Deliverable D16: Report of the course on Red Cells and

Deliverable D16: Report of the course on Red Cells and Iron

Disorders

Report version: D6

Executive summary

Project number: 2008 12 10 Project Acronym: ENERCA 3 Title: European Reference Network of Expert Centres in Rare Anaemias

Deliverable: D16

Delivery Date: October 2010

Short description:

This deliverable D16 has been developed under WP3 “Education and Training “. It aims at reporting the activities developed on course 1 “Innovative Therapies for Red Cell and Iron related Disorders”. The report includes an introduction of the WP with its tasks, the programme of the course, a summary report, abstracts of the talks, educational material and conclusions. Responsible partner: CHUM

Partners contributed: ESH Made available to: Internal

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PROJECT ENERCA 3 Workpackage 3: Education & training

Patricia Aguilar Martinez, WP3 leader

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Report on course 1

C O U R S E 1 - E D U C A T I O N & T R A I N I N G

Innovative therapies for red cell an iron related disorders

© copyright ENERCA 2010

Table of contents

Introduction .............................................................................................................................. 1

Work package n° 3 : Education and training.......................................................................................... 2

WP3 : Tasks........................................................................................................................................... 3

WP3 : Courses ........................................................................................................................................ 3

Program of the course ............................................................................................................... 4

Organization............................................................................................................................................ 4

Place and dates......................................................................................................................................... 5

Program of the course ................................................................................................................................ 5

Diffusion of the course............................................................................................................................... 7

Summary report on course 1...................................................................................................... 9

EHA-ESH Scientific Workshop “Innovative Therapies for red cell and iron related disorders”................. 9

Abstracts................................................................................................................................... 11

Selected abstracts .................................................................................................................................... 11

Educational material............................................................................................................... 24

Abstract book ........................................................................................................................................ 24

The handbook : "Disorders of erythropoiesis , erythrocytes and iron metabolism" , (2009 new edition)................................................................................................................................................... 25

Selected slide presentations from ENERCA speakers available online .................................................... 25

The curriculum on iron metabolism and related disorders ......................................................................... 25

Conclusion............................................................................................................................... 27

Acknowledgements ................................................................................................................. 28

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Introduction

NERCA Workpackage 3 (WP3) is responsible for the organization of educational activities, among which, 3 courses on rare anemia (RA). This report gives the details of the organization of the first ENERCA course that was held in April 2010 in Portugal.

WP3 partners consist in 4 associated partners (WP leader CHUM) and 5 collaborating partners. All the other WPs also contribute to the project educational activities.

For this specific course the main work package partner was WP6 (group of Clara Camaschella on rare iron disorders).

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E W P 3 P A R N E R S

WP Leader: CHUM Associated partners: ESH KINGS CHC Other WPs UNIMILANO UKNEQAS ERASME UNISR TIF UULM UMCU Collaborating partners: WHO IUH BRF ITHANET UMFVB

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Work package n° 3 : Education and training

-- GGeenneerraall WWPP oobbjjeeccttiivveess::

OBJ 2: To promote harmonization of procedures for diagnosis, treatment and follow-up of patients for RA

OBJ 4: To improve continuous medical education in order to insure the provision of the highest quality services for patients with RA.

OBJ 5: To increase patients and public awareness about RA.

-- SSppeecciiffiicc WWPP oobbjjeeccttiivvee((ss))::

To disseminate harmonized comprehensive up-to-date knowledge on RA to European health professionals, practitioners and patients by establishing close collaborative links with recognized European organizations with professional education and training activities

European School of Haematology (ESH): co-organisation of meetings on training in diagnostic procedures (haematology laboratory specialization) and clinical care of patients with rare anaemias. ENERCA will also be associated with ESH training courses also in the field of the rare disorders related to iron metabolism.

European Haematology Association (EHA): participation in the EHA congresses and annual meetings by organizing satellite symposia in collaboration with the "red cells metabolism and its disorders" scientific working group. This will facilitate the involvement of large number of young European haematologists in the diagnosis and treatment of rare anaemias

World Health Organization (WHO): Establishment of a close collaboration with the WHO programme of Human Genetics and other issues involving rare anaemias in Europe, in order to develop joint documents, reports and recommendations for the prevention, diagnosis and treatment of these diseases throughout Europe, especially for the less economically developed and new incoming EU members

National Societies of Haematology Biochemistry or Paediatrics will be also directly implicated in Education and Training activities developed by ENERCA in each EU country.

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WP3 : Tasks Task 1. Courses for Continuous Medical Education and Training (Organization ESH, CHUM, partners involved (elaboration of the program, course and diffusion): KINGS, HC, UNIMILANO, UKNEQAS, ERASME, UNISR, TIF, UULM, UMC, advice and diffusion: WHO, IUH, BRF, ITHANET, UMFVB)

The main objective of these courses is to promote harmonization of procedures for diagnosis, treatment and follow-up of patients with RA.

WP3 : Courses

1.1. Courses with emphasis on biology and diagnosis (organized with ESH):

Course 1 – Thalassaemia and other Haemoglobin disorders

Course 2 – Rare red cell disorders: Diagnosis of red blood cell membrane disorders and CDA, Red Blood Cell Enzymopathies & other rare red cells disorders (organized with ESH):

1.2. Courses with emphasis on clinical management:

Course 1 - Red cells and iron disorders (co-organised with the ESH)

Target groups: haematologists, paediatricians, Medical students in specialization for haematology

Main objective: promoting harmonization of procedures for diagnosis, treatment and clinical follow-up of patients with Red cells and iron disorders.

