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1. Allgemeine Angaben

1.1 Antragsteller

- Prof. Dr. Norbert Gattermann, Heinrich-Heine-Universität Düsseldorf - Dr. Mayka Sanchez, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona,

Spain.

1.2 Thema:

Myelodysplastische Syndrome (MDS): Ursache der mitochondrialen Eisenüberladung bei sidero-

blastischer Anämie (MDS: Cause of mitochondrial iron overload in sideroblastic anemia).

1.3. Kennwort: MDS: mitochondriale Eisenüberladung

1.4. Fachgebiet und Arbeitsrichtung des Antragstellers:

Gattermann: Hämatologie/Onkologie, MDS, Epigenetische Behandlung, Refraktäre Anämie mit

Ringsideroblasten (RARS), Mutationen der Mitochondrien-DNA, Eisenüberladung

Sanchez: Regulation des Eisenstoffwechsels, IRP/IRE (iron regulatory proteins / iron response

elements), Verbindung zwischen Eisenstoffwechsel und Krebsentstehung, Entdeckung neuer

Gene mit ursächlicher Bedeutung für Erkrankungen mit gestörter Eisenbilanz.

1.5. Gesamtdauer des Projektes: 3 Jahre

1.6. Förderzeitraum: 01.06.2014 bis 01.06.2017

1.7. Gewünschter Beginn der Förderung: 01.06.2014

2. Abstract

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by

dysplasia and ineffective hematopoiesis. A subgroup of patients, namely those with sideroblastic

anemia, have abnormal erythroid precursors (ring sideroblasts) showing massive mitochondrial

iron accumulation. These subtypes are called “refractory anemia with ring sideroblasts” (RARS),

“RARS with thrombocytosis” (RARS-T), and “refractory cytopenia with multilineage dysplasia and

ringed sideroblasts” (RCMD-RS). MDS patients with sideroblastic anemia develop systemic iron

overload, partly from increased intestinal iron absorption and partly from chronic transfusion

therapy.

In the majority (60-80%) of patients with RARS and RARS-T, somatic mutations are detectable in

SF3B1, a component of the spliceosomal U2snRNP complex. However, the molecular

mechanisms by which mutations in SF3B1 lead to mitochondrial iron accumulation are still unclear.

To dissect the underlying mechanism in SF3B1 mutated sideroblastic MDS, we will study the

cellular iron regulatory system (IRP/IRE system), in particular the possible implications of newly

identified mRNAs controlled by this system. Consequences of altered gene expression and

altered exon usage will be investigated with regard to the IRP/IRE regulatory system, the iron-

sulfur-cluster biogenesis and the cellular iron speciation in MDS patients with sideroblastic anemia.

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As MDS patients with sideroblastic anemia have a low rate of leukemic transformation, their main

problem is chronic anemia. Understanding the mechanism responsible for deranged mitochondrial

iron metabolism may lead to novel treatment strategies aimed at more effective erythropoiesis and

less iron-related cell and tissue damage.

3. State of the art

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell diseases characterized by

dysplasia, ineffective hematopoiesis, and the risk of progression to acute myeloid leukaemia

(AML). Shortened survival in MDS patients is due to the consequences of bone marrow failure [1].

Since MDS is an age-related disease, with a median age of 72 years at diagnosis, the prevalence

is rising steadily in our aging population.

A subgroup of patients have sideroblastic anemia, which is characterized by at least 15% ring

sideroblasts in the bone marrow, i.e. pathological erythroblasts showing gross mitochondrial iron

accumulation. These cases are classified as refractory anaemia with ring sideroblasts (RARS),

RARS with thrombocytosis (RARS-T), and refractory cytopenia with multilineage dysplasia and ring

sideroblasts (RCMD-RS). MDS patients with sideroblastic anemia develop systemic iron overload,

partly from increased intestinal iron absorption and partly from regular transfusion of red blood

cells. Iron overload contributes to shortened survival in these patients [2]. The observation that iron

chelation therapy improves erythropoiesis in about 20% of transfusion-dependent MDS patients [3]

suggests that iron overlaod aggravates the bone marrow failure in MDS.

Recently, whole genome sequencing revealed that spliceosome mutations are detectable in

approximately 45% of all MDS patients [4]. In particular, 60-80% of patients with RARS and RARS-

T present somatic heterozygous mutations in the SF3B1 gene [5]. SF3B1, splicing factor 3b,

subunit 1, is a component of the U2-small nuclear ribonucleoprotein (U2-snRNP) complex needed

for normal RNA splicing. Several studies with material from RARS patients as well as an Sf3b1

heterozygous knockout mouse model found a strong correlation between SF3B1 mutations and the

sideroblastic phenotype [6-8]. SF3B1 mutations are infrequently found in non-myelodysplastic

bone marrow failure, but, if present, are always associated with the presence of ring sideroblasts

[9]. Since SF3B1 mutations are an independent predictor of a favourable clinical course, they may

be incorporated into risk stratification systems for MDS [8]. It is still unclear how mutant SF3B1

leads to the formation of ring sideroblasts, and some discrepancies remain unresolved. For

instance, Boultwood et al [10] showed down-regulated mRNA of the iron-sulphur cluster exporter

ABCB7 in patients with RARS, but Visconte et al [6] found no difference in exon-usage or gene

expression in ABCB7, a gene mutated in X-linked congenital sideroblastic anemia with ataxia. Mitochondrial iron accumulation can be due to impaired synthesis/export of heme or impaired

synthesis/export of iron-sulphur clusters (ISCs). However, the differential usage of exons and the

down-regulation of many genes in hematopoietic cells carrying SF3B1mutations have not revealed

any obvious gene involved in mitochondrial iron, heme or ISC metabolism that might explain the

sideroblastic phenotype.

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We have hypothesized that SF3B1 mutations may compromise a hitherto unknown physiologic role

of SF3B1 in mitochondrial iron metabolism or create a novel, unphysiologic effect on mitochondrial

iron handling (Annex 1) [11]. This may involve one or more of the novel mRNAs that were identified

by Dr. Sanchez (IJC, Badalona) to be regulated by the iron regulatory proteins IRP1 and IRP2 [12].

Cellular iron homeostasis is controlled post-transcriptionally by the IRP/IRE regulatory system[13].

The iron-regulatory proteins (IRP1 and IRP2) can recognize cis-regulatory mRNA motifs called

iron-responsive elements (IREs), i.e. conserved RNA elements located in the untranslated regions

(UTR) of mRNAs that encode proteins involved in iron metabolism (TFR1, DMT1, ferritin L and H,

ferroportin, ACO2 and ALAS2)[13]. IRP1 and IRP2 IRE binding activities are regulated by iron, and

IRPs only bind to IREs under iron-limited conditions. IRP1 is a dual-function protein that acts as

aconitase when containing an iron-sulfur cluster (ISC) in iron replenished conditions, whereas it

shows IRE-binding activity when intra-cytoplasmatic levels of iron are low. IRP2 is degraded in the

presence of high cytoplasmatic iron levels by a proteosomal E3 ubiquitin ligase complex containing

the FBXL5 protein, a protein that itself requires iron and oxygen for its function. Heme has been

also implicated in the degradation of IRP2[14]. Both IRPs inhibit translation initiation when bound to

5’UTR IREs, while their association with the 3’UTR IREs of the TFR1 mRNA decreases its turnover

by preventing mRNA degradation via an as yet unidentified endoribonuclease. Disruption of both

copies of the IRP1 and the IRP2 genes in mice is embryonic lethal, indicating that the IRP/IRE

regulatory network is essential[15]. While IRP1-deficient mice display no overt phenotype in steady

state conditions, IRP2 knock-out mice develop mild microcytic hypochromic anemia with altered

body iron distribution[16], and mice with an inducible gain of IRP1 function show abnormal body

iron distribution and erythropoiesis[17].

A defect in ISC biosynthesis/export or in the heme pathway will affect the IRE binding activity of

IRP1 and IRP2. Indeed, abnormally high IRP1-IRE activity has been found in patients affected by

congenital sideroblastic anaemia (CSA) linked to deficiency of GLRX5 (glutaredoxin 5 plays a role

in ISC assembly)[18]. Furthermore, the expression of ALAS2 (aminolevulinic acid synthase), which

is mutated in X-linked CSA, is controlled by the IRP/IRE system[19]. These findings in congenital

sideroblastic anemias suggest that the IRP/IRE regulatory system may also be disrupted in

MDS-related sideroblastic anemia. As this hypothesis has not been adequately studied to date,

we wish to test it in the proposed project (see objective 1).

