PBS. All data are expressed as the mean mean the as expressed are data All PBS. or RAP-011 d with 30 or 10 5, for treated mice thalassemic of serum from ELISA by measured levels ( PBS. with or subcutaneously) ( (RDW) width ( levels 1 Figure in increase volume, mean corpuscular hemoglobin mean corpuscular an revealed mice thalassemic RAP-011–treated in parameters RBC circulating of analysis Time-course mice. PBS-treated to compared concentration ( higher RBC count ( sotatercept, of origin. mouse of is IgG Fc domain the version which in RAP-011, mouse a used we domain, Fc IgG1 human the against xenoresponse a To avoid overload. iron progressive and RBC aberrant splenomegaly, anemia, chronic poiesis, erythro ineffective including disease, the of features several exhibit the intermedia, of model preclinical a used we thalassemia, of poiesis erythro ineffective in trap ligand ActRIIA the of role the Tostudy ActRIIA ligand trap improves anemia in RESULTS pathway. (FasL) ligand Fas–Fas the by inhibiting erythroblasts of immature mulation and stress oxidative A  * i e Wild type P

a Treatment of thalassemic mice with RAP-011 for with mice 60 in Treatmentd a resulted of thalassemic < 0.05, ** < 0.05, MCV (fl) 6 3 RBCs (1 × 10 /mm ) s e l c i t r 10 60 40 20 0 2 4 6 8 0 c WT ), reticulocyte counts ( counts reticulocyte ),

WT RAP-011 treatment improves hematological parameters in thalassemic mice. ( mice. thalassemic in parameters hematological improves treatment RAP-011 5 0 5 0 P h < 0.01, *** < 0.01, ) in wild-type (WT) and thalassemic mice treated for 0, 5, 10, 30 or 60 d with RAP-011 (10 mg per kg body weight twice weekly weekly twice weight body kg per mg (10 RAP-011 d with 60 or 30 10, 5, 0, for treated mice thalassemic and (WT) wild-type ) in Fig. Fig. 1 Time (d) Hbb Time (d) Fig. 1 α c th1/th1 10 10 ), as well as decreased reticulocytosis ( ), as well reticulocytosis as decreased -globin precipitation and promotes the accu the promotes and precipitation -globin a P ), hematocrit ( 30 < 0.005; < 0.005; 30 * mouse 25 d i ) Morphology of RBCs in peripheral blood smears following 30 d of RAP-011 or PBS treatment. ( treatment. PBS or RAP-011 d of 30 following smears blood peripheral in RBCs of ) Morphology ), mean corpuscular volume (MCV) ( (MCV) volume corpuscular mean ),

µ m ** 60 60 * 1 8 f b Hb n MCH (pg) Hematocrit (%) . = 5 mice per group for one out of three independent experiments. independent three of out one for group per = 5 mice Hbb 10 20 30 40 50 b 10 15 20 25 0 th1/th 0 5 Fig. 1 WT 1 th1/th1 WT

b + PBS -thalassemic mice b Hbb Hbb Wild type 5 0 ) and total hemoglobin 5 0 (thalassemic) mice mice (thalassemic) th1/th1 th1/th1 ** Time (d) PB RAP-011 Time (d) β S

-thalassemia -thalassemia 10 morphology morphology 10 * * Fig. Fig. 1 ** 30 30 * e 2 ), MCH levels ( levels MCH ), 5 µ 60 d ** m 60 * ), ), - - -

g c Hb

MCHC (g/dl) Hb (g/dl) in resulting erythroblasts, immature of expansion with anemia marrow mice. bone thalassemic in expansion corrects and splenomegaly decreases RAP-011 that ( mice thalassemic PBS-treated Ter-119 erythroid of proportion decreased a and cellularity and weight had spleen mice decreased thalassemic RAP-011–treated analysis, time-course a In RAP-011 corrects pathological features of improves anemia. ameliorating mice count, RBC and content hemoglobin thalassemic in RAP-011 with treatment that show ( treatment during sustained was increase this and treatment RAP-011 of start the following rapidly increased tion ( morphology RBC blood peripheral improved also mice thalassemic of treatment increase in reticulocyte numbers ( C57BL/6 mice did not induce although polycythemia, we an observed ( width distribution RBC of normalization concurrent and concentration MCH and (MCH) 10 12 14 16 b 40 50 10 20 30 0 2 4 6 8 Ineffective erythropoiesis in in erythropoiesis Ineffective 0 th1/th WT WT 1 f

+ RAP-01 ), MCH concentration (MCHC) ( (MCHC) concentration MCH ), a 5 0 5 0 – h ) Red blood cell counts ( counts cell blood ) Red 1 Fig. 1 Fig. Time (d advance online publication online advance Time (d 10 10 * i ) ). In addition, serum erythropoietin concentra erythropoietin serum addition, In ). + ) cells in spleen and bone marrow, compared to to marrow, compared bone and spleen in cells 30 30 * Fig. Fig. 1e 2 5 µ 60 60 * * m – Fig. 2a Fig. Supplementary Fig. 1a h β h d ). RAP-011 treatment in wild-type ). RAP-011 in treatment wild-type RDW (%) 6 3 by characterized is -thalassemia a g 10 15 20 25 Reticulocytes (1 × 10 /mm ) 0. 1. 1. 2. 2. ), hematocrit ( hematocrit ), ) and red cell distribution distribution cell red ) and 0 5 5 0 5 0 5 0 j WT –

EPO (ng/ml) WT e 10 20 30 40 50 ). These data demonstrate demonstrate data These ). 0 5 0 WT 5 0 b j Fig. 1 Fig. ) Erythropoietin (EPO) (EPO) ) Erythropoietin Hb Hb Wild type -thalassemia

b b b ** Time (d nature medicine nature th1/th th1/th 5 ), hemoglobin (Hb) (Hb) hemoglobin ), Time (d Time (d j 1 1 10 ). These results results These ). PB RAP-01 10 ) – ** S ) 10 h * ) ). RAP-011 30 30 1 ± s.e.m. s.e.m. ** 30 60 * * 60 * -

© 2014 Nature America, Inc. All rights reserved. and and ( erythroblasts splenic immature/mature of ratio decreased 1m Fig. ( mice PBS-treated in to cells) Ery.C compared reticulocytes, and erythroblasts (orthochromatic cells Ter-119 of number absolute the and percentage the in increase a concomitant and cells) Ery.B erythroblasts, chromatic absolute the Ter-119 and of number percentage the both in decrease a revealed mice erythroblasts analyses immature/mature cytometry of ratio imbalanced an *** ( bilirubin direct PBS: or RAP-011 d with 60 to up for ( values. minimal and maximal and means show ( area. scatter forward FSC-A, treatment). d of (30 mice thalassemic PBS-treated and RAP-011– in distribution subset erythroblast marrow bone and spleen ( values. minimal and maximal and means show plots Box-and-whisker shown. is populations erythroblast different of percentage The distribution. (FSC) scatter forward and Ter-119 and CD71 staining by evaluated and RAP-011 with treatment d after 5–30 ( treatment). d of (30 mice PBS-treated or RAP-011– in number erythroblast spleen ( treatment). d of (60 mice thalassemic PBS-treated or RAP-011–treated of bones of cross-sections H&E-stained in observed ( cellularity marrow 2 Figure nature medicine nature arrest maturation g e a

P CD71 CD71 + 6 Spleen weight (g) 10 10 10 10 10 10 10 10 Supplementary Fig. 1o Fig. Supplementary 0.1 0.2 0.3 0.4 0.5 < 0.005; < 0.005; Ter-119 (1 × 10 Spleen 0 0 2 3 4 5 2 3 4 5 0 h cells per spleen) Spleen ) An index of ineffective erythropoiesis established by calculating the ratio of Ery.B and Ery.C percentage populations. Box-and-whisker plots plots Box-and-whisker populations. Ery.BEry.Cof and percentage ratio the calculating by established erythropoiesis ineffective of index ) An WT 200 400 600

0 0 0.251 0.784% RAP-011 treatment reduces ineffective erythropoiesis in thalassemic mice. ( mice. thalassemic in erythropoiesis ineffective reduces treatment RAP-011 . hs RP01tetd hlsei mc soe a showed mice thalassemic RAP-011–treated Thus, ). ProE ProE 0 10 10 Hb % 2 2 1 5 Ter-119 Ter-119 Hb b 10 10 ** th1/th1 n Time (d b 3 3 = 5 mice per group for one out of three independent experiments. independent three of out one for group per = 5 mice th1/th1 Ter-119 Ter-119 3 0 62 10 10 59.7 Hbb Hbb Wild-type PBS % RAP-011 c 4 4 ** 2 ) of thalassemic mice treated for 5, 10, 30 or 60 d with RAP-011 or PBS. ( PBS. or RAP-011 d with 60 or 30 10, 5, for treated mice thalassemic ) of

1 PB ** 10 10 ) 2 + th1/th th1/th . 6 0 0 advance online publication online advance 5 5 CD71 of spleens from RAP-011–treated thalassemic thalassemic RAP-011–treated from of spleens S 1 1 ** PBS RAP-011 CD71 CD71 + 6 0 10 10 10 10 10 10 10 10 Ter-119 (1 × 10 0 0 2 3 4 5 2 3 4 5 + cells per femur) b FSC 10 15 20 ), indicating correction of erythroblast erythroblast of correction indicating ), 0 0 Spleen cell number 50K 50K 0 5 6 43.4 30.1 (1 × 10 )