The present report concerns this course.

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Program of the course

Organization

he program of this course has been elaborated by the chairpersons, three of whose are ENERCA3 partners, in collaboration with the European Haematology Association (EHA).

The European School of Hematology (ESH) was responsible for the organization of the course.

ENERCA 3 Workpackages WP6 and WP3 are co-responsible of the organization of this course among the ENERCA3 project.

.

WP3 associated partners, Swee Lay Thein (London), Dimitris Loukopoulos (Athens), Leticia Ribeiro (Coimbra), as well as WP3 leader, Patricia Aguilar Martinez, and CHUM members attended the course either as speakers or as participants.

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C H A I R P E R S O N S C O U R S E 1

Clara Camaschella…… WP6 Carole Beaumont……. WP6 Yves Beuzard……….. WP3 Philippe Leboulch Mohandas Narla

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Place and dates

The course took place in Cascais (Lisbon, Portugal) in April 16th-18th 2010. It was organized by the ESH staff.

Program of the course

Friday, 16 April, 2010

08h30-10h30

Session I - Hematopoietic Stem Cell (HSC) Therapies - Chair: F. Locatelli (Pavia)

Allogeneic BMT in Sickle cell anemia F. Bernaudin (Créteil)

Hematopoietic stem cell therapy for thalassemias S. Hongeng (Bangkok)

New Trends in HSC therapies F. Locatelli (Pavia)

Panel Discussion

Selected oral presentations (15 min each)

10h30 - 11h00 Coffee break

11h00-13h00

Session II - From HSC to Erythocyte - Chair: F. Grosveld (Rotterdam)

Expansion of HSCs K. Humphries (Vancouver)

The molecular switching in erythroid differentiation S. Orkin (Boston)

Programming the hemoglobin genes for therapies F. Grosveld (Rotterdam)

Overcoming insertional adverse events of integrating gene vectors C. Baum (Hannover)

13h00 - 13h00 Lunch

15h00- 17h30

Session III - Gene Therapy - Chair: P. Leboulch (Fontenay/Boston)


Gene Therapy of hemoglobin disorders Ph. Leboulch (Fontenay/Boston)

Design of lentiviral vectors C. Frecha (Lyon)

Oncogenic safety C. Baum (Hannover)



18h00-19h00 Poster Session I: Biotherapies

Saturday, 17 April, 2010

08h30 - 10h30

Session IV - Iron regulatory pathway for therapies - Chair: C. Camaschella (Milano)

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Heme and Iron recycling C. Beaumont (Paris)

Role of Hemojuvelin in iron deficiency and overload C. Camaschella (Milano)

The essential role of BMP6 in hepcidin regulation M.P. Roth (Toulouse)



11h00 - 13h00

Session V - The hepcidin effector pathway for therapies - Chair: C. Beaumont (Paris)

Hepcidin agonists and antagonists E. Nemeth (Los Angeles)

Targeting the hepcidin binding site of ferroportin I. de Domenico (Salt Lake City)

Relocating misdistributed iron chelators I. Cabantchick (Jerusalem)


13h00 - 15h00 Lunch

15h00 - 17h30

Session VI - Innovative therapies for red cell disorders (I) - Chair: N. Mohandas (New-York)

Predictive factors of severity for therapies of hemoglobin disorders S. Fucharoen (Bangkok)

Erythrocyte acting therapies Y. Beuzard (Fontenay)

Gene activation therapies S.L. Thein (London)

EPO therapy in hemoglobin disorders F. Galactéros (Créteil)



18h00 - 19h00

Poster Session II: Iron and erythrocyte targeting therapies

Sunday, 18 April, 2010

09h00 - 10h00

Session VII - Innovative Therapies for red cell disorders (II) - Chair: F. Galacteros (Creteil)

Vascular acting therapies in Sickle cell disease P-L. Tharaux (Paris)

Heme oxygenase protects against red cell disorders M. Soares (Lisbon)


10h30 - 11h30 Keynote Lecture

Erythrocyte targeting for therapies in malaria N. Mohandas (New-York)

End of the meeting

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Diffusion of the course

Announcement of the course was done through different means.

General organization by the ESH : advertising, registration of participants, local organization, arrangement of speakers' travel and accommodation, arrangement of participants accommodation, teaching material…

Presentation of the program on the ESH and ENERCA websites.

FIGURE 1. Presentation of course 1 on the ENERCA website where a link was created with the ESH website.

Diffusion of the course to all ENERCA partners and by ENERCA partners to their respective national scientific societies (Italy, Portugal, UK, Germany, Belgium France etc).

Preparation of a poster on the ENERCA3 project for presentation during the course.

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FIGURE 2. Poster on the ENERCA3 project presented during the Course in Cascais (Portugal).

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Summary report on course 1

This report has been written by Pr Clara Camaschella chairperson of the course.

EHA-ESH Scientific Workshop “Innovative Therapies for red cell and iron related disorders”

he EHA-ESH Scientific Workshop “Innovative Therapies for red cell and

iron related disorders” organized in collaboration with ENERCA took

place in Cascais, Portugal, April 16-18, 2010. The aim of the workshop was

to discuss recent and original data that may result in new therapeutic strategies for red

cell and iron metabolism disorders.

Hot topics discussed were: HSC transplantation for beta-thalassemia with

both related and unrelated donors, the controversial indication to transplantation in

severe sickle cell anemia, the problem of gene therapy for beta-thalassemia, including

the possibility of ex vivo HSC expansion, genetic modification and modalities of

overcoming insertional adverse effect of integrating vectors. The molecular basis of

the hemoglobin switching by BCL11A was also discussed in the light of fetal

hemoglobin reactivation in hemoglobinopathies.