Recent discoveries by Dr. Sanchez led to the identification of novel mRNAs that can bind IRPs.

Using a high-throughput genome-wide approach[20], Sanchez et al. defined the whole repertoire of

mRNAs regulated by both IRPs [21-23] and identified a cohort of first-time-described IRP1- and

IRP2-specific binding mRNAs [23]. In total, 258 mRNAs were found to interact with IRPs (44

mRNAs bind both IRPs, 101 specifically bind IRP1, and 113 specifically bind IRP2), including all 9

known murine IRP-binding mRNAs [23]. These findings generate questions as to the regulation of

the novel IRP-target mRNAs via iron-dependent or -independent mechanisms and create new

opportunities to investigate the cause of mitochondrial iron overload. Thus, our objective 2 is to

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explore the role of novel IRP-target mRNAs in the pathophysiology of MDS-related sideroblastic

anemia.

Since cytokines are important in regulating differentiation of hematopoietic cells, targeting them

appears to be a rational therapeutic strategy in MDS. Various studies suggest Tumor Necrosis

Factor α (TNF-α) [24], Transforming Growth Factor β (TGF β) [25], Vascular Endothelial Growth

Factor (VEGF) [26], Activin receptor like kinase (ALK) [27], Interleukins (ILs) [28], and Interferons

(IFN) [29] regulate the bone marrow milieu in MDS. The physiologic effects of a few of these

cytokines are executed by the support of transcription regulators like the JAK-STAT pathway and

many other pathways. Studies have demonstrated over-activated signaling of myelo-suppressive

cytokines in MDS hematopoietic stem cells (reviewed in [30]). Hence strategies that can balance

the effects of the stimulatory and inhibitory cytokine pathways can potentially be of therapeutic

utility in MDS and other hematologic neoplasm [31-32]. We plan to take this concept into

consideration, knowing that cytokine derangements can have a substantial impact on iron

metabolism, as illustrated by complex dysregulation of the hepcidin-ferroportin axis in patients with

MDS [33-34]. Therefore, when studying the consequences of altered gene expression and altered

exon usage in hematopoetic cells with mutant SF3B1, we will pay special attention to cytokine

genes and study their possible role in iron homeostasis (objective 3).

4. Previous work

N. Gattermann is a hematologist with a long-standing interest in MDS-related sideroblastic anemia

who investigated the disorder using hematopoietic colony assays1 mitochondrial DNA analysis2,3,

further mutation analysis4, and iron chelation therapy. He recently summarized the mitochondrial

iron-related pathways that could be involved in the pathomechanism of ring sideroblast formation5. 1Gattermann N, Aul C., Schneider W: Two types of acquired idiopathic sideroblastic anaemia (AISA).

Br J Haematol 74: 45-52, 1990. 2Gattermann, N., Retzlaff, S., Wang, Y.-L., Hofhaus, G.,Heinisch, J., Aul, C., Schneider, W.: Heteroplasmic point

mutations of mitochondrial DNA affecting subunit I of cytochrome-c-oxidase (COX I) in two patients with acquired

idiopathic sideroblastic anaemia. Blood 90: 4961-4972, 1997. 3Wulfert M, Küpper AC, Tapprich C, Bottomley SS, Bowen, Germing U, Haas R, Gattermann N. Analysis of

mitochondrial DNA in 104 patients with myelodysplastic syndromes. Exp Hematol 2008;36:577-586 4Gattermann N, Billiet J, Kronenwett R, Zipperer E, Germing U, Nollet F, Criel A, Selleslag D. High frequency of the

JAK2 V617F mutation in patients with thrombocytosis (platelet count>600x109/L) and ringed sideroblasts more than 15%

considered as MDS/MPD, unclassifiable. Blood 109:1334-5, 2007 5Gattermann N. SF3B1 and the riddle of the ring sideroblast. Blood. 2012;120:3167-8.

M. Sanchez is an experienced basic researcher with a special interest in the regulation of cellular

iron metabolism and the genetic basis of iron-related disorders. As described above, recent

discoveries by Dr. Sanchez led to the identification of the whole repertoire of 258 mRNAs regulated

by both IRPs6-9 and identified a cohort of first-time-described IRP1- and IRP2-specific binding

mRNAs9. These findings provide a basis for detecting disturbances in cellular and mitochondrial

iron homeostasis that are closely linked to the mechanism of mitochondrial iron overload. 6Sanchez M, Galy B, Hentze MW, Muckenthaler MU. Identification of target mRNAs of regulatory RNA-binding proteins

using mRNP immunopurification and microarrays. Nat Protoc, 2007; 2:2033-42.

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7Sanchez M, Galy B, Dandekar T, Bengert P, Vainshtein Y, Stolte J, Muckenthaler MU, Hentze MW. Iron regulation and

the cell cycle: identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A

mRNA by a refined microarray-based screening strategy. J Biol Chem, 2006. 281:22865-74. 8Sanchez M, Galy B, Muckenthaler MU, Hentze MW Iron-regulatory proteins limit hypoxia-inducible factor-2 alpha

expression in iron deficiency. Nat Struct Mol Biol, 2007; 14:420-6. 9Sanchez M, Galy B, Schwanhaeusser B, Blake J, Bähr-Ivacevic T, Benes V, Selbach M, Muckenthaler MU, Hentze

MW, Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular

proteome by IRPs. Blood 2011;118:e168-79

Combining the knowledge, resources, and ideas of these two investigators provides a new

opportunity to solve the almost 60-year-old riddle of the ring sideroblast [35].

5. Aims and objectives

In order to improve diagnostic precision in patients with a sideroblastic type of MDS we will perform

mutational screening that includes not only the SF3B1 gene but also a new panel of 36 genes

involved in iron metabolism that is currently being set up in the laboratory of Dr. M. Sanchez. The

investigation will draw on stored blood and bone marrow samples from 100 MDS patients with

>15% ring sideroblasts, and, for comparison, on 50 sequential patients newly diagnosed.

Systematic screening of a panel of 36 iron-related genes in a specific cohort of patients

characterized by disturbed mitochondrial iron metabolism provides an opportunity to discover

relevant mutations (besides SF3B1) that may have been missed in large genome-wide studies

including a limited number of patients with sideroblastic anemia. We will correlate the presence of

mutations in the 36-gene panel with clinical and laboratory features like severity of anemia,

transfusion dependency, parameters of iron metabolism, and percentage of ring sideroblasts in the

bone marrow. In addition, use of the “iron gene panel” will allow us to detect cases of late-onset

congenital sideroblastic anemia, for instance with mutations in ALAS2, which can be mis-

diagnosed as MDS [36].

A candidate mechanism to explain the sideroblastic phenotype is impaired assembly or impaired

mitochondrial export of iron-sulfur clusters (ISCs), which may lead to mitochondrial iron

accumulation by way of inactivating ferrochelatase (a heme biosynthesis enzyme), impairing the

mitochondrial respiratory chain, or activating IRP1 as an IRE-binding protein. Similar to what has

been described in congenital sideroblastic anemias, it is likely that the IRP/IRE regulatory system

is disturbed in MDS-related sideroblastic anemia. To gain more insight into the principal

mechanism involved, we will study the IRP/IRE system in MDS patients with sideroblastic anemia

(objective 1), explore the role of novel IRP-target mRNAs (objective 2) and study the

consequences of altered gene expression and altered exon usage in SF3B1 mutated cells with

regard to mitochondrial iron accumulation (objective 3).

The IRP/IRE regulatory system and the iron content and subcellular iron distribution will be studied

systematically in bone marrow CD34+ cells from MDS patients with a sideroblastic phenotype.

Expression of genes well known to be regulated by the IRPs (ferritins, transferring receptor 1,

ferroportin) will be studied at mRNA as well as protein levels.

Since alterations in the cytokine milieu have been described to play a role in MDS (see above); we

will pay special attention to cytokine genes and study their possible role in iron homeostasis. One

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of the genes already shown to have a low expression in SF3B1-mutated cases is the cytokine

CCL2 [6]. Preliminary results have linked the expression of the cytokine CCL2 to iron metabolism

(ASH 2011, abstract no. 685), characterized CCL2 as a suppressor of TFR1 expression, and

shown that CCL2 knock-out mice are iron overloaded. In addition, the mRNAs of two other

cytokines (CXCL16 and CXCL12) were discovered to bind to IRPs [12] and to be down-regulated

in MDS patients with SF3B1 mutations.