100K 200 400 600 800

100K % % low FSC- FSC- 0 ** 42.9 150K 150K 29 cells (late basophilic and poly and basophilic (late cells WT % 26.2 23.9 % A A Fig. 2f Fig. 200K 200K % % **

250K

250K 5 Time (d 10 i Bone marrow , , g j

). Biochemical analysis of parameters of ineffective erythropoiesis in sera of thalassemic mice treated treated mice thalassemic of sera in erythropoiesis ineffective of parameters of analysis Biochemical ). CD71 CD71 Bone marrow and and 10 10 10 10 10 10 10 10 30 ** ) 0 0 2 3 4 5 2 3 4 5 Time (d Time (d i ) and total bilirubin ( bilirubin total ) and f 1.42 ProE 0 0 1.64% 60

ProE % cells per % cells per * 10 Supplementary Supplementary 10 %

+

2 femur spleen 2 Ter-11 Ter-11 0 1 2 3 4 5 0 1 2 3 4 5

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c Hb 3 3 b Cell number Ter-119 Ter-119 59.3 65.8 th1/th 10 6 10 9 b 9 per femur (1 × 10 ) % % 4 1 5 4 th1/th 10 20 30 40 1 1 5 − 0 10 9 1 10 Fig. 2 Fig. FSC RAP-01 . Flow Flow . 5 WT 5 1 PBS 3 0 3 0 low

CD71 CD71 Hbb Hbb Wild-type PBS 5 10 10 10 10 10 10 10 10 h 0 - 1 0 0 * 0

5 2 3 4 5 2 3 4 Time (d j th1/th th1/th ). All data are expressed as the mean mean the as expressed are data All ). 0 0 10 48.3% 50K 44.7% Ter-119 of number absolute the in increase an observe did we although ity, cellular marrow bone affect not did RAP-011 with treatment mice, healthy In spleen. the on drug the of by effect the on depends RAP-011 erythropoiesis ineffective of correction that suggest data These RAP-011 can promote splenic erythropoiesis in wild-type mice. mice. wild-type in in an reticulocyte increase we with concept, this Consistent observed erythropoiesis splenic promote can RAP-011 ( and erythroblast unchanged immature/mature were number the ratio and absolute cells Ery.C the of mice, percentage thalassemic RAP-011–treated Ter-119 of percentage 50K 20 40 60 20 40 60 100K 1 1 0 0 RAP-011 PBS

100K 30 Although we observed a decrease in the absolute number and and number absolute the in decrease a observed we Although ) FSC- FSC- * WT WT 7.43% 150K 7.16% 150K Ery.A: basophilic 41.7% 45.7% f ) Erythroblast differentiation in bone marrow and spleen harvested harvested spleen and marrow bone in differentiation ) Erythroblast 60 A A 200K 200K 1 5 a 5 * + –

pen el ( cells spleen 250K

c 250K ) Spleen weight ( weight ) Spleen 10 d 3 0 h 30 Hb

Time (d) Time (d) 0 d WT WT 30 10 30 10 ) Bone marrow erythroblast number and distribution distribution and number erythroblast marrow ) Bone b 5 5 th1/th Wild-type PBS Bone marrowmaturation Spleen maturationindex 0 0 20 40 60 80 20 40 60 1 0 0 0.5

RAP-011 Hbb Hbb Wild-type PBS 500 500 Ery.B: latebasophilic WT Ery.B/Ery.C Ery.B/Ery.C upeetr Fg 1i Fig. Supplementary WT and polychromatic + i. 2f Fig. 5 1.0 g erythroblasts in the bone marrow of of marrow bone the in erythroblasts index ) Representative flow cytometry analysis of analysis cytometry flow ) Representative th1/th th1/th µ µ a m m ), total spleen cell number ( number cell spleen total ), 5 1 5 1.5 1 1 10 RAP-011 PBS 10 2.0 Cell number – * 3 0 ± h (per field) Hb 2.5 s.e.m. * s.e.m. 15 10,00 15,00 and and ** * 30 5,000 ** b 0 th1/th 0 0 0 500 100 1 j i upeetr Fg 1n Fig. Supplementary PBS Direct bilirubin 20 40 60 20 40 60 80 0 0 µ P *** Ery.C: orthochromatic Total bilirubin m WT WT

e ( mol/l)

< 0.05, ** < 0.05, µ NS 0.5 1.0 1.5 and reticulocytes ) Bone marrow and and marrow ) Bone (µmol/l) 0 2 4 6 8 0 ** 1 5 1 5 WT WT – l s e l c i t r a ), suggesting that that suggesting ), 3 0 3 0 5 0 5 0 Hbb Hbb Wild-type PBS Hbb Hbb Wild-type PBS b Time (d Time (d P ) and bone bone ) and ** * < 0.01, < 0.01, * 0 0 th1/th th1/th th1/th th1/th 10 10 ** ** ) ) 1 1 1 1 RAP-011 PBS RAP-011 PBS 30 30 ** * 60 60 ** **

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© 2014 Nature America, Inc. All rights reserved. In addition to anemia, ineffective erythropoiesis in subjects with with subjects in β erythropoiesis ineffective anemia, to addition In RAP-011 decreases the iron overload of mice. thalassemic of spleen the in erythropoiesis ineffective correct Taken that an together, confirm results ActRIIA these trap ligand can bilirubin or lactate dehydrogenase levels ( alter not did treatment RAP-011 erythropoiesis, ineffective no ably RAP-011 administration. In wild-type mice, in which there is presum 2a Fig. compared to in those mice PBS-treated ( mice in dehydrogenase were RAP-011–treated thalassemic decreased mice. diseased and healthy in erythropoiesis splenic on effects different has RAP-011 that suggest ( populations precursor erythroid marrow bone in or cells Ery.C of numbers the Ter-119 (Ery.A, populations precursor ( erythroid splenic in increase mice an ited wild-type of 1c Fig. treatment upon numbers mean the as expressed are data All group). (CD71 immature purified in the as expressed bands, gel indicate Arrows indicated. as d, 30 for 5 animals) of out (3 RAP-011 or 5 animals) of out (4 PBS with treated mice thalassemic and wild-type from collected blood of electrophoresis gel ( group). each for 5 animals of out d (4 30 for RAP-011 or PBS with treated mice thalassemic from hemolysates respectively. erythroblasts, RAP-011–treated and PBS- represent lines red and black and cells, control unstained indicates line Gray fluorescence. DCFH for FACS histogram a representative Right, h. 48 for PBS or RAP-011 with treated erythroblasts primary in (DCFH) dichlorodihydrofluorescein using cytometry flow by evaluated n method. Drabkin’s by assayed as PBS, or 10 h with 48 for treated erythroblasts thalassemic primary in levels ( hemoglobin membrane and expression chain globin on RAP-011 of significant. not NS, RAP-011. or PBS with treated mice thalassemic or wild-type of samples liver from (qPCR) PCR quantitative by ( PBS. or RAP-011 with d 60 or 30 10, 5, 0, for treated mice in ( levels ( levels iron Systemic mice. thalassemic and wild-type in parameters iron ( mice. thalassemic in precipitates hemoglobin RBC-associated and overload iron 3 Figure A  the of levels circulating decreased and absorption iron intestinal d a = 3 mice per group. ( group. per = 3 mice

-thalassemia results in systemic iron overload, increased gastro increased overload, iron systemic in results -thalassemia ) Hepcidin ( ) Hepcidin

The serum levels of total and direct bilirubin and serum lactate lactate serum and bilirubin direct and total of levels serum The Iron (µmol/l) s e l c i t r 10 20 30 40 50 0 a b WT ), confirming that ineffective erythropoiesis is mitigated by mitigated is erythropoiesis ineffective that confirming ), – ,

) and transferrin (Tf) saturation ( saturation (Tf) transferrin ) and + d c RAP-011 treatment decreases decreases treatment RAP-011 CD71 . n diin RP01tetd idtp mc exhib mice wild-type RAP-011–treated addition, In ). ) Biochemical analysis of serum serum of analysis ) Biochemical α n -globin precipitation. Total precipitation. -globin e 5 0 = 5 mice per group. ( group. per = 5 mice Hamp ) and soluble hemoglobin ( hemoglobin soluble ) and Supplementary Fig. 1p Fig. Supplementary + FSC Time (d ) mRNA levels measured measured levels ) mRNA high n g 10 ) Left, ROS generation generation ROS ) Left, = 5 mice per group. group. per = 5 mice µ and Ery.B cells), but showed no changes in in changes no showed but cells), Ery.B and ) g/ml RAP-011 RAP-011 g/ml a + α ), transferrin transferrin ), ) erythroblasts from wild-type and thalassemic mice treated with PBS or RAP-011 for 30 d (4 out of 5 animals for each each for 5 animals of out d (4 30 for RAP-011 or PBS with treated mice thalassemic and wild-type from ) erythroblasts 30 -globin to to -globin *