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In the field of iron special emphasis was given to the regulation of the

hepcidin pathway as a potential target of therapeutic manipulation to treat iron

overload by hepcidin agonists and to treat anemia of inflammation by hepcidin

antagonists. The use of low dose iron chelators was discussed to relocate

misdistributed iron in neurodegenerative disorders and anemia of inflammation The

relevance of Genome Wide Association Studies to identify genetic modifiers of beta-

thalassemia phenotype and new approaches to the molecular pathophysiology of

sickle cell syndromes were addressed. The potential protective effect of heme

oxigenase-1 in conferring the well known selective protection against malaria in

hemoglobinopathies and other congenital hemolytic disorders raised great interest.

The ENERCA network and aims were presented in the Poster Session.

Notwithstanding the Workshop took place during the volcano ash cloud it

was unanimously considered a success. Several new advances and ideas are now

available to the ENERCA community.

Important Note

FIGURE 3. The Iceland volcano ash cloud had strong impact on the meeting, reducing the number of speakers and participants and causing serious problems with return flights (C Camaschella).

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Abstracts The abstract book of the meeting summarizes the different talks given during the course. However, we have highlighted a selection of these talks and give here a more details presentation as well as the links that will be available in the ENERCA and ESH websites for these talks.

Selected abstracts

elected abstract from the meeting oral presentations have been processed in order to give an overview of the main topics of this course. The slide presentation is available on the ENERCA website.

List of selected abstracts

Philippe LEBOULCH (presented by Yves BEUZARD)

Conversion to transfusion independence with Hmga2 activation after lentiviral gene therapy for severe human beta- thalassemia Carole BEAUMONT Heme and iron recycling

Clara CAMASCHELLA The role of hemojuvelin in iron deficiency and overload

Marie-Paule ROTH The essential role of BMP6 in hepcidin regulation

Ivana De DOMENICO Targeting the hepcidin binding site of ferroportin

Yves BEUZARD Erythrocyte acting therapies of hemoglobin disorders

Swee Lay THEIN Impact of gamma globin gene expression on haemoglobin disorders

Chapter

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Philippe LEBOULCH, * Fontenay-aux-Roses, France - Boston, Massachusetts, USA

Philippe Leboulch, M.D.1,2,3 and the LentiGlobin clinical trial study group 1CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses 92265, France,

2Inserm U962 and University Paris XI, CEA-iMETI, Fontenay-aux-Roses 92265, France,

3Genetics Division, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA.

Conversion to transfusion independence with Hmga2 activation after

lentiviral gene therapy for severe human beta-thalassemia

The β-hemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassemia is especially challenging given the requirement for massive hemoglobin production in a lineage specific manner and the lack of selective advantage for corrected hematopoietic progenitors. An 18 year old male with severe βE/β0-thalassemia and no HLA-matched sibling donor was transplanted after Busulfex-mediated myeloablation with autologous bone narrow CD34+ cells transduced ex vivo with a lentiviral vector expressing a marked βA-T87Q-globin gene. Before transplantation, the patient was dependent on monthly transfusions since age 3 (2 to 3 RBC packs each time; 157 ml RBCs/kg the year before transplant) with growth retardation and spontaneous hemoglobin (Hb) levels between 4 and 6 g/dL, splenectomized since age 6, and under iron chelation therapy since age 8. Hydroxyurea therapy was ineffective. Thirty four months after gene therapy, Hb levels are between 9 and 10 g/dL, of which one third contains vector-encoded βA-T87Q-globin, the remainder being HbE and HbF. No transfusion has been provided for the last 22 months, and there is near physiological levels of βA-T87Q-globin expression on a per gene basis. Most of the therapeutic benefit results from a partially dominant, yet homeostatic, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of Hmga2 in erythroid cells and further amplification of a truncated Hmga2 mRNA insensitive to degradation by let-7 microRNAs. While hematopoietic homeostasis is currently maintained, this observation together with recent results of deep sequencing analysis of other gene therapy clinical trials question whether therapeutic potency can be currently entirely dissociated from vector-mediated genomic effects. * Dr Leboulch was unable to attend the meeting due to the volcano ash cloud. This talk was given by Pr Yves Beuzard who is a co-author of this work.

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Carole BEAUMONT, Paris France