Comparison of the genes affected by altered splicing in SF3B1 mutated cells on the one hand, and

the new IRP target mRNAs detected by us on the other hand, yielded 6 genes that are present in

both lists (CRAMP1L, DNAJB6, ELF2, KIF24, PARP14 and PML). We will study how these genes

are regulated by the IRPs and how they may contribute to the sideroblastic phenotype.

In addition, from the list of 258 mRNAs that can interact with IRPs we will select those genes that

are highly or exclusively expressed in erythroid precursor cells, according to BIOGPS and GEO

expression data. For 50 of those genes (including the 8 genes previously mentioned to be affected

by mutant SF3B1 and known to bind IRPs) we will determine by qRT-PCR whether their

expression is altered in SF3B1-mutated cases compared to controls. Protein levels will be

analyzed by Western blot or immunoassays in those genes found to be differentially expressed.

We will then focus on characterizing the novel IRP-target mRNAs by identifying the RNA binding

motif, using our bioinformatics web server for prediction of IREs [37] and performing experiments

with competitive electrophoretic mobility shift assays (EMSAs). We will address the mechanistic

IRP-dependent regulation of those mRNAs, conducting sucrose gradient and mRNA stability

assays. The response of those genes to iron and oxidative stress will be assessed in tissue culture

with iron donors, iron chelators, and H2O2.

Hypothesis:

Our goal is to clarify how SF3B1 mutations interfere with mitochondrial and cellular iron

metabolism and thus cause the sideroblastic phenotype in a proportion of MDS patients. Mutant

SF3B1 may compromise a hitherto unknown physiologic role of SF3B1 in mitochondrial iron

metabolism or create a novel, unphysiologic effect on mitochondrial iron handling through

abnormal splicing and disturbed gene expression or through an abnormal protein-protein

interaction, independent of the splicing machinery. Since the IRP/IRE regulatory system is pivotal

for iron homeostasis, it is most likely that this system is affected in MDS-related sideroblastic

anemia. We hypothesize that the new target-mRNAs associated with this system will provide us

with crucial evidence regarding the iron-related metabolic pathway that is deranged as a direct

consequence of SF3B1 mutations.

Specific objectives of the proposal:

1. Study the IRP/IRE regulatory system, the iron-sulfur-cluster biogenesis and the iron speciation in

MDS patients with sideroblastic anemia

2. Explore the role of novel IRP-target mRNAs in the pathophysiology of sideroblastic MDS

3. Study the consequences of altered gene expression and altered exon usage in SF3B1 mutated

cells with regard to mitochondrial iron accumulation

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The knowledge acquired through these studies will contribute to a better understanding of

mitochondrial iron accumulation in patients with MDS-related sideroblastic anemia and may

provide targets for novel therapeutic approaches to this refractory anemia.

General objectives: horizontal transfer

A general objective of this proposal is to provide molecular genetic reports on mutations related

to disturbed iron metabolism to the physicians in charge of patients with MDS, thus contributing to

improved diagnosis and clinical decision making. We will also make our findings available to the

research community by publications in high-impact journals such as Blood or Haematologica.

6. Methodology and Research Plan

6.1. Recruitment of patients and samples.

Blood and bone marrow (BM) aspirates from MDS patients with sideroblastic anemia will be

obtained from the Department of Hematology, Oncology and Clinical Immunology at Heinrich-

Heine-University, Düsseldorf, Germany, and the Spanish registry of MDS patients inside the

Spanish Group of Myelodisplastic Syndrome (GESMD). The Düsseldorf MDS Registry includes

more than 5000 cases with clinical follow-up data. For about a quarter of the patients, samples

have been frozen (unfractionated bone marrow aspirates, BM mononuclear cell fractions, extracted

DNA, and/or serum samples). New MDS patients are constantly referred to the MDS outpatient

clinic at the university hospital in Düsseldorf by collaborating hospitals and office-based physicians.

The Spanish MDS Registry is the largest collection of MDS patients worldwide and has 9409

cases, 1300 of them present ring sideroblasts. We collaborate with the Hospital Germans Trias i

Pujol and the Hospital Clinic of Barcelona, providing further opportunities to obtain fresh samples. Blood and bone marrow (BM) aspirates from controls will be acquired from the Biobank of the

Barcelona Blood and Tissue Bank (BST) and from Hospital Germans Trias i Pujol (HGTiP).

Frameworks agreement between the BST, HGTiP and IJC will be signed for that purpose. The

cession of samples will be approved by the BST and the HGTiP ethics committees.

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The project will be submitted to the ethics committee of the Medical Faculty of Heinrich-Heine-

University Düsseldorf, which has already approved blood and bone marrow sampling for molecular

genetic studies aimed at unravelling the molecular pathology of MDS. Written informed consent will

be obtained from all patients included in this project. 6.2. Clinical characterization of patients

Classification of MDS patients will be done according to the WHO 2008 classification. Correct

cytomorphological diagnosis of MDS is ensured by central morphology review, conducted by

recognized experts for each case included in the Düsseldorf MDS Registry and the Spanish MDS

Registry. Further characterization is provided by the structured clinical data set of these registries

and the regular clinical follow-up activities of the registry’s data managers. 6.3. Mutational screening of SF3B1 gene in sideroblastic MDS patients using the Iron panel.

We will use a novel method (Haloplex technology, Agilent) coupled to the next-generation

sequencing platform MiSeq (Illumina) to screen for SF3B1 mutations in 150 MDS patients with

sideroblastic anemia. Illumina’s sequencing by synthesis technology is a widely-adopted next-

generation sequencing platform that is currently being introduced for clinical genetic diagnostics

using fully integrated personal sequencers (MiSeq). For screening our patient samples we will use

the Iron panel, which is currently being set up in the laboratory of Dr. M. Sanchez. This panel will

improve the genetic diagnostics in sideroblastic anemia and may be commercialized for this

purpose. Briefly, a DNA sample is fragmented using restriction enzymes, and denatured. The

probe library (designed by SureDesign) is added and hybridized to the targeted fragments. Each

probe is an oligonucleotide designed to hybridize to both ends of a targeted DNA restriction

fragment, thereby guiding the targeted fragments to form circular DNA molecules. The HaloPlex

probes are biotinylated and the targeted fragments can therefore be retrieved with magnetic

streptavidin beads. The circular molecules are then closed by ligation and targeted fragments are

amplified with PCR. The enriched and barcoded circular DNA targets are then ready for

sequencing. We will multiplex 12-24 samples to be sequenced in 1 run of MiSeq (2x250bp read

length). Custom design for our panel (containing all exons of the 36 genes involved in iron

metabolism diseases) has been supervised by bioinformatics experts in Dr. Sanchez’s team in

addition to being based on the by-default SureDesign online tool provided by Agilent Technologies.

Regions of interest will be defined by chromosomal coordinates. Our 36 gene targetable region has

an approximate size of 208 Kb with a target coverage of 99.5%.

6.4. Cell cultures. Fresh blood and bone marrow CD34+ cells from MDS patients and healthy

controls will be isolated as follows. Briefly, erythrocytes will be removed by ficoll-paque density

gradient centrifugation and CD34+ cells will be isolated by magnetic cell selection using anti-hCD34

magnetic microbeads (Miltenyi) and frozen immediately after purification. Cells are cultured

according to a method developed to study the generation of erythroblasts [38]. The purity of CD34+

separated cells will be assessed by flow cytometry using anti–CD34 monoclonal antibodies (BD

Biosciences). The cultures will be kept at 37 ºC with 5% CO2, 20% O2.

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Iron chelators (DFO, Deferasirox) and iron donors (heme, ferric ammonium citrate) will be used in

established erythroblast/erythroleukemia human cell lines such as TF-1 and HEL 92.1.7 (ATCC) to

perform experiments characterizing new genes involved in MDS pathophysiology.

6.5. Assessment of the IRP/IRE regulatory system and the ISC status. The amount of apoIRP1

and ISC-IRP1 will be determined in native/nondenaturing polyacrilamide gels. IRE binding activities

will be measured by competitive EMSAs using cell extracts and the H-Ferritin IRE probe.