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Supplementary Supplementary RAP-011 Supplementary Supplementary Time (d th1/th α β i 10 -globin -globin 1 ) RAP-011 30 g DCFH fluorescence

h (median) 60 P 10,000 15,000 20,000 ) Determination of total and soluble hemoglobin by Drabkin’s method in blood blood in method Drabkin’s by hemoglobin soluble and total of ) Determination – Hb 5,00 < 0.01 for one out of three independent experiments. independent three of out one for < 0.01 d ). ). ­ - - c 0 0 Tf saturation (%) 20 40 60 WT 0 from our our expected from As PBS. or RAP-011 of presence the in from mice (ProE) thalassemic proerythroblasts cultured we treatment, RAP-011 of effects the underlying mechanisms cellular the into insight Togain RAP-011 decreases cytotoxic membrane GDF15. of independently overload iron decreases and expression ActRIIA trap reverses ineffective derepresses erythropoiesis, hepcidin ( promoter functional reporter assays Smad2/3 using in the CAGA RAP-011 box by from the humaninhibited PAI-1not was activity GDF15 that ing that GDF15 not does bind previous observations ActRIIA thalassemic mice ( However, samples. RAP-011- and PBS-treated did between not expression GDF15 differ spleen and marrow bone mouse in expression ulator of expression hepcidin in ( controls in PBS-treated to those compared mice in RAP-011–treated lassemic mice ( 2e Fig. ( mice wild-type in homeostasis iron modulate not hepcidin hormone hypoferremia-related Sp Fig. Fig. 3 Hbb Hbb WT le * en Hbb th1/th th1/th j ) Expression of of ) Expression PBS 5 0 d – th1/th 1 1 ). Because GDF15 secreted by erythroblasts is by). GDF15 a secreted Because reg erythroblasts potential PBS RAP-011 g ), it reduced serum iron and transferrin saturation in tha in saturation transferrin and iron serum reduced it ), in vivo in 1 Ti upeetr Fg 2j Fig. Supplementary me Hbb RAP-011 10

Fig. Fig. 3a (d advance online publication online advance ) th1/th1 observations, RAP-011 treatment increased total total increased treatment RAP-011 observations, 0 Supplementary Fig. 2h Unstained 30 Hbb-a1 ** 1 0 – c 60 ). ). Hepatic hepcidin mRNA levels were higher ** 0 2 adult globin mRNA evaluated by qPCR, qPCR, by evaluated mRNA globin adult fluorescence d 10 DCFH Hamp mRNA expression α-globin/β-globin ratio (OD) 3 i 15 10 β PBS (normalized by RPS3) ) Left, triton–acetic acid–urea (TAU) acid–urea triton–acetic ) Left, 0 5 0 1 2 3 -thalassemia Hbb Wild type Hbb 10 ). These results argue that the the that argue results These ). th1/th1 th1/th1 4 NS * ** RAP-011 10 PBS RAP-011 , 2 i 5 ** 2 ). Moreover, we confirmed . Although RAP-011 did did RAP-011 Although . a -globin precipitates 2 3

, , we analyzed GDF15 j e h nature medicine nature Hbb-a1 mRNA expression Soluble Hb/total Hb Hemoglobin (mg/ml) 0. 1. 1. 0. 0. 0. 0. 1.

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4 ** * by show * 1 1 PBS RAP-011 ** - - -

© 2014 Nature America, Inc. All rights reserved. of of expression addition, In mice. of thalassemic of effects spleen the the in in RAP-011 participate not do B activin and A activin ligands ( detectable ( controls wild-type from those to compared mice thalassemic from higher in Ter-119 by encoded lassemic and mice. wild-type Activin A and activin B are homodimers TER-119 in expression their in differences ActRIIA bind to shown previously been erythroblasts spleen and marrow bone TGF- modulate to ( mice wild-type from Fig. 3g those to compared thalassemic from mice erythroblasts cultured in higher was expression ment with our observations using freshly isolated cells, ( erythroblasts marrow–derived Acvr2a from wild-type mice ( those to relative mice thalassemic from erythroblasts marrow bone that strated ( mice control erythroblasts from thalassemic mice compared to cells from wild-type lation of ( erythroblasts TGF- other of upregulation noted also We in erythroblasts, decreased is thalassemic of samples Fig. three 3a Supplementary of two was in increase (this observed mice control wild-type from cells to compared with of mice (E14.5)) day 14.5 onic of TGF- the set data ( expression gene published a of analysis retrospective a In Aberrant GDF11 expression in RBCs. of correction the to contribute decreased exhibit trap ligand Fig. 2l embryonic or in adult difference compared to that in controls ( α in adult a decrease revealed mice thalassemic from RAP-011–treated mice thalassemic membrane–associated RBC of level the and erythroblasts thalassemic cultured in levels ROS in survival RBC reduced and erythropoiesis ineffective with associated are precipitates these ( cells α thalassemic untreated to compared erythroblasts thalassemic cultured in levels hemoglobin soluble and hemoglobin nature medicine nature Ter-119 Supplementary Fig. 4a Fig. Supplementary GSE3412 -globin precipitation and the increased ROS levels associated with with associated levels ROS increased the and precipitation -globin -globin ( -globin Globin Globin gene expression analysis in purified immature erythr Because activin A, activin B, GDF-8, GDF-11 and BMP-10 have have BMP-10 and GDF-11 GDF-8, B, activin A, activin Because We confirmed increased expression of in vivo Mstn ) was apparent. Thus, thalassemic mice treated with the ActRIIA , and and h (the gene encoding GDF8) and and GDF8) encoding gene (the + Acvr2b ). ). Thus, the observations, suggesting that environmental conditions may bone marrow– and spleen-derived cells (data not shown). shown). not (data cells spleen-derived and marrow– bone 5 Hbb-a1 ), we found that that found we ), Supplementary Fig. Supplementary 4b β β Acvr1b Acvr2b Inhba superfamily in erythroblasts from fetal liver (at liver embry from fetal in erythroblasts superfamily family type I or type II receptor genes, in in genes, receptor II type or I type family β Supplementary Fig. 3c Fig. Supplementary Supplementary Fig. 3b Fig. Supplementary expression in Ter-119 β -like globin ( globin -like Hbb + family receptor expression, leading to changes in in changes to leading expression, receptor family in vivo in ) and an increase in in increase an and ) bone marrow cells, but not in Ter-119 and and β

and and was similar between cultured spleen- and bone bone and spleen- cultured between similar was minor chain ( in vitro th3/th3 advance online publication online advance Supplementary Fig. 3e ). ). In addition, we found that Inhbb Tgfbr1 ( ), whereas whereas ), β Fig. 3h Fig. erythroblasts ( erythroblasts -thalassemia. RAP-011 treatment lowered lowered treatment RAP-011 -thalassemia. erythroblast culture results are in contrast Gdf11 Fig. 3 Hbb-bh1 α , respectively , expression was higher in splenic and and splenic in higher was expression -globin gene expression, which could could which expression, gene -globin α Supplementary Fig. 3g Supplementary Acvr2a b -globin precipitation in circulating circulating in precipitation -globin , i Hbb-b2 j -thalassemia ). is the only upregulated member member upregulated only the is ). ). These data suggest that ActRIIA and β Inhbb -thalassemia major ( major -thalassemia + splenic (but not bone marrow) ) expression ( expression ) ). , ε in vivo in d gene expression, but not of of not but expression, gene Supplementary Fig. 2m y globin ( globin y Acvr2a Bmp10 ). Further analysis demon analysis Further ). expression was minimally minimally was expression ) ) ( 2 Supplementary Fig. 3a Fig. Supplementary + 2 4 Supplementary Fig. 2k Supplementary , 4 erythroblasts from tha from erythroblasts , we explored potential potential explored we , α f . . ). Expression of . -globin precipitates in in precipitates -globin Inhba and revealed upregu was undetectable in in undetectable was Hbb-y in vitro in Tgfb1 Supplementary Supplementary Supplementary Supplementary expression was was expression

Acvr2a + – ) expression expression ) spleen cells, i expression ). ). In agree Hbb Fig. 3e Fig. Hbb ( Fig. 3 Fig. Acvr1b mRNA o th3/th3 th3/th3 blasts blasts ). No , f g ). ). ). ). - - - - - ) ) ) ,