INSERM U773, CRB3, Paris, France

ENERCA WP6 Heme and iron recycling

Iron homeostasis relies mostly on the efficient and tightly controlled recycling of iron by tissue macrophages following phagocytosis of senescent red blood cells. This recycling of heme iron provides most of the iron required daily for bone marrow erythropoiesis. Throughout their life span, circulating erythrocytes will accumulate biochemical changes at their surface, such as peroxydation of membrane-bound lipoproteins, loss of syalic acid residues and formation of senescence neoantigens, or will undergo eryptosis, a particular programmed cell death characteristic of red blood cells. These modifications will allow the tissue macrophages to identify the red blood cells to be eliminated, through interactions with specific receptors. After this initial recognition step, the red blood cell is internalised by phagocytosis, red cell constituents will be degraded and the heme molecule will be catabolised by an enzymatic complex anchored in the endoplasmic reticulum membrane and comprising an NADPH-cytochrome c reductase, heme oxygenase 1 and biliverdin reductase. Iron released from heme catabolism is either recycled back to the plasma through ferroportin, a membrane-bound Fe (II) export molecule, or retained within the ferritin molecules, to be released at later stages. Erythrophagocytosis induces changes in macrophage gene expression, including changes in HO1, ferroportin and ferritin expression, through heme-mediated transcriptional regulations and iron-mediated post-transcriptonal regulations through the IRE/IRP system. Recently, it has been shown that transcription of ferroportin gene is inhibited by Bach1 and activated by Nrf2 in a heme-dependant mechanism involving a MARE/ARE element located 7 Kb upstream of the FPN1 promoter. However, the actual amount of iron exported from macrophages to the plasma is directly controlled by hepcidin through its interaction with ferroportin. Hepcidin is a peptide hormone acting as a negative regulator of iron homeostasis and allowing a fine-tuning of the amount of iron recycled by macrophages to the iron demand of bone marrow erythropoiesis. Modifications in erythrophagocytosis contribute to the pathogenesis of several disorders. Increased eryptosis of erythrocytes has been described in iron deficiency, in sickle cell disease, or in patients with non-alcoholic steatohepatitis, contributing to reduced life span of red blood cells, severity of the anemia and abnormal liver iron deposits. Activation of macrophages by cytokines and increased erythrophagocytosis is a hallmark of the anemia of chronic diseases and of the hemophagocytic syndrome. Therefore, erythrophagocytosis and recycling of heme iron appears as a central process in iron homeostasis.

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Clara CAMASCHELLA, Milan, Italy

ENERCA WP6 (UNISR) Clara Camaschella, Alessia Pagani, Antonella Nai, Laura Silvestri

Vita-Salute University and IRCCS San Raffaele, Milan, Italy

The role of hemojuvelin in iron deficiency and overload Hemojuvelin (HJV), the protein mutated in 1q-linked juvenile hemochromatosis (OMIM #602390) is a GPI-anchored protein, homologous to Repulsive Guidance Molecules (RGMa and RGMb) of the central nervous system, but its expression is restricted to liver, skeletal muscles and heart (1). As the other RGMs, HJV is a co-receptor for Bone Morphogenetic Proteins (BMPs), a function indispensable to activate transcription of the iron regulatory hepatic peptide hepcidin (2). BMPs bind to specific receptors and to the co-receptor HJV on the hepatocyte surface to activate signaling through SMAD proteins. Although several BMP activate hepcidin transcription in vitro the physiological activator in vivo is BMP6 (3-4). Liver specific inactivation of Smad4 in mouse causes iron overload with extremely low hepcidin levels (5), a phenotype similar to that of Hjv- (6-7), hepcidin - (8) and Bmp6- (3-4) deficient mice HJV matures through a complex processing, involving glycosylation and autoproteolytic cleavage that are essential for the membrane targeting of the protein. The role of BMP co-receptor is lost in several HJV mutations associated with juvenile hemochromatosis. HJV mutants of the C-terminal portion of the protein do not undergo autoproteolysis and are ER or Golgi retained (9). Proteins mutated at the N-terminus are targeted to the plasma membrane, but are unable to properly activate the hepcidin promoter in vitro (10). As the other RGMs HJV releases a soluble component (s-HJV) that increases in iron-deficiency and chemically induced hypoxia (11-12). s-HJV ,is a decoy molecule produced by furin cleavage that downregulates hepcidin transcription by binding to and sequestering BMPs. Chronic administration of s-HJV causes iron overload, impairing hepcidin production in mice (13). s-HJV shows a transient increase upon induction of iron deficiency in rats (14); nevertheless the tissue origin of s-HJV and its physiological role remain unclear. To make the issue more complex neogenin, when inactivated in mice, has been shown to regulate s-HJV and to cause iron overload likely through uncontrolled s-HJV production (15). Hemojuvelin plays also a passive role in iron deficiency, since its membrane-associated form is the substrate of the proteolytic activity of TMPRSS6, which encodes the transmembrane serine protease matriptase-2. TMPRSS6 strongly inhibits hepcidin in vivo, since its genetic inactivation causes excessive hepcidin production and consequent iron deficiency anemia in mice (16) and iron-refractory iron deficiency anemia (IRIDA) in humans (17). Matriptase-2 controls hepcidin by cleaving the BMP6 coreceptor HJV from plasma membrane, thus inhibiting its activatory pathway (18). These results are confirmed by the double Tmprss6 and Hjv knock out mouse, which shows the same (iron overload) phenotype of Hjv -/- mouse, confirming a genetic interaction between the two proteins (19). TMPRSS6 function prevails over all hepcidin regulatory pathways for intestinal iron uptake, when iron requests are high. The other hepcidin inhibitors, as s-HJV and GDF15, expected to be released in iron deficiency, do not compensate for the lack of matriptase-2 in IRIDA patients. HJV inactivation causes juvenile hemochromatosis (1) as hepcidin inactivation (20); HJV hyperactivity causes iron deficiency (16-17) as hepcidin overexpression (21). These findings

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points to the HJV-related pathway as essential for hepcidin modulation: how the other hepcidin activators/inhibitors relate to this pathway remains to be clarified.