Supershifts will be done to determine the contribution of IRP2 to the band shifted, as IREs

complexed with IRP1 and IRP2 migrate at the same size in humans. H and L ferritins, transferrin

receptor 1, ferroportin and other well characterized IRP targets will be quantified by Western Blot

or ELISA at the protein level and by qRT-PCR at the mRNA level.

Cytosolic and mitochondrial aconitase activity will be measured spectrophotometrically and

normalized on mitochondrial malate dehydrogenase (MDH) activity in erythroid cells from MDS

patients with a sideroblastic phenotype. Other activities of Fe-S cluster proteins, including

succinate dehydrogenase and ferrochelatase, will be also determined.

6.6. Sucrose gradient and mRNA stability experiments. For those mRNAs with a 5´ or a CDS

IRE-like motif the effect of IRP binding on translational repression will be studied by sucrose

gradient centrifugation to obtain polysome and non-polysome fractions with the methodology

described previously[22]. If the IRPs efficiently bind and repress the expression of IRE-containing

mRNAs, the mRNAs should mostly be detected in the non-polysome fraction under iron deprivation

conditions (i.e. when IRPs are actively binding the IREs), and redistributed to the polysome

fractions under high iron conditions. For those mRNAs with a 3´ or a CDS IRE-like motif we will

perform mRNA stability studies using transcriptional blockers such as actinomycin D or DRB [21].

The mRNA decay will be followed in time course experiments under different iron conditions

(control treatment, iron chelation and iron supplementation). In all of these experiments mRNA

levels will be assessed by qPCR.

6.7. Measurement of iron content by atomic emission spectroscopy and further

characterization of the iron deposits. Iron content will be determined in mitochondrial fractions

and cytosolic fractions of erythroid cells by inductively coupled plasma atomic emission

spectroscopy (ICP-AES) in collaboration with the group of Dr. Puerto Morales at Insituto de

Ciencia de Materiales (ICMM-CSIC), Madrid. Briefly, ICP-AES is a very sensitive analytical

technique used to determine the elemental composition of a material. It uses inductively coupled

plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths

characteristic of a particular element that are then captured by a detector. These measurements

will inform about the total amount of iron in the different fractions (mitochondrial or cytosolic) from

the erythroid cells. Not only the quantity of iron but also the form in which iron is present in the

different cell fractions, the iron speciation, is important to be determined to develop disease-

specific treatments. In particular, the composition of mitochondrial iron deposits in the context of

MDS disease has not yet been adequately characterised. We will use electron energy-loss

spectroscopy and energy-filtered transmission electron microscopy to reveal the elemental

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composition of the aggregates observed inside the mitochondria. Results from Mössbauer

spectroscopy will inform us about the crystalline structure of the iron deposits, in particular about

the presence of Fe2+ and Fe3+. Highly specific magnetic measurements, such as AC

susceptibility measurements, will further help to determine the chemical form of these

compounds, allowing the simultaneous identification of ferritin iron and other mineral species.

6.8. RNA isolation and quantitative real-time RT-PCR. RNA isolation will be performed using

TRIzol (LifeTechnologies) or RNA extraction kits (PreAnalitix; Qiagen). DNAseI (Invitrogen) DNAse

treatment will eliminate contaminating genomic DNA. Reverse transcription will be performed using

the Reverse Transcription System (Promega), following the manufacturer’s recommendations.

Quantitative real-time–PCR (qRT-PCR) is based on the SYBR Green PCR Master Mix (Applied

Biosystems) and the Applied Biosystems Model 7900HT Sequence Detection System. Absolute

expression values of each gene will be determined by using the relative standard curve method.

Briefly, a standard curve is established by serial dilutions of a human reference total RNA

(Clontech). The expression level of each gene is calculated by interpolation from the standard

curve. Alternatively, relative gene expression will be calculated by using the 2DeltaCt method, in

which Ct indicates cycle threshold, the fractional cycle number where the fluorescent signal

reaches the detection threshold. Genes will be normalized with at least 2 housekeeping controls.

6.9. Measurements of cytokines/chemokines. Plasma or serum samples will be assayed for

cytokines and chemokines using commercially available enzyme-linked immunosorbent assays

(ELISA) and following the manufacturer’s instructions. The samples will be diluted to optimize each

assay and the assays will be done in triplicate.

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8. Malcovati, L., et al., Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. Blood, 2011. 118(24): p. 6239-46.

9. Visconte, V., et al., SF3B1 mutations are infrequently found in non-myelodysplastic bone marrow failure syndromes and mast cell diseases but, if present, are associated with the ring sideroblast phenotype. Haematologica, 2013. 98(9): p. e105-7.

10. Boultwood, J., et al., The role of the iron transporter ABCB7 in refractory anemia with ring sideroblasts. PLoS One, 2008. 3(4): p. e1970.

11. Gattermann, N., SF3B1 and the riddle of the ring sideroblast. Blood, 2012. 120(16): p. 3167-8.

11

12. Sanchez, M., et al., Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins. Blood, 2011. 118(22): p. e168-79.

13. Muckenthaler, M.U., B. Galy, and M.W. Hentze, Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr, 2008. 28: p. 197-213.

14. Goessling, L.S., D.P. Mascotti, and R.E. Thach, Involvement of heme in the degradation of iron-regulatory protein 2. J Biol Chem, 1998. 273(20): p. 12555-7.

15. Hentze, M.W., et al., Two to tango: regulation of Mammalian iron metabolism. Cell, 2010. 142(1): p. 24-38.

16. Galy, B., et al., Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2). Blood, 2005. 106(7): p. 2580-9.

17. Casarrubea, D., et al., Abnormal body iron distribution and erythropoiesis in a novel mouse model with inducible gain of iron regulatory protein (IRP)-1 function. J Mol Med (Berl), 2013.

18. Camaschella, C., et al., The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic anemia and iron overload. Blood, 2007. 110(4): p. 1353-8.

19. Dandekar, T., et al., Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J, 1991. 10(7): p. 1903-9.

20. Sanchez, M., et al., Identification of target mRNAs of regulatory RNA-binding proteins using mRNP immunopurification and microarrays. Nat Protoc, 2007. 2(8): p. 2033-42.

21. Sanchez, M., et al., Iron regulation and the cell cycle: identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy. J Biol Chem, 2006. 281(32): p. 22865-74.

22. Sanchez, M., et al., Iron-regulatory proteins limit hypoxia-inducible factor-2alpha expression in iron deficiency. Nat Struct Mol Biol, 2007. 14(5): p. 420-6.

23. Sanchez, M., et al., Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by IRPs. Blood, 2011.

24. Mundle, S.D., et al., Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes. Cancer Lett, 1999. 140(1-2): p. 201-7.

25. Zhou, L., et al., Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS. Blood, 2008. 112(8): p. 3434-43.

26. Savic, A., et al., Angiogenesis and survival in patients with myelodysplastic syndrome. Pathol Oncol Res, 2012. 18(3): p. 681-90.

27. Cunha, S.I. and K. Pietras, ALK1 as an emerging target for antiangiogenic therapy of cancer. Blood, 2011. 117(26): p. 6999-7006.

28. Kordasti, S.Y., et al., IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br J Haematol, 2009. 145(1): p. 64-72.

29. Sharma, B., et al., Protein kinase R as mediator of the effects of interferon (IFN) gamma and tumor necrosis factor (TNF) alpha on normal and dysplastic hematopoiesis. J Biol Chem, 2011. 286(31): p. 27506-14.

30. Bachegowda, L., et al., Signal transduction inhibitors in treatment of myelodysplastic syndromes. J Hematol Oncol, 2013. 6: p. 50.

31. Greenberg, P., Treatment of myelodysplastic syndrome with agents interfering with inhibitory cytokines. Ann Rheum Dis, 2001. 60 Suppl 3: p. iii41-2.

32. Gupta, D., et al., Role of plasmapheresis in the management of myeloma kidney: a systematic review. Hemodial Int, 2010. 14(4): p. 355-63.

33. Santini, V., et al., Hepcidin levels and their determinants in different types of myelodysplastic syndromes. PLoS One, 2011. 6(8): p. e23109.

34. Zipperer, E., et al., Serum hepcidin measured with an improved ELISA correlates with parameters of iron metabolism in patients with myelodysplastic syndrome. Ann Hematol, 2013.