compared to control mice ( mice control to compared as thalassemic in affected minimally was mice, control from spleens in detected readily both were which B, and A activin of expression ( chemistry immunohisto control using mice from to wild-type compared those We found higher GDF11 expression in spleens from mice thalassemic GDF11 overexpression in thalassemic erythroblasts mice. thalassemic in trap ligand ActRIIA the of effects spleen-specific the mediating for ligand candidate a is mice, whose expression is upregulated in splenic erythroblasts of thalassemic a in suggested previously as engagement, receptor by modulated be can Fig. 4d ( ligands trap ActRIIA of ligand expression the reduced that in results showed RAP-011 the of absence or the in presence cultured erythroblasts thalassemic derived bone-marrow in 4c Fig. marrow (but not spleen) cells from thalassemic mice ( ( not bone marrow) cells from compared thalassemic to mice wild-type Gdf11 cultured erythroblasts positive for the erythroblast developmental marker developmental glycophorin A (GPA) from subjects with erythroblast the for positive erythroblasts cultured ( control mice to compared mice thalassemic of sera in higher was concentration ( controls healthy to relative thalassemia but not A activin or B, activin concentration is higher in with subjects GDF11, that revealed subjects control healthy 8 and major lassemia ( expression GDF11 reduced considerably cells the of treatment RAP-011 that and erythro blasts spleen–derived thalassemic in overexpressed or is A B, activin activin not but GDF11, that confirmed cultures erythroblast ( spleen the in observed levels high the to compared as mice, thalassemic row from mar bone in levels low very only at expressed was GDF11 addition, precursors (CD71 erythroid immature relatively to confined is overexpression GDF11 showed that populations of erythroid different scrutiny Careful cells. dendritic or cells T cells, B by produced not is GDF11 that gesting not white pulp cells, produced GDF11 ( ( macrophages in not but roblasts β conditions of erythropoiesis more GDF11 may with the be ineffective associated anemia hemolytic or overexpression. GDF11 reverses treatment RAP-011 that cells that GDF11 concentration is increased in marrow spleen thalassemic cells and bone data demonstrate These cells. in spleen in expression ActRIIA ligand wild-type and ( thalassemic between sion 4e Fig. marrow bone in in vivo concentration GDF11 decreased also treatment RAP-011 model. mouse this in overexpression GDF11 reverse can much a lesser to extent, activin and, A and GDF11 activin B of ( expression the decreased RAP-011 with Fig. 4 Supplementary Fig. 4f Fig. Supplementary -thalassemia than with stress erythropoiesis. stress with than -thalassemia Comparison of of Comparison ActRIIA ligand concentration in sera from 16 subjects with tha with subjects 16 from sera in concentration ligand ActRIIA in Ter-119 was overexpressed GDF11 mice, In thalassemic hypoxic under mice in upregulated not was expression GDF11 Xenopus expression was strongly upregulated in Ter-119 in upregulated strongly was expression a , as determined by immunoblotting analyses ( analyses by immunoblotting , as determined ), confirming previous gene expression data set results. set data expression gene previous confirming ), ). This finding suggests that ). suggests the This of finding expression ActRIIA ligands ), ), whereas . hr ws ite ifrne n cRI lgn expres ligand ActRIIA in difference little was There ). Fig. Fig. 4 model Fig. 4 Fig. i. 4 Fig. b + Gdf15 ), confirming ), the confirming gene expression data. In contrast, Acvr2a Ter-119 25 i Supplementary Fig. 4g Fig. Supplementary ). Confocal microscopy analyses confirmed that confirmed analyses microscopy ). Confocal , 2 g 6 . Thus, . Thus, ). Flow cytometric analysis from primary primary from analysis cytometric Flow ). expression was upregulated in Ter-119 ), in contrast to the differences we observed observed we differences the to contrast in ), , , low/− Inhba Fig. 4 Fig. Gdf11 and Ter-119 , , b Inhbb Fig. 4 Fig. ). Treatment of thalassemic mice mice thalassemic of Treatment ). 2 Fig. 4 7 , , a TGF- ( Supplementary Fig. 5a Supplementary i. 4c Fig. and and e Fig. 4 Fig. ). Only red pulp cells, and and cells, pulp red Only ). b ), indicating that RAP-011 + ). CD71 Gdf11 β superfamily member member superfamily h , d β ). Similarly, GDF11 GDF11 Similarly, ). -thalassemia major -thalassemia ), indicating that that indicating ), s e l c i t r a + transcript levels levels transcript cells ( Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary + spleen (but (but spleen Fig. Fig. 4 + eryth + ), ), sug bone f ). ). In  ------­

© 2014 Nature America, Inc. All rights reserved. number number TUNEL increased an exhibited mice thalassemic RAP-011 expected, As of apoptosis. on impact the investigated we erythropoiesis, ineffective of characteristic classic a is which splenomegaly, reverses treatment the bypass to atapoptotic crisis stage the polychromatophilic unable erythroblasts immature of proliferation sive in erythropoiesis Ineffective GDF11 contributes to immature erythroblast pool expansion with humans and mice both in ropoiesis eryth ineffective of characteristic is erythroblasts immature in sion Fig. 5b GPA in expressed minimally only and cells GPA in undetectable is GDF11 whom (in subjects control healthy from erythroblasts to compared expression GDF11 increased have ** ( wild-type from sera in GDF11 ligand Fc–bound ( 50 bars, Scale blue. in (DAPI) nuclei and green in shown is expression protein GDF11 mice. thalassemic of marrow bone and spleen from sections Ter-119 green, in shown is expression protein GDF11 ( anemia hemolytic and hypoxia normoxia, ( (aRBC). anemia hemolytic and hypoxia normoxia, of conditions under mice C57BL/6 wild-type from sections spleen of staining immunohistochemical ( d. 30 for RAP-011 or PBS with treated mice thalassemic and wild-type from sections spleen of staining immunohistochemical GDF11 B and activin A, activin ( units. arbitrary AU, ( mice thalassemic and type wild- from erythroblasts marrow bone and spleen in qPCR by evaluated levels mRNA ( erythropoiesis. ineffective β 4 Figure A  erythroblasts. mouse thalassemic maturing of apoptosis with h d c a d -thalassemia and is associated with with associated is and -thalassemia e

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2– 0.05 0.10 0.15 e 200 200 200 4 + 0 Fig. Fig. 5 . Because RAP-011 . Because f erythroblasts in blue and F4/80 and blue in erythroblasts µ µ µ Detection anti-GDF11 Control spleen m m m Coating RAP-011 Supplementary Supplementary e GDF11 a ,

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RAP-011 = 5). All data are expressed as the mean mean the as expressed are data All = 5). b Thalassemia + ). In RAP-011–treated mice, Fas expression was upregulated upregulated was Fas expression mice, In RAP-011–treated ). ( 200 200 200 Hb e CD71 ) and CD71 ) and b µ µ µ m m m th1/th Supplementary Fig. 5c advance online publication online advance 5 Ter-11 c 1 0

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© 2014 Nature America, Inc. All rights reserved. mediated by the Smad2 pathway. As expected, Smad2 and/or Smad3 Smad3 and/or Smad2 pathway. Smad2 As by expected, the mediated ( phosphorylation. Smad1/5 affect not did it whereas pathway, Smad2 the of activation decreased showed mice thalassemic RAP-011–treated in cells spleen Moreover, activation. pathway Smad2 in results mice thalassemic of ( mice wild-type from those to comparison in mice thalassemic from sec tions spleen in higher were (Smad1/5) Smad5 and/or Smad1 ylated phosphor degree, lesser a to and, Smad2 phosphorylated of with levels associated erythropoiesis ineffective β the to lead could that We next investigated the molecular pathways downstream of ActRIIA GDF11 inhibits terminal erythropoiesis in in expansion erythroid to contribute may that erythroblasts previously immature a is in expression pathway Fas-FasL regulating overexpression mechanism undescribed GDF11 that demonstrate ( results erythroblasts cultured of tosis apop in resulted also siRNA by expression GDF11 of Knockdown nature medicine nature Ter-119 to restricted was activation (Smad2/3) -thalassemia. Immunohistochemical analysis demonstrated Immunohistochemical that the -thalassemia. d b c a

β + + + % Fas cells % Fas cells

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100 (bone marrow) (spleen) 20 40 60 80 10 15 20 20 40 60 80 20 40 60 80 Fig. 6 Fig. Hbb 0 0 5 0 0 Hbb th1/th1 ** ProE th1/th1 ProE ** a

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ne f lcig niois ietd gis atvn , cii B, activin A, activin A, activin to but not GDF11, to activin Antibodies or GDF11. GDF8 against directed antibodies blocking of ence pres the in cells ProE thalassemic cultured we maturation, roblast with associated ( or rGDF15, increased ROS levels in primary thalassemic erythroblasts ( levels ROS reduced in resulted erythroblasts thalassemic primary of treatment RAP-011 with cultured ( dithiocarbamate pyrrolidine erythroblasts antioxidant the thalassemic in increased maturation gates membrane-bound cytotoxic and ROS to expression in expression mice. thalassemic of erythroblasts splenic in vated signals GDF11 that pathway Smad2/3 the observations through previous confirm results these luciferase reporter assays ( CAGA- functional in activity rGDF11 and rGDF8 inhibited bodies anti blocking GDF11 or GDF8 and RAP-011 expected, as Finally, th1/th1 0 Fig. 6 Fig. 3 10 To further define the role of ActRIIA ligands in terminal eryth terminal in ligands ActRIIA of role the define further To GDF11 regulating mechanisms molecular the into To insight gain

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2 th1/th1 µ 5 9 m h Hbb Hbb . GDF11 expression decreased and terminal erythroblast erythroblast terminal and decreased expression GDF11 . Ery. Ery. 0 Hbb ), suggesting that GDF11 contributes to the oxidative stress stress oxidative the to contributes GDF11 that suggesting ), ActB Ab Count

th1/th1 Fas C th1/th th1/th C 1 0 AV AV GDF11 Ab L 1 1 PBS RAP-011 β + + 0 7AAD -thalassemia, we evaluated the relationship of GDF11 the relationship we evaluated -thalassemia, 3 α 10 4 -globin precipitates. -globin 10 + 5 Fig. 6 Fig. mice. ( mice. thalassemic in apoptosis erythroblast stage 5 Figure luciferase activity ( activity luciferase (ALK4/5) inhibitor) abolished CAGA-GDF11-induced ALK5 and ALK4 (an SB505124 and inhibitor) (an ALK5 SB431542 addition, In activation. pathway Smad1/5 does induce not GDF11 that idea the with consistent ( respectively) aling, gene (specific for Smad2/3 and Smad1/5 sign BRE a (BMP responsive not element)-luciferase but reporter box- CAGA a of activation the induced (rGDF11) GDF11 recombinant ( spleen GDF11, activin A or activin B (one out of three three of out B (one activin A or activin GDF11, against antibodies neutralizing with treated mice thalassemic from erythroblasts cultured in staining) V (AV)7-AAD and (annexin apoptosis n experiments, independent three of out (one Ab) B (ActB activin or Ab) A (ActA activin Ab), (GDF11 GDF11 for specific antibodies neutralizing with or without treatment after differentiation of stages indicated the at cells erythroblast thalassemic cultured in cells FasL ( shown. also are cells Ery.Bin spleen staining FasL and Fas of FACS histograms Representative group). each RAP-011 d ( 30 for PBS with or spleenand of thalassemic mice treated with marrow bone (Ery.A, Ery.BEry.C) from and Fas ( red. in Ter-119 green, in shown TUNEL d. 30 for PBS or RAP-011 ( mice thalassemic and wild-type from sections showing micrographs Confocal erythroblasts. mean mean All data as are expressed the group). each experiments, independent n = 3 mice for each group). ( group). each for = 3 mice = 3 mice for each group) treated with with treated group) each for = 3 mice Supplementary Fig. Supplementary 5h g + ). Most notably, rGDF11, but not rGDF8 but rGDF8 not Most notably,). rGDF11, and FasL and ± a s.e.m. * s.e.m. 2 b ) TUNEL staining of CD71 of staining ) TUNEL