References. 1. Papanikolaou G, Samuels ME, Ludwig EH, MacDonald ML, Franchini PL, Dubé MP, Andres L, MacFarlane J, Sakellaropoulos N, Politou M, Nemeth E, Thompson J, Risler JK, Zaborowska C, Babakaiff R, Radomski CC, Pape TD, Davidas O, Christakis J, Brissot P, Lockitch G, Ganz T, Hayden MR, Goldberg YP. Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet. 2004;36(1):77-82. 2. Babitt JL, Huang FW, Wrighting DM et al. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet. 2006;38:531-9. 3. Meynard D, Kautz L, Darnaud V et al. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet. 2009;41(4):478-81. 4. Andriopoulos JB, Corradini E, Xia Y et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet. 2009;41(4):482-7. 5. Wang RH, Li C, Xu X, Zheng Y, Xiao C, Zerfas P, Cooperman S, Eckhaus M, Rouault T, Mishra L, Deng CX. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell Metab. 2005;2(6):399-409. 6. Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload. J Clin Invest. 2005;115(8):2180-6. 7. Huang FW, Pinkus JL, Pinkus GS, Fleming MD, Andrews NC. A mouse model of juvenile hemochromatosis. J Clin Invest. 2005;115(8):2187-91 8. Lesbordes-Brion JC, Viatte L, Bennoun M, Lou DQ, Ramey G, Houbron C, Hamard G, Kahn A, Vaulont S. Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis Blood. 2006;108(4):1402-5. 9. Silvestri L, Pagani A, Fazi C, Gerardi G, Levi S, Arosio P, Camaschella C. Defective targeting of hemojuvelin to plasma membrane is a common pathogenetic mechanism in juvenile hemochromatosis. Blood. 2007;109(10):4503-10 10. Pagani A, Silvestri L, Nai A, Camaschella C. Hemojuvelin N-terminal mutants reach the plasma membrane but do not activate the hepcidin response. Haematologica. 2008;93(10):1466-72.11. 11. Silvestri L, Pagani A, Camaschella C. Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis.Blood. 2008;111(2):924-31. 12. Valore EV, Ganz T. Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells Mol Dis. 2008;40(1):132-8. 13. Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance. J Clin Invest. 2007;117(7):1933-9. 14. Zhang AS, Anderson SA, Meyers KR, Hernandez C, Eisenstein RS, Enns CA. Evidence that inhibition of hemojuvelin shedding in response to iron is mediated through neogenin. J Biol Chem. 2007;282(17):12547-56. 15. Lee DH, Zhou LJ, Zhou Z, Xie JX, Jung JU, Liu Y, Xi CX, Mei L, Xiong WC. Neogenin inhibits HJV secretion and regulates BMP induced hepcidin expression and iron homeostasis. Blood. DOI 10.1182/blood-2009-11-251199. 16. Du X, She E, Gelbart T et al. The serine protease TMPRSS6 is required to sense iron deficiency. Science 2008;320:1088-1092. 17. Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA, Hartman KR, Mayo MM, Samuel SM, Strouse JJ, Markianos K, Andrews NC, Fleming MD. Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet. 2008;40(5):569-71. 18. Silvestri L, Pagani A, Nai A et al. The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin

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activation by cleaving membrane hemojuvelin. Cell Metab. 2008;8:502-511. 19. Truksa J, Gelbart T, Peng H et al. Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6. Br J Haematol. 2009;147:571-581. 20. Roetto A, Papanikolaou G, Politou M, Alberti F, Girelli D, Christakis J, Loukopoulos D, Camaschella C. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet. 2003;33(1):21-2. 21. Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, Sirito M, Sawadogo M, Kahn A, Vaulont S. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.Proc Natl Acad Sci U S A. 2002;99(7):4596-60

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Marie-Paule ROTH, Toulouse, France

Inserm U563, Toulouse, France

The essential role of BMP6 in hepcidin regulation Hepcidin, a small peptide produced primarily by hepatocytes, plays a major role in the regulation of iron metabolism. It interacts with the cellular iron exporter ferroportin, causing its degradation and preventing release of iron from macrophages or intestinal cells into the plasma (1). The consequent increase in splenic iron and decrease in dietary iron absorption lead to decreased circulating iron levels. As expected, a feedback relationship exists between body iron status and hepcidin expression: hepcidin is upregulated in response to iron loading and decreased in response to iron deficiency. Interestingly, hepcidin production is regulated by the BMP signaling pathway (2). BMPs are members of the TGF-β superfamily of ligands and initiate an intracellular signaling cascade by binding to two type I and two type II serine-threonine kinase receptors. The activated receptor complex phosphorylates cytoplasmic effectors, the Smad1, 5 and 8 proteins, which then form heteromeric complexes with Smad4 and translocate to the nucleus to modulate gene expression. Mutations in the BMP coreceptor, hemojuvelin, are associated with inappropriately low hepcidin expression and massive iron overload in both humans and mice (3, 4). Suppression of hepatic BMP signaling by a liver-specific conditional knockout of Smad4 has similar consequences (5). BMP signaling can be inhibited in vivo with soluble hemojuvelin which binds to endogenously secreted BMP ligands and prevents their interaction with cell surface receptors (6), or with dorsomorphin which blocks the ability of activated BMP type I receptors to phosphorylate Smad1/5/8 (7). The inhibition of BMP signaling reduces hepcidin expression and increases serum iron. Conversely, BMP administration in vivo increases hepcidin expression and reduces serum iron. In vitro, numerous BMP ligands, including BMP2, BMP4, BMP5, BMP6, BMP7 and BMP9, can regulate hepcidin when added exogenously. However, the observations made in cell culture do not always translate to the situation in vivo and, until last year, the exact nature of the endogenous ligand that activates the BMP/SMAD signaling cascade in response to iron was unknown. We first observed that a systemic iron challenge induces hepatic Smad1/5/8 phosphorylation, which indicates that BMP signaling is involved in the feedback regulation of hepcidin transcription by iron. The analysis of the liver transcriptomes of mice kept on a low- or high-iron diet then showed that the levels of Bmp6 mRNA, but not that of other Bmps, were concordant with changes in hepcidin mRNA concentrations, suggesting that BMP6 could be the endogenous regulator of iron metabolism (8). Our group and that of J. Babitt recently confirmed this possibility. Indeed, targeted disruption of Bmp6 in mice causes a rapid and massive iron accumulation in the liver, pancreas, heart and kidney. Despite their severe iron overload, these mice have low levels of hepatic Smad1/5/8 phosphorylation and markedly reduced hepcidin synthesis, indicating that BMP6 is critical for iron homeostasis and functionally nonredundant with other members of the BMP subfamily of ligands (9, 10). Impaired regulation of hepcidin expression in response to iron loading appears to be the pathogenic mechanism for hereditary hemochromatosis. The molecular function of the HFE protein, involved in the most common form of hereditary hemochromatosis, is still unknown. We have used Hfe-deficient mice to test whether HFE has a role in the signaling cascade induced by BMP6. At weaning, these mice have normal Bmp6 mRNA levels, but inappropriately low hepcidin mRNA. This leads to increased iron