35. Bjorkman, S.E., Chronic refractory anemia with sideroblastic bone marrow; a study of four cases. Blood, 1956. 11(3): p. 250-9.

36. Cotter, P.D., et al., Late-onset X-linked sideroblastic anemia. Missense mutations in the erythroid delta-aminolevulinate synthase (ALAS2) gene in two pyridoxine-responsive patients initially diagnosed with acquired refractory anemia and ringed sideroblasts. J Clin Invest, 1995. 96(4): p. 2090-6.

37. Campillos, M., et al., SIREs: searching for iron-responsive elements. Nucleic Acids Res, 2010. 38 Suppl: p. W360-7.

38. Tehranchi, R., et al., Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors. Blood, 2003. 101(3): p. 1080-6.

12

Research Plan

Team 1: Group of the Prof. Dr. Norbert Gattermann, Heinrich-Heine-University Dusseldorf, Germany Team 2: Group of the Dr. Mayka Sanchez, Josep Carreras Leukaemia Research Institute (IJC), Barcelona, Spain. Collaborators: MDS registries: Düsseldorf MDS Registry at Universitätsklinikum Düsseldorf (UKD), GESMD: Spanish Group of MDS (patient registry), HGTiP: Hospital German Trias i Pujol. Team 3: group of Dr. Puerto Morales (ICMM-CSIC, Madrid). TC= tissue culture experiments, 7. Ethical issues and data protection

The research proposal will be submitted to the Ethics Committee of the Medical Faculty of

Heinrich-Heine-University Düsseldorf and to the Ethics Committee of Hospital Germans Trias i

Pujol, Barcelona, Spain. We will follow the following German, Spanish and EU legislation

concerning management of patients’ samples and data protection:

- Helsinki Declaration for ethical principles regarding human experimentation

- EU Data Protection Directive 95/46/EC

- EU proposal for a Data Protection Regulation (GDPR, COM (2012) 11 final)

- Spanish Data protection law: “La Ley Orgánica 15/1999 de 13 de diciembre de Protección de

Datos de Carácter Personal, (LOPD)”

- Bundesdatenschutzgesetz (BDSG); Landesdatenschutzgesetz NRW (LDSG-NRW)

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8. Beantragte Mittel

8.1. Gesamtkosten des Vorhabens

8.1.1. Personalmittel: 700.000 €

8.1.2. Investitionsmittel: 1.000.000 €

8.1.3. Verbrauchsmaterial: 150.000 €

8.1.4. Gesamtkosten: 1.850.000 €

8.2. Beantragte Mittel:

8.2.1. Personalmittel: 241.377 €

- Gehalt eines Postdoktoranden: 112.377 €

- Gehalt 2 Doktoranden: 48.000+81.000 = 129.000 €

8.2.2. Investitionsmittel: werden nicht beantragt, da Geräte bereits vorhanden sind (siehe 9.1)

8.2.3. Verbrauchsmaterial 106.000 €

- SF3B1-Mutationstest sowie Mutationsanalyse anderer relevanter Gene („Eisen-Panel“): 52.500 €

- Kulturmedien, Zytokine, Antikörper, Kulturgefäße: 15.000 €

- Intrazelluläre Eisenmessungen: 13.500 €

- Quantitative RT-PCR; Analysen des IRP/IRE-System und der ISC-Biosynthese: 15,000 €

- Zytokin-Assays, ELISAs, Messung von Enzymaktivitäten und sonstige Analysen: 10,000 €

8.2.4. Total: 427.433 € (einschließlich Overhead) bzw. 347.377 € (ohne Overhead)

8.3. Kostenübernahme durch Institutionen der Antragsteller oder andere Drittmittel

8.3.1. Personalmittel: ca. 459.000 €

Düsseldorf: - Referenz-Zytomorphologie durch Prof. Germing und/oder Prof. Gattermann

- Patientenrekrutierung durch Arzt der hämatologisch-onkologischen Ambulanz

Barcelona: - Referenz-Zytomorphologie durch Dr. B. Xicoy and Dr. F. Milla.

- Dr. Sanchez (Arztgehalt)

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- MTA am Institut der Antragstellerin in Barcelona wird vom IJC finanziert.

- Patientenrekrutierung durch Arzt der hämatologisch-onkologischen Ambulanz

8.3.2. Investitionsmittel: Die Investitionen, die in den beteiligten Instituten bereits für die Geräte

getätigt wurden, die bei dem geplanten Projekt genutzt werden, belaufen sich auf ca. 1.000.000 €.

8.3.3. Verbrauchsmaterial: Probentransport zwischen den Krankenhäusern, Publikationskosten,

Versenden der molekulargenetischen Diagnoseberichte, Basismaterialien (Puffer, milliQ-Wasser):

ca. 44.000.

8.3.4. Gesamtbetrag: ca. 1.500.000

9. Research Facilities and Resources

9.1. Available Resources

All resources required to achieve the objectives of the proposed project are available in the

institutes participating in this study. The required technologies have been set up and all mentioned

infrastructures are established. Specifically, the participating institutions have state-of-the-art

facilities and technologies appropriate for cell biology, genomics, molecular biology, and material

sciences. These include: cell culture rooms, microbiology laboratories, licensed radioactivity

installations, cold rooms, cryopreservation rooms, freezer rooms, microscopy rooms (bright field,

dark field, phase contrast, fluorescent, confocal), dark rooms for Western blot detection, animal

facilities, conventional PCR instruments, real-time PCR instruments, capillary Sanger sequencing

and MiSeq platform, electrophoresis areas and photodocumentation instruments, spectrophoto-

meters (including ICP atomic emission spectrometer), transmission electron microscopes, vibrating

sample magnetometer, freeze-dryers, fume hoods, plate readers (spectrophotometers, fluorimeters

and luminometers), sonicators, homogenizers, tissue disruptors, electroporation systems,

crosslinking instruments, bench-top, high speed and ultracentrifuges, bioanalyzers, microvesicle

generation system, mass spectrometry platform. In addition, the main two participant groups are part of their respective national registries for

samples of MDS patients and are recognized experts in the iron metabolism and/or MDS field.

9.2. Primary Institution/Department

University Hospital (UKD) of Heinrich-Heine-University (HHU), Düsseldorf, Germany The UKD (Universitätsklinikum Düsseldorf) provides 1,100 beds and admits 42,000 patients per

year, with a proportion of approx. 20% cancer patients. In the outpatient clinics, about 140,000

individual patients are seen per year, including a proportion of about 10% patients with cancer.

Both the UKD and the Medical Faculty of HHU are strongly committed to the University Cancer

Center (Universitätstumorzentrum, UTZ), which has recently been included by the Deutsche

Krebshilfe (German Cancer Aid) in their Program for the Development of 12 Interdisciplinary

Oncology Centers of Excellence in Germany. An important component of the UTZ is the Depart-

ment of Hematology, Oncology and Clinical Immunology, which performs ca. 200 hematopoietic

stem cell transplantations (HSCT) per year, approx. 40% allogeneic. HSCT activities in adult and

pediatric hematology/ oncology are supported by the José Carreras Cord Blood Bank, which keeps

15

about 24.000 CB units and has provided material for more than 1000 transplantations worldwide.

The main clinical research interests in adult hematology/oncology in Düsseldorf are multiple

myeloma and MDS. The site runs numerous clinical trials for MDS patients and is certified as ’MDS

Center of Excellence’ by the MDS Foundation. Düsseldorf plays an important part in the German-

Austrian-Swiss (D-A-CH) MDS Working Group, and two pivotal projects, i.e. Central Registry (U.

Germing) and Central Biobanking (N. Gattermann) of the German MDS collaborative research

project “Myelodysplastic syndromes as an age-related clonal disorder of the hematopoietic stem

cell”, funded by Deutsche Krebshilfe, are located in Düsseldorf.