4 ) Flow cytometric quantification of quantification cytometric ) Flow i. 6b Fig. ActRIIA trap therapy promotes early- promotes therapy trap ActRIIA and show that this pathway is acti is pathway this that show and + erythroblast populations populations erythroblast P c < 0.05 and ** ) The percentage of Fas and and Fas of percentage ) The – d Supplementary Fig. 5g Fig. Supplementary ). In C2C12 myoblasts, myoblasts, C2C12 In ). Supplementary Fig. 5f Supplementary Fig. 6e Fig. + in blue and CD71 and blue in n = 3 mice for for = 3 mice ) s e l c i t r a d 2 4 ) Quantification of ) Quantification . . Taken together, α n , P f = 5 mice for for = 5 mice -globin aggre -globin < 0.01. ). In addition, addition, In ). + + staining is staining

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© 2014 Nature America, Inc. All rights reserved. are expressed as the mean mean the as expressed are staining and FSC distribution. The percentages of immature (Ter-119 immature of percentages The distribution. FSC and staining (Ter-119 (Ter-119 ProE as classified were Cells shown. is distribution FSC and Ter-119 and CD71 staining using differentiation erythroblast of analysis cytometry Flow propeptide. GDF11 B or activin A, activin against antibodies neutralizing with treated ( DCFH. FACSby using measured was generation ROS ng/ml). 50 or (5 rGDF8 ( PDTC. or RAP-011 with (Ter-119 immature ( RAP-011. or PDTC with treatment after levels GDF11 intracellular (5 (PDTC) dithiocarbamate pyrrolidine with treated mice, Ter-119 and (green) expression protein p-Smad2/3 F4/80 Ter-119 (green), expression protein p-Smad2/3 mice. thalassemic and wild-type from pulp red spleen of sections showing ( d. 30 for RAP-011 or PBS with treated mice thalassemic and wild-type from samples spleen in ActRIIB and ActRIIA and 6 Figure in maturation erythroid terminal regulates atively Supplementary Fig. 6a ( maturation erythroblast terminal promoted GDF8, or B A 

b d c a s e l c i t r Spleen Spleen p-Smad2/3 p-Smad1/5 ActRII +

( p-Smad2/3 DAPI p-Smad2/3 CD71 Ter-119 p-Smad2/3 F4/80 Ter-119

Wild type Wild type b Spleen + GDF11 inactivation promotes terminal erythropoiesis. ( erythropoiesis. terminal promotes inactivation GDF11 CD71 ) and CD71 ) and Wild type Hb − ) erythroblasts. ( ) erythroblasts. b th1/th + CD71 1 200 200 200 + ( c µ µ µ m m m , ) cells (red) are shown. ( shown. are (red) ) cells h b + ) ROS production after treatment of erythroblast cultures from bone marrow of thalassemic mice with rGDF11, rGDF15 or rGDF15 rGDF11, with mice thalassemic of marrow bone from cultures erythroblast of treatment after production ) ROS ) or mature (Ter-119 mature ) or ), supporting the hypothesis that GDF11 neg ± 50 50 50 s.e.m. * s.e.m. µ µ µ m m m Hbb j Hbb ) Erythroblast differentiation in samples treated with rGDF11 (100 ng/ml) for 48 h, as evaluated by CD71 and Ter-119 and CD71 by evaluated as h, 48 for ng/ml) (100 rGDF11 with treated samples in differentiation ) Erythroblast Hbb Bone marrow th1/th1 th1/th P th1/th < 0.05, ** < 0.05, PB 1 1 S 200 200 200 µ µ µ Hbb m m m + CD71 th1/th d P Hbb ) Confocal micrographs showing sections of spleen and bone marrow from thalassemic mice. mice. thalassemic from marrow bone and spleen of sections showing micrographs ) Confocal + < 0.01; < 0.01; 1 erythroblasts (blue) are shown. ( shown. are (blue) erythroblasts 50 50 50 th1/th − ) erythroblasts. ( ) erythroblasts. µ µ β µ m m m 1 -thalassemia.

RAP-011 µ M), RAP-011 (10 (10 RAP-011 M), n = 3–5 mice per group for one out of three independent experiments. independent three of out one for group per mice = 3–5 200 200 200 Fig. 6 Fig. a µ µ µ ) Immunohistochemical staining of phosphorylated Smad2/3 (p-Smad2/3), p-Smad1/5 p-Smad1/5 (p-Smad2/3), Smad2/3 phosphorylated of staining ) Immunohistochemical m m m f ) Erythroblast differentiation after treatment with PDTC. Cells were classified as classified were Cells PDTC. with treatment after differentiation ) Erythroblast i + i and CD71

CD7110 10 10 10 10 10 10 10 g e ) ROS levels in bone marrow–derived thalassemic erythroblasts after treatment treatment after erythroblasts thalassemic marrow–derived bone in levels ) ROS 0 0 2 3 4 5 2 3 4 5 GDF11 (MFI) - Hb Ter-119 Hbb 2,000 4,000 6,000 8,000 h 0 0

Cells (%) 25.1 ProE 27.9% + b ProE 10 10 100 120 ROS (MFI) ) and mature (Ter-119 mature ) and th1/th µ th1/th 20 40 60 80 10,000 15,000 0 i ees f cii rcpo epeso ( expression receptor activin of levels similar showed erythroblasts spleen-derived and ­marrow–derived 2 2

Hbb % 0 ) Erythroblast cultures, derived from bone marrow of thalassemic mice, mice, thalassemic of marrow bone from derived cultures, ) Erythroblast 5,000 g/ml) or PBS as a vehicle for 48 h. ( h. 48 for a vehicle as PBS or g/ml) Hbb Vehicle th1/th1 1 1 10 10 th1/th1 that observation the with agreement In controlisotype activinAAb 0 3 3 GDF11 Ab PDTC * 10 10 Immature Immature e

Hbb 5 0 56.7 rGDF11 4 4 – Mature Mature * 13.5%

(ng/ml) RAP-011 12.6% th1/th1 61.9 g 10 10 ) Erythroblast cultures, derived from cells from thalassemic thalassemic from cells from derived cultures, ) Erythroblast % * % 5 5 Hbb ActA Ab 10 10 10 10 10 10 10 10 50 th1/th1 0 0 2 3 4 5 2 3 4 5 Hb Hb advance online publication online advance

5 0 0 0 18.0% ProE ActB Ab rGDF15 24.5 b (ng/ml) ProE 10 10 b dim f th1/th th1/th 2 2 %

+ % of cells CD71 100 120 CD71 1 10 10 1 20 40 60 80 GDF11Ab activinBAb 50 0 3 3 Vehicle 10 10 Immature Immature Mature Immature ProE 5 0 + 58.9 − 4 4 Mature 64.0 Mature 14.9% 15.5 ), immature (Ter-119 immature ), rGDF8 ) erythroblast populations are shown. All data data All shown. are populations ) erythroblast (ng/ml)

PDTC * 10 10 % % % 5 5 50

e ) Flow cytometry analysis showing showing analysis cytometry ) Flow Mature Immature + upeetr Fg 3g Fig. Supplementary b j erythroblasts (blue) and and (blue) erythroblasts

, CD71 c 10 10 10 10 10 10 10 10 ) Confocal micrographs micrographs ) Confocal 0 0 2 3 4 5 2 3 4 5 n vitro in g + Ter-119 CD71 ROS (MFI) rGDF11 (100ng/ml

% of cells 81.4 ±2.9% Immature 93.9 Immature 0 Control 0 10,000 15,000 18.6 Mature 6.1 Mature 100 120 5,000 10 20 40 60 80 10 nature medicine nature 0 ± 2

Control 2 ± 1.7% ± 0 + 1.7% 2.9%

Vehicle bone –cultured ) and mature mature ) and Immature Mature 10 10 rGDF11 3 3 ** PDTC * 10 10