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absorption, progressive accumulation of iron in the liver and, as expected, increased Bmp6 mRNA and protein. However, this is followed by only a slight increase in hepcidin mRNA, insufficient in regard to the levels of Bmp6. Lack of HFE therefore impairs signal transduction induced by the BMP6 ligand (11). Potential of BMP6 agonists and antagonists for the treatment of genetic hemochromatosis or iron-refractory iron deficiency anemia will be discussed at the workshop.

References 1. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090-2093. 2. Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA, Campagna JA, et al. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet 2006;38:531-539. 3. Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload. J Clin Invest 2005;115:2180-2186. 4. Papanikolaou G, Samuels ME, Ludwig EH, MacDonald ML, Franchini PL, Dube MP, Andres L, et al. Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet 2004;36:77-82. 5. Wang RH, Li C, Xu X, Zheng Y, Xiao C, Zerfas P, Cooperman S, et al. A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell Metab 2005;2:399-409. 6. Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance. J Clin Invest 2007;117:1933-1939. 7. Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, Lin HY, et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol 2008;4:33-41. 8. Kautz L, Meynard D, Monnier A, Darnaud V, Bouvet R, Wang RH, Deng C, et al. Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood 2008;112:1503-1509. 9. Andriopoulos B, Jr., Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, Knutson MD, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet 2009;41:482-487. 10. Meynard D, Kautz L, Darnaud V, Canonne-Hergaux F, Coppin H, Roth MP. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet 2009;41:478-481. 11. Kautz L, Meynard D, Besson-Fournier C, Darnaud V, Al Saati T, Coppin H, Roth MP. BMP/Smad signaling is not enhanced in Hfe-deficient mice despite increased Bmp6 expression. Blood 2009;114:2515-2520.

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Ivana De DOMENICO, Salt Lake City, USA

Department of Internal Medicine, School of Medicine, University of Utah, Salt Lake City, Utah USA

Targeting the Hepcidin Binding Site of Ferroportin

Hepcidin is a peptide hormone secreted in response to iron loading and inflammation. Hepcidin regulates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation. Internalization of ferroportin results from the activation of the protein kinase Jak2 and phosphorylation of ferroportin. Hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Treatment of ferroportin expressing macrophages with hepcidin results in changes in a wide variety of genes. Changes in transcript levels for many of the affected genes is a direct effect of hepcidin and is dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulate endotoxin induced transcription in both cultured macrophages and in vivo, suppressing IL-6 and TNFa. Hepcidin-mediated transcription in mice suppresses toxicity and morbidity due to single doses of endotoxin, poly(I:C) or turpentine. These results suggest a new function for hepcidin in modulating acute inflammatory responses.

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Yves BEUZARD, Fontenay-aux-Roses, France

ENERCA WP3 (IUH)

CEA-iMETI and GenetiX-France, CEA, Fontenay-aux-Roses, 92265 France

Erythrocyte acting Therapies of Hemoglobin Disorders

The most important factors in the pathophysiology of severe β thalassemia are the excess of “free” and unstable α globin and α hemoglobin chains, heme derivatives and iron toxicity being responsible for increased hemolysis and ineffective erythropoiesis. The relative excess of α chains is initiated by the imbalance ratio between α and β + γ globin chains and the rate of protein synthesis. The clinical benefit and reduction of unpaired α hemoglobin chains is well demonstrated in β-thalassemic patients upon additional α thalassemic determinants or heterocellular forms of the hereditary persistence of fetal hemoglobin. Other factors are less explored, such as the inadequate capability of erythroid cells to prevent the instability of the soluble α chains and to improve proteolysis, upon the ubiquitin/proteasome system or using other proteases. The proteolytic process is efficient enough to remove the excess of unpaired α chains in erythroid cells of β-thalassemic trait carriers, but it is oversaturated upon additional α gene triplication, dominant mutation and in E/β-thal.compound heterozygotes. The group of Yelena Ginzburg (Huihui Li et al., Nature Medicine, 2010) demonstrates the benefit of slowing down the iron loading in erythroid cells in mouse β-thalassemia, by using daily injections of human transferrin. The amount of precipitated α hemoglobin is highly decreased as well as hemolysis. The erythrocyte survival and the hemoglobin level in blood are well improved. This encouraging proof of concept indicates that inhibitors of erythroid transferrin receptors may be beneficial. Other agents, such as the intracellular iron scavengers and antioxidants could be also effective, alone or in combination. Sickle cell anemia is a mixt to various extents of two different syndromes: the “hyperhemolytic vasculopathy” and the “vaso-occlusive events”. The primum movens for both syndromes is the formation of polymers of hemoglobin S in erythrocytes, highly dependant upon the hemoglobin S concentration in erythrocytes and deoxygenation. The mechanism of intravascukar hemolysis in sickle cell anemia is poorly understood. However, it is known that inhibitors of the Gardos channel (calcium dependant potassium channel Kca 3.1), like clotrimazole and the derivative Senicapoc (ICA-17043-05) and magnesium pidolate which inhibits the erythrocyte K+Cl- co-transport system are preventing or reverting dehydration of sickle cells and reduce hemolysis, both in sickle mice and in patients. Up to now, the clinical benefits were not adequately evaluated in a phase II study of Senicapoc (KI Ataga et al. Blood 2008), because the increase in hemoglobin improved the hyperhemolytic syndrome but the frequency of the painful crisis was maintained or even increased in patients in some patients. An ongoing phase III trial of Senicapoc has to take separately these two syndromes into