9.3. Other Participant Institutions

The Josep Carreras Leukaemia Research Institute (IJC)

IJC is a newly established Institute of Research (December 2009). This is a public entity of the

Generalitat of Catalunya/Spain, with a significant participation of a private foundation, the Josep

Carreras Foundation. The main objective of the IJC is to promote translational research and

applied basic research in the field of hematological malignancies. The IJC has three physical

campuses, the Campus Clinic, Campus Germans Trias i Pujol, and Campus Sant Pau. In these

campuses hematologists of national and international prestige are involved in healthcare and

science. Thus, each year approximately 4,200 new patients are seen and over 27,000

chemotherapy sessions are administered, and more than 250 bone marrow transplantations are

performed. In 2011, 210 scientific articles indexed were published with a total impact factor of

1050. At present, the IJC has 500 square meters of research laboratory, and a new building of

more than 3,000 square meters is under construction. The three campuses are equipped with

modern laboratory equipment and support platforms for research: genomics, transcriptomics,

proteomics, bioinformatics, cytometry, biobank, cryopreservation, and tissue culture rooms. The

IJC pursues competitive research, benchmarked for excellence, and fosters postgraduate teaching

of high quality. Main research objectives of the Campus Germans Trias i Pujol of IJC are to

understand the origin and development of leukemia and other hematological malignancies,

including MDS, and to develop more accurate and less toxic treatments for these diseases.

Specific objectives of the Campus Germans Trias i Pujol of IJC in relation with the proposal:

1. - Biologic characterization of MDS and CMML (Coordinator Dr. Francesc Sole)

2. - Identification of specific therapeutic targets against MDS (Coordinator Dr. Francesc Sole)

Ongoing clinical trials in the Campus Germans Trias i Pujol of IJC related to the proposal:

- Observational study of patients treated with Exjade (an iron chelator) (CICL670A2301, Novartis).

PI: Blanca Xicoy, ICO-HGTP

- European Register of newly diagnosed MDS patients with low or intermediate-1 risk score

(EUMDS). PI: Blanca Xicoy, ICO-HGTP

- Patients diagnosed with MDS 5q- without transfusion needs (SINTRA-REV, Salamanca). PI:

Blanca Xicoy, ICO-HGTP

Dr. Blanca Xicoy is currently spending a research stay in the Heinrich-Heine-University Dusseldorf,

Germany, with Dr. Ulrich Germing and Dr. Norbert Gattermann.

16

9.4. Third-party Resources

Iron measurements and iron speciation using special techniques such as inductively coupled

plasma atomic emission spectroscopy (ICP-AES), electron energy-loss spectroscopy,

energy-filtered transmission electron microscopy, and Mössbauer spectroscopy will be done

in collaboration with the group of Dr. Puerto Morales at Insituto de Ciencia de Materiales (ICMM-

CSIC), Madrid. This group has expertise in the use of these techniques and their application to

biological samples, with important publications: 1. Whitnall M, Suryo Rahmanto Y, Huang ML, et al. Identification of nonferritin mitochondrial iron deposits in

a mouse model of Friedreich ataxia. Proc Natl Acad Sci USA. 2012 Dec 11;109(50):20590-5.

2. Mejías R, Pérez-Yagüe S, Gutiérrez L, et al., Dimercaptosuccinic acid-coated magnetite nanoparticles for

magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy. Biomaterials. 2011

Apr;32(11):2938-52.

3. Cohen LA, Gutierrez L, Weiss A, et al., Serum ferritin is derived primarily from macrophages through a

nonclassical secretory pathway. Blood. 2010 Sep 2;116(9):1574-84.

4. Gutiérrez L, Lázaro FJ, Abadía AR, et al. Bioinorganic transformations of liver iron deposits observed by

tissue magnetic characterisation in a rat model. J Inorg Biochem. 2006 Nov;100(11):1790-9.

10. Declaration

„Ein Antrag auf Finanzierung dieses Vorhabens wurde bei keiner anderen Stelle eingereicht.

Sollten wir einen solchen Antrag stellen, werden wir die Deutsche José Carreras Leukämie-

Stiftung e.V. unverzüglich benachrichtigen. Die Deutsche José Carreras Leukämie-Stiftung e.V. ist

unabhängig davon berechtigt, bei anderen Fördereinrichtungen anzufragen, ob bereits Förder-

anträge mit ähnlicher oder gleicher Themenstellung vorgelegt wurden.“

„Wir versichern hiermit, dass alle Angaben zu eigenen und fremden Vorarbeiten, zum Arbeits-

programm, zu Kooperationen, zu laufenden Drittmitteln und zu allen anderen für das Vorhaben und

dessen Begutachtung wesentlichen Tatsachen korrekt und nach bestem Wissen und Gewissen

gemacht wurden.“

11. Signatures

Düsseldorf/Barcelona,

October 25th, 2013

Dr. Norbert Gattermann Dr. Mayka Sanchez

17

12. Annexes

12.1. Currículum vitae of all applicants21

Prof. Dr. Norbert Gattermann Department of Hematology, Oncology, and Clinical Immunology and University Cancer Center (Universitätstumorzentrum, UTZ) Heinrich-Heine-University Düsseldorf. Moorenstr. 5, D-40225 Düsseldorf, Germany. Tel.: +49-211-81-16500, Fax: +49-211-81-18853. e-mail: gattermann@med.uni-duesseldorf.de Qualifications and Employment 1978 -1985 Medical School (Düsseldorf [Germany], London [UK], Boston [USA]) as follows: 1978 -1981 Medical School at Unversity of Düsseldorf, Düsseldorf (Germany) 1981 -1982 Studies at Royal Free Hospital School of Medicine, London (U.K.) 1983 Clinical courses at Massachusetts General Hospital, Harvard Medical School, Boston (USA) 1984 -1985 Medical School at Unversity of Düsseldorf, Düsseldorf (Germany) 1979 -1985 Fellow of "Studienstiftung des Deutschen Volkes" (German National Scholarship Foundation) 12/1985 Approbation as physician (Der Regierungspräsident, Düsseldorf) 1986-1998 Resident at the Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine University Düsseldorf 1989 Doctoral thesis "Two types of acquired idiopathic sideroblastic anemia“ (summa cum laude) 04/87-03/95 Rotational training at Düsseldorf University: Cardiology, Gastroenterology, Intensive Care, Ultrasound, Diagnostic Radiology 04/93 Board Certification Internal Medicine by Ärztekammer Nordrhein, Düsseldorf 1997 Habilitation/Lecturer in Internal Medicine at Düsseldorf University 1997 Senior Lecturer; Dept. of Hematology, Oncology and Clinical Immunology; 08/1998 Board Certification Hematology/Medical Oncology 1999 Assistant Professor, Heinrich-Heine-University Düsseldorf since 12/2002 Professor of Internal Medicine 2000-2008 Vice Head, Dept. of Hematology, Oncology and Clinical Immunology; Heinrich-Heine-University Düsseldorf (Head: Prof. Dr. Rainer Haas) since 2007 Managing Director, University Cancer Center Memberships Association of German Internists (BDI), German Society of Hematology and Oncology (DGHO), American Society of Hematology (ASH), ELN European Leukemia Net, MDS Foundation, German-Austrian-Swiss (D-A-CH) MDS Study Group ASH Scientific Committee for Iron and Heme (since 2012) Research Fields Epidemiology, pathogenesis and treatment of myelodysplastic syndromes Mitochondrial DNA mutations. Iron overload. Treatment of chronic myeloid leukemia Current funding: Leader of subproject B (central biobanking) of the German MDS collaborative research project “Myelodysplastic syndromes as an age-related clonal disorder of the hematopoietic stem cell”, funded by Deutsche Krebshilfe. 378.000,- € over 3 years (2013-2016) Referee for scientific journals: Blood, J Clin Oncol, Leukemia, Haematologica, Leuk Res, Ann Hematol Publications 180 peer-reviewed publications (36 as first author; 31 as last author). Average citations per article: 28; H-index: 40 12 selected references 1. Malcovati L, Hellström-Lindberg E, Bowen D, Adès L, Cermak J, Del Cañizo C, Della Porta MG, Fenaux P, Gattermann N, Germing U, Jansen JH, Mittelman M, Mufti G, Platzbecker U, Sanz GF, Selleslag D, Skov-Holm M, Stauder R, Symeonidis A, van de Loosdrecht AA, de Witte T, Cazzola M. Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet. Blood. 2013 Aug 26. [Epub ahead of print] 2. Gattermann N, Finelli C, Della Porta M, Fenaux P, Stadler M, Guerci-Bresler A, Schmid M, Taylor K, Vassilieff D, Habr D, Marcellari A, Roubert B, Rose C. Hematologic responses to deferasirox therapy in transfusion-dependent patients with myelodysplastic syndromes. Haematologica. 2012;97:1364-71