RAP-011 *

4 4 ) 10 10 5 5 , h ), ), © 2014 Nature America, Inc. All rights reserved. Our results identify TGF- identify results Our erythroblasts. immature of apoptosis and differentiation erythroid pathway Fas-FasL the of activation to lead ROS Notably, erythroblasts. ture GDF11 using blockade an ActRIIA ligand trap results in fewer imma apoptosis, and pathway Fas-FasL the by inducting that, demonstrate FAS conditions stress and steady-state both in esis, in poiesis gesting that the molecular mechanisms controlling ineffective erythro conditions of stress erythropoiesis induced by hypoxia or anemia, sug under or conditions steady-state under mice wild-type of spleen the GDF11 is overexpressed in the spleen of mouse thalassemic but not in erythropoiesis responses tion stimula marrow BMP4 areto bone unresponsive adult from derived ineffective to in contributing erythropoiesis thereby stress, oxidative and pathway Fas-FasL the modulates signaling, ALK4/5–Smad2/3 through acting in splenomegaly, anemia of of amelioration and MCH increased reversal counts, RBC in increased resulting erythropoiesis, ineffective maturation. Together, erythroblast amelioratedterminal effects these cytotoxic of and amount levels pathway–dependent ROS the reduced and Fas-FasL erythroblasts immature in induced apoptosis concentration, GDF11 decreased administration trap ligand ActRIIA pathogenesis. disease to contributes that arrest maturation erythroid stress, terminal in resulting oxidative increased and erythroblasts immature of number increased an to leads pathway this of activation and tissue-specific (ActRIIA and ActRIIB) TGF- immature splenic erythroblasts along with type I (ALK4/5) and type II defined. poorly are erythropoiesis ineffective in role erythropoiesis stress and oxidative specific pathways their regulating molecular ineffective consequent and stress oxidative increased with are associated precipitates membrane ( cells wild-type not but thalassemic in maturation blast bone of treatment rGDF11 marrow– blocked and erythro terminal erythroblasts spleen-derived observation, this with agreement In tha in phosphorylation ( erythroblasts Smad2/3 but not wild-type lassemic in increase dose-dependent a two these ( types in cell similarly signaling pathway Smad2 induced rGDF11 nature medicine nature pathway. Fas-FasL the of elh erythroblasts in healthy arrest maturation erythroid terminal promotes stress Oxidative DISCUSSION pathway. Fas-FasL the of inhibition to due roblasts eryth immature of expansion concomitant a and terminal arrest in maturation results pathway this of activation Chronic expression. and stress GDF11 oxidative between loop amplification an autocrine in erythropoiesis ineffective that tion ( rGDF11 treatment had no or on effect survival prolifera erythroblast addition, In cells. thalassemic in erythropoiesis ineffective promote 7a Fig. Supplementary The Fas-FasL pathway is critical for the modulation of erythropoi for the modulation pathway The Fas-FasL is critical In during conditions, steady-state mice, hematopoietic progenitors in overexpressed is GDF11 that demonstrate we study, this In β -thalassemic -thalassemic mice. Taken together, our findings show that GDF11, 3 can result in ineffective erythropoiesis in humans in erythropoiesis ineffective in result can 5 Supplementary Fig. 7c Supplementary . However, BMP4 has a major role in the control of hypoxic stress 4 0 β , suggesting that oxidative stress is linked to both terminal terminal to both is linked stress oxidative that , suggesting Supplementary Fig. 6c Fig. Supplementary -thalassemia and in stress erythropoiesis areand in different. stress erythropoiesis -thalassemia β

-thalassemia. advance online publication online advance 30 10 , 3 , b 1 , 3 β . In thalassemic erythroblasts, erythroblasts, thalassemic In . ), suggesting that GDF11 is sufficient to to sufficient is GDF11 that suggesting ), 6 family members as negative modulators modulators negative as members family . The data presented here demonstrate that α , β d -globin aggregates, thus promoting promoting thus aggregates, -globin family receptors in ). Based on these findings, we ). on propose Based findings, these β ). Furthermore, rGDF11 induced induced rGDF11 Furthermore, ). -thalassemia is a consequence of of consequence a is -thalassemia Supplementary Fig. 6c Fig. Supplementary 22 37 , 32– , 3 β 8 3 , and mutation of of mutation and , -thalassemia. The -thalassemia. 4 . However, the the However, .

3 9 Fig. 6 Fig. . Our data data Our . α -globin -globin j and and , d ). ). ------­

our RAP-011 data, we propose that sotatercept will have the com the have will sotatercept that propose we data, RAP-011 our of light In RBCs. circulating mature fewer and erythroblasts late of resulting in expanded immature erythroblasts, suboptimal production maturation, erythroblast ment of of progression terminal the normal impair the to factors contributing key are overexpression receptor the accumulation of early erythroblasts. Therefore, GDF11 and activin to contributes that pathway Fas-FasL the modulate to negatively acts signaling GDF11-ActRIIA addition, In erythropoiesis. ineffective of stress, suggesting that an autocrine amplification loop is characteristic by oxidative induced is itself expression GDF11 Furthermore, arrest. tive stress and humans) provides strong evidence that this signaling promotes oxida in modulator of may be different from those involved in in subjects with with subjects in administration of sotatercept and tolerability safety the to determine ( are studies underway currently Clinical production. erythropoietin on sotatercept of effects the investigate to needed is ( anemia ameliorate and sion expres hepcidin derepress splenomegaly, and erythropoiesis fective inef to reverse be would expected mature to immature erythroblasts ing apoptosis of immature Increasing the erythroblasts. proportion of induc and maturation erythroid terminal of promoting effect bined suggesting that the ActRIIA ligands involved in healthy erythropoiesis under steady-state conditions in the absence of GDF11 overexpression, uration effect of stromal cells on human erythroblasts, promoting ocrit erythroid mat hematand levels hemoglobincount, RBC rapidlyincreases istration experiments, experiments, analyzed the data and wrote the manuscript. the manuscript. O.H. the and overall I.C.M. project, supervised performed the and editing of the manuscript. Y.Z.G. contributed to the writing and editing of contributed to the development of the project and contributed to the writing participated in project planning, provided RAP-011 and ACE-011, actively analysis. J.-A.R. and J.-B.A. provided human samples. T.O.D., andR.C. V.S. mice, thalassemic intellectual input forand technical expertise the hemoglobin G.C. performed experiments and analyzed data. E. Payen, P.L. and Y.B. provided data and helped write the manuscript. J.V., C.C., F.C., D.G., O.N., E. Paubelle and M.D., T.T.M. and A.F. anddesigned performed all experiments, analyzed the antibody was provided by S. Izui (University Medical Center, University of Geneva). N. Gadessaud for assistance in histological sample processing. 34-3C IgG2a for assistance with confocal microscopy and O. Thibaudeau, F. Watier and assistance, C. Brouzes for advice, M.G. cytological Traore and N. Goudin ANR-11-IDEX-0005-02, respectively). We thank S. Nelson and J. forBex technical d’avenir’ of the French National Research Agency (ANR-10-IAHU-01 and the Laboratory of Excellence GR-Ex are funded by the program ‘Investissements Fugain and Association pour la Recherche sur le Cancer. The Imagine Institute and Fondation pour la Recherche Médicale, Fondation de France, Association Laurette Translationnelle, Institut National du Cancer, Cancéropôle Île-de-France, Assistance Publique Hôpitaux de Paris–CNRS Contrats Hospitaliers de Recherche JCJC-1108, ANR-12-BSV1-0039, ANR-11-LABX-0051 and ANR-10-BLAN-1109), This work was supported by Agence Nationale de la Recherche (grants ANR-10- online version of the pape Note: Any Supplementary Information and Source Data files are available in the the t of version in available are references associated any and Methods M AUTHOR CONTRIBUTIONS A c et In summary, our data support a role for activin and BMP signaling as a Clinical Clinical studies in healthy subjects revealed that sotatercept admin β k -thalassemic, but not in normal, erythroblasts (in both mice and mice both (in erythroblasts but normal, not in -thalassemic, no 1 h 7 . Consistent with this finding, sotaterceptinhibitory the finding, Consistentblocks this . with ods 2 w 8 . Therefore, ActRIIA ligand traps can promote erythropoiesis ledgments he pape he β α -thalassemia pathophysiology. GDF11 overexpression -thalassemia -globin precipitation, resulting in terminal maturation β -thalassemia intermedia. -thalassemia r . r . Supplementary Fig. 8 Fig. Supplementary β -thalassemia. s e l c i t r a ). Further study study Further ). NCT0157163

online online 5 2  8 ------)

© 2014 Nature America, Inc. All rights reserved. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/ at online available is information permissions and Reprints version of t The authors declare competing interests:financial details are available in the A 1 COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING 0