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consideration. In addition, it will be useful to evaluate the inhibitors of Ca2+ permeable cation conductance, active specifically upon deoxygenation and stretching of sickle red blood cells. It is inhibited by carbone monoxide 25 ppm pretreatment, dipyridamole, DIDS and mechanotoxine-4 (DH Vandorpe et al. Plos one 2010). The combination of these approaches and with the hydroxycarbamide therapy could be beneficial.

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Swee Lay THEIN, London, UK

ENERCA WP3 (KINGS)

Kings College London School of Medicine and Kings College Hospital, London, UK

Impact of gamma globin gene expression on haemoglobin disorders Inter-individual variation in fetal haemoglobin (HbF, α2γ2) expression contributes substantially to disease severity in the two major β haemoglobin disorders – β thalassaemia and sickle cell disease (SCD) - and much effort has been spent on understanding itsregulation in adults. The beneficial effect of HbF on both disorders has been known for decades and exploited in the development of HbF reactivating agents including hydroxyurea, the only agent approved by FDA (USA) and EMEA (Europe) for the treatment of SCD. Inter-individual HbF variation is largely genetically controlled but the inheritance patterns are not clear. Our understanding of haemoglobin control, including the persistence of HbF synthesis in adults, is historically based on Mendelian models of inheritance of natural mutants. Indeed, a series of mutations at the β globin cluster that impairs the switch from fetal to adult haemoglobin have been characterised in a syndrome termed hereditary persistence of fetal haemoglobin (HPFH). The talk will focus on our understanding of the persistence of fetal haemoglobin in adults as a quantitative trait. From a genetics perspective, the story of HbF quantitative genetics uncannily mirrors the changing focus of human gene mapping, from candidate genes through positional cloning to genome-wide association studies (GWAS). Recent genome-wide studies have shown that common genetic polymorphisms in three major loci (Xmn1-HBG2, HBS1L-MYB intergenic region (HMIP) on chromosome 6q23, and BCL11A on chromosome 2p16) account for a relatively large proportion (20%-50%) of the phenotypic variation in HbF levels, not only in healthy adults but also in patients with these β haemoglobinopathies in diverse ethnic groups. Among African-American patients with sickle cell anaemia, the three loci account for up to 20% of the HbF variation with a corresponding reduction in acute pain rate. In Sardinia, BCL11A and HBS1L-MYB with α thalassaemia account for up to 75% of the variable phenotypic severity in β thalassaemia. The three loci are also associated with both disease severity and HbF levels in patients with HbE/β thalassaemia in Thailand. Two of the major QTLs include oncogenes emphasising the importance of cell proliferation and differentiation as an important contribution to the HbF phenotype. These genetic results have already provided remarkable insights into molecular mechanisms that underlie the haemoglobin ‘switch’. References. 1. Galanello, R., Sanna, S., Perseu, L., Sollaino, M.C., Satta, S., Lai, M.E., Barella, S., Uda, M.,

Usala, G., Abecasis, G.R. & Cao, A. (2009) Amelioration of Sardinian beta-zero thalassemia by genetic modifiers. Blood, 114, 3935-3937.

2. Lettre, G., Sankaran, V.G., Bezerra, M.A., Araujo, A.S., Uda, M., Sanna, S., Cao, A.,

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Schlessinger, D., Costa, F.F., Hirschhorn, J.N. & Orkin, S.H. (2008) DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease. Proc Natl Acad Sci U S A, 105, 11869-11874.

3. Menzel, S., Garner, C., Gut, I., Matsuda, F., Yamaguchi, M., Heath, S., Foglio, M., Zelenika, D., Boland, A., Rooks, H., Best, S., Spector, T.D., Farrall, M., Lathrop, M. & Thein, S.L. (2007) A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15. Nat Genet, 39, 1197-1199.

4. Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel, B., Mikkola, H.K., Hirschhorn, J.N., Cantor, A.B. & Orkin, S.H. (2008) Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A. Science, 322, 1839-1842.

5. Sedgewick, A.E., Timofeev, N., Sebastiani, P., So, J.C., Ma, E.S., Chan, L.C., Fucharoen, G., Fucharoen, S., Barbosa, C.G., Vardarajan, B.N., Farrer, L.A., Baldwin, C.T., Steinberg, M.H. & Chui, D.H. (2008) BCL11A is a major HbF quantitative trait locus in three different populations with beta-hemoglobinopathies. Blood Cells Mol Dis, 41, 255-258.