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3. Neukirchen J, Fox F, Kündgen A, Nachtkamp K, Strupp C, Haas R, Germing U, Gattermann N. Improved survival in MDS patients receiving iron chelation therapy - a matched pair analysis of 188 patients from the Düsseldorf MDS registry. Leuk Res. 2012;36:1067-70 4. Schildgen V, Wulfert M, Gattermann N. Impaired mitochondrial gene transcription in myelodysplastic syndrome and acute myeloid leukemia with myelodysplasia-related changes. Exp Hematol 2011;39:666-675 5. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A, Schoch R, Gattermann N, Sanz G, List A, Gore SD, Seymour JF, Bennett JM, Byrd J, Backstrom J, Zimmerman L, McKenzie D, Beach C, Silverman LR; International Vidaza High-Risk MDS Survival Study Group. Efficacy of azacitidine compared with that of conventional care regimens in the treatment of high-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 2009; 10:223-32 6. Wulfert M, Küpper AC, Tapprich C, Bottomley SS, Bowen, Germing U, Haas R, Gattermann N. Analysis of mitochondrial DNA in 104 patients with myelodysplastic syndromes. Exp Hematol 2008;36:577-586 7. Gattermann N, Billiet J, Kronenwett R, Zipperer E, Germing U, Nollet F, Criel A, Selleslag D. High frequency of the JAK2 V617F mutation in patients with thrombocytosis (platelet count>600x109/L) and ringed sideroblasts more than 15% considered as MDS/MPD, unclassifiable. Blood 2007; 109:1334-5 8. Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A, Powell BL, Gabrilow JL, Rousselot P, reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson B, Taylor K, Baccarani M, So C, Letvak L, Larson RA, IRIS Investigators. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355:2408-17 9. Kuendgen A, Strupp C, Aivado M, Bernhardt A, Hildebrandt B, Haas R, Germing U, Gattermann N: Treatment of myelodysplastic syndromes with valproic acid alone or in combinatin with all-trans retinoic acid. Blood 2004; 104:1266-1269 10. Gattermann N, Wulfert M, Junge B, Germing U, Haas R, Hofhaus G.: Ineffective hematopoiesis linked with a mitochondrial tRNA mutation (G3242A) in a patient with myelodysplastic syndrome. Blood 2004; 103:1499-1502 11. Aivado M, Gattermann N, Bottomley S: X chromosome inactivation ratios in female carriers of X-linked sideroblastic anemia. Blood 2001; 97:4000-4002 12. Gattermann N, Retzlaff S, Wang Y-L, Hofhaus G, Heinisch J, Aul C, Schneider W: Heteroplasmic point mutations of mitochondrial DNA affecting subunit I of cytochrome-c-oxidase (COX I) in two patients with acquired idiopathic sideroblastic anemia. Blood 1997; 90: 4961-4972 Dr. Mayka (Maria Carmen) Sanchez Fernandez Date of Birth: 21/10/1974 (38 years). Nationality: Spanish Married, 1 child born (2012) Qualifications 1996 Degree in Biochemistry (University Barcelona) 2002 PhD in Biology-Genetics (University Barcelona and Hospital Clinic of Barcelona) Positions and Employment 2002-2009 Staff and postdoctoral researcher in Heidelberg, Germany (EMBL, University of Heidelberg, MMPU) Nov 2009 Junior Group Leader at IMPPC, Badalona, Barcelona, Spain Jan 2010-now Head of the Advanced Genetic Diagnostics for Rare Iron Metabolism Disorders – UDGAEMH-IMPPC, Badalona, Barcelona, Spain Sept 2013-now Group Leader at Josep Carreras Leukaemia Research Institute (IJC), Badalona,

Barcelona, Spain. Publications Total peer-reviewed publication: 24 (12 as 1st author, 2 as last author) (8 as independent group, 8 as postdoc, 8 as predoc). Publications as last author: 2 Number of publications in the 1st quartil (Q1): 16/24 (66,6%) Total citations: 492 (JCR-WoK, Oct 2013). Sum of Impact factors: 135.57. H-index: 10 Average citations per Article: 22.36 Other publications: Chapter of books: 4. Divulgative medical publications: 6 Honors April 2009 Josep Maria Sala Trepat Prize for outstanding young researchers by the Catalan Society of Biology. The prize is awarded for outstanding research on gene expression that has been mainly carried out abroad the country of origin. Sept 2006 2nd Prize for the best Oral presentation. The European Iron Club (EIC) Annual Meeting. Barcelona, Spain. May 2005 Antoni Caparrós Award by the Social Council of the University of Barcelona, 31 May 2005 Best thesis work that makes a significant contribution to the transfer of knowledge to society.

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For the work entitled “Screening of C282Y and H63D mutations of the hemochromatosis gene (HFE) in 5,370 blood donors from the Spanish population” Research Funds as Co-applicant or Principal Investigator (since Nov 2009 with my independent group) 2013-2016 e-ENERCA network. New e-Health Services for the European Reference Network on Rare Anaemias. Coordinator: Dr. JL Vives-Corrons 2009-2014 Ramón y Cajal 5-year re-integration contract. Amount: 192.480 €. 2012-2015 Ramón Areces Private Foundation grant CIVP16A1857. Amount: 101.725 € 2013-2016 Spanish National grant SAF2012-40106. Amount: 117.000 € 2010-2015 CONSOLIDER-INGENIO National Spanish grant. Junior partner. PI: Dr. J. Varcarcel (CRG). An integrated approach to post-transcriptional regulation of gene expression and its role in human disease. Amount: 4.700.000 €. 2009-2012 ERARE grant HMA-IRON. EU.Co-applicant. Coordinator: Dr. C Beaumont (INSERM France). Amount: 510.613 €. 2009-2012 Spanish National grant FIS PS09/00341. Molecular studies in rare genetic iron diseases: congenital anaemias and non-HFE Hereditary Hemochromatosis. Amount: 78.335,40 € 2 contracts for other members of my lab: Technician’s Carlos III contract and FEBS postdoctoral fellowship. Participation in Clinical Trials: ClinicalTrials.gov Identifier: NCT01797055 (Apotransferrin in Atransferrinemia) Sanquin Blood Supply Summary Congress participation/organization: Total events (period 1998-2012): 37. International events: 26/37. Invited speaker: 14. Co-chairwoman: 3. Congress Organizer: 3. Oral presentation: 11. Posters: 22 Elected member of Scientific /Executive Board Committees 2010-now Spanish ORPHANET Scientific Committee in haematology and paediatric haematology. Expert collaborator (reviewer of IRIDA and CDA rare diseases) 2008-now European Federation of Associations of Patients with Hemochromatosis (EFAPH). 2006-now Spanish Hemochromatosis Association (AEH) Referee for Scientific journals Haematologica, PLoS One, Human Mutation, Human Molecular Genetics and RNA Biology 12 selected publications 1. Athiyarath R, Arora N, Fuster F, Schwarzenbacher R, Ahmed R, George B, Chandy M, Srivastava A, Rojas AM, Sánchez M, Edison ES. Two Novel Missense Mutations in Iron Transport Protein Transferrin Causing Hypochromic Microcytic Anaemia and Haemosiderosis: molecular characterisation and structural implications. Brit J Haematol 2013. Accepted. IF: 4.941 (2011 JCR). Q1D1 2. De Falco L*, Sánchez M*, Silvestri L, Kannengiesser C, Muckenthaler MU, Iolascon A, Gouya L, Camaschella C, Beaumont C. Iron Refractory Iron Deficiency Anemia. Haematologica 2013 Jun;98(6):845-53. IF: 6.424 (2011 JCR). *equal contributor authors.Q1D1 3. Luscieti S, Tolle G, Aranda J, Benet-Campos C, Risse F, Morán M, Muckenthaler MU, Sánchez M. Novel mutations in the Ferritin-L iron-responsive element that only mildly impair IRP binding cause Hereditary Hyperferritinaemia Cataract Syndrome. Orphanet Journal of Rare Diseases 2013. Feb 19;8(1):30. IF: 5.074 (2011 JCR). Q1D1 4. Sánchez M, Galy B, Schwanhaeusser B, Blake J, Bähr-Ivacevic T, Benes V, Selbach M, Muckenthaler MU andHentze MW. Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by IRPs. Blood 2011 Nov 24;118(22):e168-79. Epub 2011 Sep22. IF: 9.898. Cites: 10 (Oct 2013 WoK) Q1D1 5. Kannengiesser C*, Sanchez M*, Sweeney M*, Hetet G, Kerr B, Moran E, Fuster-Soler JL, Maloum K, Matthes T, Oudot C, Lascaux A, Pondarré C, Sevilla-Navarro J, Vidyatilake S, Beaumont C, Grandchamp B, May A. Missense SLC25A38 gene variations play an important role in autosomal recessive inherited sideroblastic anaemia.*equal contributor authors. Haematologica. 2011 Jun;96(6):808-13. Epub 2011 Mar 10. PubMed PMID: 21393332. IF: 6.424. Cites: 6 (Oct 2013 WoK). 6. Campillos M, Cases I, Hentze MW, Sánchez M. SIREs: Searching for Iron-Responsive Elements. Nucleic Acids Research 2010 Jul 1;38 (Web Server Issue)W360-7. Epub 2010 May 11. PubMed PMID: 20460462. IF: 7.836. Cites: 11 (Sep 2013 WoK). Q1 7. Ramsay AJ, Quesada V, Sánchez M, Garabaya C, Sardà MP, Baiget M, Remacha A, Velasco G, López-Otín C. Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms. Human Molecular Genetics. 2009 Oct 1;18(19):3673-83. Epub 2009 Jul 10. PubMed PMID: 19592582. IF: 7.386. Cites: 34 (Oct 2013 WoK) Q1D1 8. Sánchez M, Galy B, Hentze MW., and Muckenthaler MU. Identification of target mRNAs of regulatory RNA binding proteins using mRNP immunopurification and microarrays. Nature Protocols 2007, 2(8):2033-2042. IF: 1.671. Cites: 7 (Oct 2013 WoK) Q1D1