s e l c i t r

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© 2014 Nature America, Inc. All rights reserved. cultured for 7 d with interleukin-6 (IL-6) (100 ng/ml), IL-3 (10 ng/ml) and ng/ml) (10 IL-3 ng/ml), (100 (IL-6) interleukin-6 with d 7 for cultured were Kit) Isolation Cell Progenitor CD34 (Miltenyi blood cord from isolated CD34 II”). “Ile-de-France personnes des protection de (Comité the from consent HôpitalatNecker board informed review institutionalapproval after the bydonorsand with obtained were samples Subject Necker-Enfants Malades. Hôpital Biotherapy, of Department the from obtained was France. Peripheral blood from adult subjectsParis, with Malades, Necker-Enfants Hôpital of Unit Obstetrics the from mother, the from consent informed after obtained, were deliveries full-term normal In vitro tion of protein concentration ProteinBCA (Thermo Scientific Assay Kit). fraction of sample loaded on the TAU gel was adjusted to 40 turing conditions TAU gel electrophoresis, which resolves (TAU-PAGE). of Analyses medium partial changes. 10 × 2 to adjusted concentrationwas cell and (Peprotech). Live cells were determined by Trypan blue (Gibco/BRL) exclusion, B activin or (MAB659, R&D Systems) Systems) blocking antibodies and R&D rGDF11, rGDF8 or rGDF15 (AF338, A activin Abcam), (ab71347, proform GDF8 Abcam),(ab71808, GDF8 Santaorand GDF11 GDF11 Cruz) (sc28910, 10 with not or supplemented d, 2–3 for 2 U/ml erythropoietin and 1 mg/ml human iron-saturated transferrin (Sigma)) with supplemented (StemPro-34 medium differentiation to transferred were ­streptomycin (Pen/Strep; Invitrogen). After 5 d of culture, the nonadherent cells (PeproTech), (SCF) tor 1 fac cell stem ng/ml 100 (Roche), recombinanthuman erythropoietin U/ml 1 with supplemented TechnologiesGibco-BRL) (Life supplement nutrient plus ‘erythroidmedium’ expansion StemPro34of consisting serum-free in pended tured as described elsewhere Invitro anti-mouse HRP-coupled antibodies, respectively. activin B (BAM3381, R&D Systems) at 2 Santa Cruz Technologies) or antibodies to A activin (AF338, R&D Systems) or (sc28910, GDF11 and GDF8 to antibody by Proteinsinterestdetected wereof major, 1/50,000 dilution, as well as from (5 controltively and thalassemic mice, 1/2 dilution. activin B was performed by coating plates with RAP-011 or sotatercept, respec manufacturer’s procedures. Sandwich forELISA GDF11 and for A and activin using the Quantikine Mouse Epo Immunoassay (R&D Systems) according to the Serum ELISA. stained with May-Grünwald-Giemsa. Retic-Count Kit). Blood smears fromcollected treated mice were air dried and Biosciences (BD Reagent Retic-COUNT with determined Analyzer were Reticulocytes Blood MS9-5 a on manufacturer’s the to according Laboratories) measured instructions. (MeletSchloesing were counts blood complete and parameters. Blood are representative of one out of three independent experiments. 60, day to 5 (day points time different at analyzed is a version of sotatercept in which the Fc region is of mouse origin. Mice were RAP-011 at a dose of 10 mg per kg body weight in PBS, twice a week. RAP-011 Treatment with RAP-011. mice. female with a of model deletion of occurring the gene encoding CommitteeCare of INSERM. Animal The the by approved were protocols All School. Medical Bichat of Mice. ONLINE MET doi: 10.1038/nm.3468 C57BL/6 mice were bred and housed in the pathogen-free facilities facilities pathogen-free the in housed and bred were mice C57BL/6 generation of CD36 erythroblast cultures. erythroblast µ g/ml). Sera from control subjects and patients with with patients and subjects control from Sera g/ml). β -thalassemia intermedia. All intermedia. -thalassemia a To visualize soluble as well as membrane-bound globins, we used Erythropoietin concentration was determined from mouse sera - and and - H 5 , 3 ODS 4 . 0.5% Triton X-100 was added to the washing solution. The Blood samples were collected in EDTA-coated tubes, tubes, EDTA-coated in collected were samples Blood b -globin chains in plasma and on RBC membranes membranes RBC on and plasma in chains -globin µ Thalassemic mice were treated subcutaneously with M dexamethasone (D2915; Sigma) and penicillin- and Sigma) (D2915; dexamethasone M 4 + Hbb 1 erythroid cells. . . Briefly, bone marrow or spleen cells were resus Cells from bone marrow and spleen were cul were spleen and marrow bone from Cells th1/th1 mouse model originated from a natural α µ β in in vivo - - and g/ml g/ml and developed by anti-rabbit or µ -globin g/ml RAP-011, antibodies against against antibodies RAP-011, g/ml 6 Umbilical cord blood units from total cells per ml daily through through daily ml per cells total β experiments were performed were performed experiments β -globin -globin subunits under dena 1 -thalassemia major ( 8 n . . = 5 per time point). Data Data point). time per 5 = Hbb th1/th1 µ g after determina mice constitute β + -thalassemia -thalassemia progenitors β 0-TM) ------

ware (Tree Star). For intracellular staining, bone marrow and spleen cells were soft FlowJo using Biosciences) (FACScanto; BD cytometry flow by analyzed further were Cells Biosciences). (BD labeling 7-AAD byby excluded were cells detected was which dilution), streptavidin-conjugated 1/200 V450 (#560797, BD Biosciences; Biosciences; 1/200 dilution). Dead BD (#555292, FasL 1/100 dilution), Fas (#557653, BD Biosciences; 1/200 dilution) and biotinylated BD Biosciences; 1/100 dilution), mouse TfR1/CD71 (#553266, BD Biosciences; 10 × (1 Cells suspensions were incubated with anti-Fc Immunofluorescence analysis by flow cytometry. ing for analysis. further thereafter. Five hundred microliters of blood were taken by retro-orbital bleed for oxygen) 10 h for 3 (20% d consecutively in a hypoxicreoxygenation animal chamber and by immediatelykilled followed h 14 for hypoxia Messer) oxygen; to (8% exposed were mice hypoxia, chronic intermittent For Geneva). of University Center, Medical University Izui, S. by provided IgG2a, (34-3C 100 of injection intraperitoneal single a by Hypoxia and chronic anemia models. the ofblood (2 U/ml). Cytospins were performed on CD36 erated in the presence of IL-3 (10 SCF ng/ml), (100 ng/ml) and erythropoietin 9500 (STEMCELL Technologies). CD36 BIT 15% with supplemented culture) cell (Gibco IMDM in ng/ml) (100 SCF hemoglobin hemoglobin was measured. For analysis, blood hemolysates were generated by by centrifugation at 14,000 by membranes absorbance was determined at Cell nm. were540 pelleted then 200 with assay. Hemoglobin software at magnification ×400. images wereConfocal acquired on a LSM Zeiss 510 and analyzed using Imaris Technologies;(Life antibodies secondary dilution)1/200 and with DAPI.then (Roche TUNEL with wereSlides incubated AlexaFluor-conjugatedwith anti-rabbit and anti-mouse first antibodies. labeled anti-CD71 anti–Ter-119 and primary with were then and Diagnostic) sections paraffin cells, TUNEL- of positive detection For Aldrich). (Sigma DAPI with counterstained and antibodies secondary AlexaFluor-conjugated with incubated were slides ysis, (Leica DM2000, Leica Microsystems) at magnification ×200. For confocal anal medium (Electron Microscopy andSciences) read with an upright microscope tion (Dako, Carpinteria, USA). Slides were mounted with the Eukitt mounting was visualized by addition of 3,3 reaction immunoperoxidase the USA), Burlingame, Vector Laboratories, kit; ABC (Vectastain, min 30 for streptavidin-HRP and h 1 for dilution) (1/250 antibodies secondary species-specific biotinylated with incubation Following 1/200 dilution) or F4/80 (sc-377009, Santa Cruz 1/50 Biotechnology; dilution). Technologies; Life (136800, CD71 dilution), Ter-119 1/100 Biosciences; BD (#553673, dilution), 1/100 Signaling; Cell (#3101, phospho-Smad2 dilution), 1/100 Signaling; Cell (#9511, phospho-Smad1/5 dilution), 1/100 Cruz; Santa activin B (MAB659, R&D Systems; 1/100 dilution), dilution), 1/100 Systems; activin R&D receptor (AF338, A II activin (sc-25451, dilution), 1/20 Cruz; Santa (sc28910, GDF11 mature dilution), 1/100 Abcam; proform(ab71347, GDF11 by each manufacturer and incubated overnight with primary antibodies against and confocal analysis, sections were processed for antigen retrieval as indicated were stained with H&E for morphological analysis. For immunohistochemistry Immunohistology. conjugated with AlexaFluor 488 (Life Technologies; 1/200 dilution). 1/100 dilution) was detected by respective species-specific secondary antibodies (AF338, R&D Systems; A 1/100 dilution) and activin B (MAB659, R&D Systems; activin dilution), 1/50 Abcam; (ab71347, proform GDF11 dilution), 1/50 Binding of primary antibodies against GDF8 and GDF11 (sc28910, Santa Cruz; with fixed paraformaldehyde and markers, resuspended in permeabilization buffer (Ebiosciences). surface cell for antibodies with stained then were Cells 2 containing medium culture in h 4 for incubated µ l Drabkin’s reagent (Sigma), and total hemoglobin concentration concentration hemoglobin total and (Sigma), Drabkin’sreagent l β 6 0-TM 0-TM subjects at day 8 of culture. ) were then stained with antibodies against Ter-119 (#553673, Ter-119(#553673, against antibodies with stained then were ) Paraffin-embedded bone marrow or spleen sections (5 rtrbat ( × 10 × (5 Erythroblasts g for 30 min, supernatants were collected and soluble ′ -diaminobenzidine (DAB) in chromagen solu Immune-mediated anemia was induced + γ erythroid progenitors were then gen R R mAb 2.4G2 to block IgG receptors. 6 µ cells/condition) were incubated incubated were cells/condition) g of purified antibodies to RBCs RBCs to antibodies purified of g + cells from cord blood and from Bone marrow and spleen cell µ g/ml brefeldin A (Sigma). (Sigma). A brefeldin g/ml nature medicine nature µ m) - - - - -