6. So, C.C., Song, Y.Q., Tsang, S.T., Tang, L.F., Chan, A.Y., Ma, E.S. & Chan, L.C. (2008) The HBS1L-MYB intergenic region on chromosome 6q23 is a quantitative trait locus controlling fetal haemoglobin level in carriers of beta-thalassaemia. J Med Genet, 45, 745-751.

7. Thein, S.L. & Menzel, S. (2009) Discovering the genetics underlying foetal haemoglobin production in adults. Br J Haematol, 145, 455-467.

8. Thein, S.L., Menzel, S., Peng, X., Best, S., Jiang, J., Close, J., Silver, N., Gerovasilli, A., Ping, C., Yamaguchi, M., Wahlberg, K., Ulug, P., Spector, T.D., Garner, C., Matsuda, F., Farrall, M. & Lathrop, M. (2007) Intergenic variants of HBS1L-MYB are responsible for a major quantitative trait locus on chromosome 6q23 influencing fetal hemoglobin levels in adults. Proc Natl Acad Sci U S A, 104, 11346-11351.

9. Thein, S.L., Menzel, S., Lathrop, M., Garner, C. Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Human Molecular Genetics. Oct 2009; 18(R2): R216-23

10. Uda, M., Galanello, R., Sanna, S., Lettre, G., Sankaran, V.G., Chen, W., Usala, G., Busonero, F., Maschio, A., Albai, G., Piras, M.G., Sestu, N., Lai, S., Dei, M., Mulas, A., Crisponi, L., Naitza, S., Asunis, I., Deiana, M., Nagaraja, R., Perseu, L., Satta, S., Cipollina, M.D., Sollaino, C., Moi, P., Hirschhorn, J.N., Orkin, S.H., Abecasis, G.R., Schlessinger, D. & Cao, A. (2008) Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia. Proc Natl Acad Sci U S A, 105, 1620-1625.

11. Wahlberg, K., Jiang, J., Rooks, H., Jawaid, K., Matsuda, F., Yamaguchi, M., Lathrop, M., Thein, S.L. & Best, S. (2009) The HBS1L-MYB intergenic interval associated with elevated HbF levels shows characteristics of a distal regulatory region in erythroid cells. Blood, 114, 1254-1262.

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Educational material Educational material used for this course included :

Abstract book

t contains an abstracts of all the talks with a selected of bibliography. It also gives the names, address and e-mails of all invited speakers and participants in order to allow wide discussion during the course and further contacts.

Chapter

5

EE DD UU CC AA TT II OO NN AA LL MM AA TT EE RR II AA LL

The abstract book of the course distributed to all participants

The handbook : "Disorders of erythropoiesis , erythrocytes and iron

metabolism" , (2009 new edition) freely distributed by the ESH to participants

Slides from speakers who accepted to give their presentation (will be

implemented on ENERCA website

The curriculum on iron metabolism and related disorders (self

education tool available freely, upon registration on the ESH website)

I

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The handbook : "Disorders of erythropoiesis , erythrocytes and iron metabolism" , (2009 new edition)

After a successful 2006 first edition, this very comprehensive hand book is now re-edited in a 2009 revised version. Editors are Carole Beaumont (WP6) , Photis Beris, Yves Beuzard (WP3) and Carlo Brugnara. It has been launch under the hospices of the French red cell club (Club du globule rouge et du fer, present president Carole Beaumont, past president, Yves Beuzard). It contains very useful contributions on red cells and iron physiology and pathology.

This handbook is freely distributed thanks to an educational grant from Novartis oncology, Vifor international and Amgen.

Selected slide presentations from ENERCA speakers available online Speakers have accepted to select the most significant slides from their talk that will help people interested in the field to learn more on the state of the art on red cell and iron related disorders and iron metabolism. The slides will be implemented on the ENERCA website in the "professional section". The presentation will comprise a the abstract of the talk , a picture of the speaker and a clickable link that will brings to the pdf slide presentation of each speaker.

Philippe LEBOULCH (presented by Yves BEUZARD), Carole BEAUMONT (WP6), Clara CAMASCHELLA (WP6), Marie-Paule ROTH, Ivana De DOMENICO, Yves BEUZARD (WP3) and Swee Lay THEIN (WP3 associated partner) are contributors of these slide presentation. .

The curriculum on iron metabolism and related disorders ESH has developed with the help of several scientist an original educational online tool for self medical education. It can be accessed by using the link : http://www.ironcurriculum.esh.org/index.cfm?thspage=curriculum

Figure 4.: screen print of the Curriculum in Iron metabolism and related disorders developed on the ESH website.

http://www.ironcurriculum.esh.org/index.cfm?thspage=curriculum

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.

Four sessions (Curriculum 1 to 4) are now available. A " Ask the Experts" module allows welcoming questions, comments, and suggestions by using a form to be emailed. In order to receive a response, an email address must be provided.

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Conclusion ourse 1 was a successful scientific and educational event despite difficulties due to the unexpected occurrence of the volcano ash cloud during the course.

Objectives of the ENERCA courses are fulfilled and dissemination of the educational material through various means (books, websites) will help diffusing this updated knowledge.

C

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Acknowledgements We would like to acknowledge all the people who helped preparing, diffusing and holding this course.

Among these, chairpersons (WP3 and WP6 members), all the ESH staff , all WP3 partners, ENERCA WP leaders who gave advice and helped diffusing the announcement of the course, ENARCA coordination team and ENERCA3 leader, Prof Joan Lluis Vives Corrons, CHUM staff members.

We also warmly thank Sponsors and the European Commission grant N°.2008 12 10

Documents

Deliverable D16: Report of the course on Red Cells and