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9. Percy MJ*, Sánchez M*, Swierczek S*, McMullin MF, Mojica-Henshaw MP, Martina U Muckenthaler MU, Prchal JT, and Hentze MW. Is congenital Secondary Erythrocytosis/Polycythemia caused by activating mutations within the HIF-2α iron responsive element? Blood 2007; 110(7):2776-2777. * equal contribution authors. PubMed PMID: 17881647. IF: 10.896. Cites: 1 (Oct 2013 WoK) Q1D1 10. Sánchez M, Galy B, Muckenthaler MU, and Hentze MW. Iron-regulatory proteins limit hypoxia-inducible factor 2α expression in iron deficiency. Nature Structural & Molecular Biology. 2007;14(5):420-426. PubMed PMID: 17417656. IF : 11.085. Cites: 89 (Oct 2013 WoK) Q1D1 11. Sánchez M, Galy B, Dandekar T, Bengert P, Vainshtein Y, Stolte J, Muckenthaler MU, and Hentze MW. Iron regulation and the cell cycle: Identification of an Iron-Responsive Element in the 3’unstranslated region of human CDC14A mRNA by a refined microarray-based screening strategy. Journal of Biological Chemistry 2006; 281(32):22865-74. PubMed PMID: 16760464. IF: 5.808. Cites: 46 (Jan 2013 WoK) Q1 12. Roy CN , Custodio AO, de Graaf J, Schneider S, Akpan I, Montross LK, Sánchez M, Gaudino A, Hentze MW, Andrews NC and Muckenthaler M. An Hfe-dependent pathway mediates hyposideremia in response to lipopolysaccharide-induced inflammation in mice. Nature Genetics 2004 ;36(5):481-5. IF: 24.695. Cites: 78 (Oct 2013 WoK) Q1D1

12.2. Collaboration Statement.

By signing this document we formalize our agreement to collaborate in the project proposed here

and commit to develop the objectives in the timeline outlined, provided funds are granted.

Dr. Norbert Gattermann Dr. Mayka Sanchez

12.3. Assessment by the Institute Directors22

Please see attached documents.

12.4. Ethical Issues. As detailed above.

12.5. Lay Abstract. See below, in German

12.6. Bestätigung der Verwaltung über die Rechtsform der Institution1

The Josep Carreras Leukaemia Research Institute (IJC) and the Univerisitätsklinikum Düsseldorf

(UKD) are providing separate letters informing about this.

Please see attached documents.

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Allgemein verständliche Zusammenfassung

Projekt: Molekularer Mechanismus der mitochondrialen Eisenüberladung, die bei Patienten mit myelodysplastischen Syndromen (MDS) das Phänomen der sideroblastischen Anämie (refraktäre Anämie mit Ringsideroblasten, RARS) hervorruft. Kurzbezeichnung: Mechanismus der sideroblastischen Anämie bei MDS

Institute / Abteilungen: Klinik für Hämatologie, Onkologie & Klinische Imunologie, Heinrich-Heine-Universität Düsseldorf, Moorenstr. 5, 40225 Düsseldorf

Institute of Predictive and Personalized Medicine of Cancer (IMPPC) Crta Can Ruti, Camí de les Escoles s/n 08916 Badalona, Barcelona. Spain Schwerpunkt: Klinisch orientierte Grundlagenforschung

Inhalt des Antrags:

Myelodysplastische Syndrome sind präleukämische Erkrankungen des Knochenmarks, die vor

allem bei älteren Menschen auftreten und zu Transfusionsabhängigkeit und Abwehrschwäche

führen. Bei einem Teil der Patienten finden sich im Knochenmark sogenannte Ringsideroblasten.

Dies sind pathologische Vorläuferzellen der roten Blutkörperchen, die in ihren Mitochondrien große

Mengen an Eisen anhäufen, anstatt es in Häm, den zentralen Bestandteil des roten Blutfarbstoffs

Hämoglobin, einzubauen. Die Formen der Blutarmut, die mit mitochondrialer Eisenüberladung

einhergehen, werden als sideroblastische Anämien bezeichnet. Es gibt angeborene und

erworbene sideroblastische Anämien (SA). Vor zwei Jahren wurden bei etwa 75% der MDS-

Patienten mit SA erworbene Mutationen im SF3B1-Gen entdeckt. Wie diese Mutationen eine

mitochondriale Eisenüberladung bewirken, ist noch unklar.

Da das SF3B1-Protein Bestandteil des zellulären Spleiß-Apparates ist, der neu synthetisierte RNA

in funktionierende Messenger-RNA (mRNA) zurechtspleißt, wird vermutet, daß falsch gespleißte

mRNA-Moleküle die Eisenregulation durcheinanderbringen. Dr. Sanchez hat vor kurzem mehr als

200 neue mRNA-Moleküle identifiziert, die mit IRP1 und IRP2, also den Regulator-Proteinen des

zellulären Eisenstoffwechsels, reagieren und daher wohl ebenfalls an dessen Steuerung beteiligt

sind. Falls eine oder mehrere dieser mRNAs infolge von SF3B1-Mutationen falsch gespleißt wird,

könnte dies zur Fehlregulation eines Stoffwechselweges und damit zur mitochondrialen Eisen-

akkumulation bei SA führen (siehe auch beigefügte Abbildung).

Im beantragten Forschungsprojekt sollen die neu entdeckten eisenregulierenden mRNAs bei MDS

mit sideroblastischer Anämie genauer untersucht werden. Im Vergleich mit normalen Kontrollen

sollen nicht nur Veränderungen der Genexpression, sondern auch die Auswirkungen auf das

IRP/IRE-Eisenregulationssystem, die Eisen-Schwefel-Cluster-Biosynthese und die Verteilung

verschiedener intrazellulärer Eisenspezies analysiert werden. Ein besseres pathogenetisches

Verständnis der sideroblastischen Störung ist Voraussetzung für neue Therapieansätze zur

Behebung der chronischen Anämie und Vermeidung der transfusionbedingten Eisenüberladung.

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Maßgebliche Mitarbeiter:

Prof. Dr. Norbert Gattermann

Klinik für Hämatologie, Onkologie u. Klinische Immunologie,

Geschäftsführender Leiter des Universitätstumorzentrums

Dr. Mayka Sanchez

Principal Investigator, Iron and Cancer Group, Josep Carreras Leukaemia Research Institute (IJC)

Head of the Advanced Genetic Diagnostic Unit of Rare Iron Disorders (UDGAEMH-IJC)

Ort: Düsseldorf und Barcelona

Dauer der Förderung: 01.06.2014-31.06.2017

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ANNEX 1

N. Gattermann. SF3B1 and the riddle of the ring sideroblast. Blood 2012;120:3167-8