© 2014 Nature America, Inc. All rights reserved. ACAGTCA-3 GTTCGACCTCATCTGC-3 Bmp10 TGATCCATCGGTTG-3 5 ( ALK4 TGGCAGAGTGAAC-3 5 AGTTCC-3 5 5 (antisense) ( A Activin Germany). the following sequences were obtained from Eurofins MWG Operon (Ebersberg, PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad). Primers with iScript reverse transcription Supermix (Bio-Rad, Hercules, CA, USA). Real-time phoresis. Reverse transcription was carried out from 500 ng total RNA using the RNA quality was assessed by 260:280 nm absorbance ratio genitorsand (RNEasyPlusagarose Mini gelkit, electroQiagen) and quantified with a Nanodrop system. Quantitative real-time RT-PCR. (Ab8227-50, Abcam, 1/5,000 dilution) was used as a loading control. (Millipore). Westernsubstrate Forte Luminata HRP with minescence HRP- using conjugated antibody, secondary detection dilution,1/50,000 andby visualized chemolu by followed dilution, 1/1,000 Cruz), Santa (sc28910, antibodies using to GDF11 immunoblotting proform by (ab71347, Abcam) detected and mature were GDF11proteins and GDF8 Specific Healthcare). (GE tricine-PAGE gel (Life Technologies) and transferred onto a PVDF membrane SDS and 2% 5% glycerol, 10% 6.8, pH Tris-HCl mM (62 buffer sample Laemmli in (Pierce). The concentrations were equalized, and theby the bicinchoninicextracts acid method according to the weremanufacturer’s boiled instructions for 5 min at 4 °C and tocentrifuged obtain the soluble Proteinsfraction. were quantified phosphatase and protease inhibitor cocktail (P0044, Sigma-Aldrich) for 10 min Immunoblotting. flow cytometry. by analyzed further were Cells transfection. after h 48 PCR real-time titative HiPerFect using siRNA specific Transfection with Reagent (Qiagen). Knockdown transfected efficiency was monitored were by quan erythroblasts ated (SI01010863). Qiagen from purchased were B activin or oligonucleotides. siRNA nm, 529 and nm respectively, and ROS 495 levels were measured by flow cytometry. at were spectra emission and excitation fluorescence that measures hydroxyl, peroxyl and other ROS within the activity cell. DCFH 2 reagent 10 permeant with cell incubator CO2 5% humidified a in °C 37 at out carried oxygenReactive species. 30 min at 4 °C and measuring absorbance at 540 nm. centrifugationat after wereSoluble levels 18,000 hemoglobin determined 10 were determined by measuring absorbance at 540 nm (TECAN infinite 200) on Total min. levels 3 hemoglobin every vortex by homogenizing and min 10 for 10 mixing nature medicine nature ′ ′ ′ -ACAGGCCCTTTGTAGTGGTG-3 -AACCAAGACCGTTCTTCACG-3 -GTTGGTTCTGTCTCTTTCCCAAT-3 µ L of hemolysate mixed with 190 190 ofhemolysatewith L mixed : (sense) 5 (sense) : β Acvr1b -mercaptoethanol). The (30 extract µ ′ ; L of blood (EDTA) with 90 (EDTA)90 with blood of L ′ Acvr2a ′ -CCGTCACTCCCATCTTTCTT-3 , (antisense) 5 : sne 5 (sense) ): ′ Cells were lysed in 1 ml of RIPA buffer supplemented with a -GACCCTTTGCTGGTTGTGTT-3 : (sense) 5 Inhba ′ ′ (nies) 5 (antisense) , ′ ,7 ROS levels were evaluated by incubation for 15 min, Four individual siRNAs that target mouse GDF11 GDF11 mouse target that siRNAs individual Four , (antisense) , 5 (antisense) ′ ′ ′ dclrfursen ictt, furgnc dye fluorogenic a diacetate, -dichlorofluorescein -CGCTCCAGGATCTCGTCTAC-3 -TGGGGGCATCGGCAGAAGG-3 ′ ): (sense) 5 (sense) ): ; ; ′ -GCGTTCGCCGTCTTTCTTATC-3 Gapdh Total RNA was extracted from erythroid pro µ : (sense) 5 (sense) : ′ L ofDrabkin’s L reagentAldrich). (Sigma ′ , , (antisense) 5 ; ALK5 ( ALK5 ; ′ ′ ′ µ -TTCCTGTTGGCTGAGTTGTG-3 -TGGTGCCAGTCTAGTGCTTC-3 -GGAGCCCTTGTCGTGGAAG-3 L of MilliQ water, incubating on ice ice water,on MilliQ incubating of L ′ ; ; µ g g per lane) was loaded on a 16% Acvr2b ′ -ACGGCAAATTCAACGGC Tgfbr1 ′ ; activin B ( B activin ; ′ - - GCTGCCCTCACAGT (es) 5 (sense) : ′ ): (sense) 5 (sense) ): , (antisense) 5 (antisense) , In vitro In Inhbb ′ ; ′ , (antisense) (antisense) , Gdf8 ′ ′ ′ , (antisense) -CAGTCG differenti -ACCTTC µ ): (sense) (sense) ): M of the the of M : (sense) ′ β -TCAT -actin -actin g for ′ ′ ′ - - - - - ; ; ,

Hbb-y CTTCTGCCCTG-3 TGGCTTACATCAAAGTG-3 5 (sense) GGGCTGGAG-3 5 ATAATCC-3 5 42. 41. portion) with Lipofectamine LTX Plus (Invitrogen) following the manufacturer’spGL3-BRE–luc or pGL3-CAGA-luc vectors and pcDNA3.1-GFP (9:1 mass pro Gene reporter assays. nlm.nih.gov/geo/geo2r GSE3 available at the Gene Expression Omnibus database under the accession number and analysis. wild-type microarray CD71 retrospective Mouse RPS3 values. using the CFX96 PCR System Data were(Bio-Rad). to normalized GAPDH or CATAAAGCTCTACGCTG-3 TC-3 TCTCCTGCTTCTCCTCCT-3 TTCAAGGAACAGTCCAGTATTC-3 0.05 0.05 (*), less than 0.01 (**) or less than 0.001 (***). (Bonferroni test). Differences were considered bysignificant at a followed test variance of analysis two-way using made tests were used to compare two groups, whereas multigroup comparisons were n (version 5.0; GraphPad software). The data are expressed as the mean Statistical analyses. instructions. manufacturer’sOlympusAU400 an the with to automatedaccording analyzer quantified were bilirubin) (conjugated) direct and bilirubin total iron, serum centrifuged (15 min, 4 °C, 3,000 Iron, bilirubin and transferrin quantification. units (RFU). were expressed as relative light units (RLU) normalized by relative Results reader.fluorescence plate M200 Infinite an on acquired were luminescence firefly plate reader (Tecan). After addition of Bright Glo reagent (1:1 vol/vol, Promega), Glo lysis buffer (Promega), and fluorescence was acquired on an Infinite M200 TGF- or R&D) ng/ml, (20 BMP2 systems), R&D ng/ml, 100 to 3 (from GDF11 centrationsof 10 10 and 1 (0.1, (SelleckBio) instructions. After 24 h, cells were incubated with the ALK4/5 inhibitor SB505124 ′ ′ determinations unless noted otherwise. Student’s -AGCATGTTGATTGGGGACAT-3 -CTGTAACCTTCCCAGGACCA-3 µ

hnrs E, hm CS, ’oz, . Wis MJ Itgae poen quality- protein Integrated M.J. Weiss, & J. D’Souza, C.S., Thom, E., Khandros, Dolznig, H. oto ptwy rglt free regulate pathways control (2001). arrays. cDNA by characterization molecular progenitors: 5265–5275 (2012). 5265–5275 M) (Sigma-Aldrich). After 30 min, cells were treated with different con different with treated were cells min, 30 After (Sigma-Aldrich). M) ′ 412 ; ; + : (sense) 5 (sense) : Ter-119 Rps3 5 ′ . Analysis was performed using GEO2R software ( software GEO2R using performed was Analysis . -CCTGGAAAGGATGTTTGCTAGCT-3 (es) 5 (sense) : ′ ; et al. Gdf11 + Hbb β FSC 1 (5 ng/ml, Sigma-Aldrich). After 16 h, cells were lysed with with lysed were cells h, 16 After Sigma-Aldrich). ng/ml, (5 1 ′ -CTCTAGCTGTCCAGCAATCCTG-3 ′ , (antisense) 5 Establishment of normal, terminally differentiating mouse erythroid th3/th3 Statistical analyses were with performed GraphPad Prism ′ high : (sense) 5 , , (antisense) 5 C2C12 cells (ATCC) were transiently co-transfected with / ′ -GACCGCAGTTGTCCAGAA-3 ). ace eyholss rm 1. ftl ies of livers fetal E14.5 from erythroblasts matched embryos have been described previously described been have embryos µ M) or the ALK5 inhibitor SB431542 (0.1, 1 and 1 (0.1, SB431542 inhibitor ALK5 the or M) ′ ′ . Gene quantification was performed in duplicate -CTAAGCGCTACAAGGCCAAC-3 ′ ; ; ′ g ′ , (antisense) 5 (antisense) , -TAAGAACCACCGGGGTGTAG-3 Hbb-bh1 ). Serum biochemical parameters (transferrin, α ′ goi i murine in -globin -TGTTCTTAACCCCCAAGCCCAAG-3 ′ ′ ; ; (nies) 5 (antisense) , ′ Hpii ( Hepcidin ; Gdf15 : (sense) 5 (sense) : ′ Blood was drawn into tubes and -TGGGGAAGTTGGTGTCTC (es) 5 (sense) : Microarray data from from data Microarray t -test or the Mann-Whitney ′ -AGTTTGGAAACCTCT ′ β (nies) 5 (antisense) , Hamp -thalassemia. FASEBJ. ′ -TCTTTTGGGTGCG ′ , (antisense) 5 (antisense) , doi: ′ ′ (nies) 5 (antisense) , -CTTGAAGACTT : sne 5 (sense) ): post hoc post 10.1038/nm.3468 http://www.ncbi P

value less than 15 , 1442–1444 , ′ , (antisense) Blood 4 ± ′ 2 ; analysis analysis ′ s.e.m. of and are and Hba-a1 -GCCG ′ -GCT

′ ′ 119 -TG -TG ′ - - , . ; :