WO2018185341A1

WO2018185341A1 - Regulator of bmp-smad signaling and uses thereof

Info

Publication number: WO2018185341A1
Application number: PCT/EP2018/059040
Authority: WO
Inventors: Laura SILVESTRI; Clara Camaschella; Antonella NAI; Silvia COLUCCI; Alessia PAGANI
Original assignee: Ospedale San Raffaele S.R.L.; Fondazione Centro San Raffaele
Priority date: 2017-04-07
Filing date: 2018-04-09
Publication date: 2018-10-11

Abstract

The present invention relates to a regulator of the BMP-SMAD signaling for use in the treatment and/or prevention of a disease selected from the group consisting of: a disease characterized by ineffective erythropoiesis, anemia, a disease characterized by low hepcidin and by iron overload or a combination thereof. Preferably said regulator is an inhibitor of erythroid TFR2 or an inhibitor of hepatocyte FKBP12 or a combination thereof. The present invention also refers to relative pharmaceutical compositions and uses thereof.

Description

Regulator of BMP-SMAD signaling and uses thereof

Technical field

Background art

Beta thalassemias are autosomal recessive disorders caused by mutations in the β-globin gene or its regulatory elements, resulting in reduced or even absent β-globin chain synthesis. The unbalanced synthesis between normal a- and reduced/absent β-globin chains is the pathogenic clue of the disease and causes massive expansion of the erythroid marrow. Ineffective erythropoiesis, extramedullary hematopoiesis, anemia and iron overload are the hallmark of β-thalassemia. The balance between erythroid progenitors' production, differentiation and apoptosis is undermined: the early erythroid cell pool is abnormally expanded while maturation of late erythroblasts is limited (Libani et al., 2008) due to increased apoptosis (Mathias et a., 2000).

Erythropoiesis is governed by erythropoietin (EPO), a hormone primarily produced by the kidney in adulthood and whose levels, stimulated by hypoxia, are high in β-thalassemia. EPO binds to its receptor (EPOR) on the surface of erythroid cells, activating the JAK2/STAT5 signaling pathway and the transcription of several genes involved in proliferation, differentiation, and survival of erythroid progenitors (Silva et al., 1999) (Socolovsky et al. 1999) (Kashii et al., 2000) (Digicaylioglu et al., 2000) (Socolovsky et al., 2001 ) (Liu et al., 2006) (Singh et al., 2012). However, due to the globin chain defect, erythroid differentiation is blocked in thalassemic marrow and excess EPO stimulation results in ineffective erythropoiesis (IE) and other complications including secondary iron overload. Despite the central role of EPO, other players are involved in the pathological events that sustain IE in thalassemia.

A fundamental control of erythropoiesis is exerted by iron, whose metabolism is dys regulated in thalassemia. Iron is essential for heme/hemoglobin production and iron and erythropoiesis are reciprocally regulated. Indeed, erythropoiesis controls iron homeostasis regulating the transcription of hepcidin (HAMP), the master regulator of iron metabolism. To signal iron needs to the liver, erythroid cells release in the circulation erythroferrone (ERFE), an EPO target gene, member of the C1 q-TNF protein family, that inhibits HAMP through a still unknown mechanism (Kautz et al., 2014). ERFE contributes to iron loading in β-thalassemia (Kautz et al., 2015). Viceversa, iron exerts a regulatory role on erythroid development: indeed iron-deficiency induces the production of EPO. Moreover reduction of iron supply to the marrow by decrease of the circulating iron carrier transferrin (TF) (Raja et al., 1999) or inactivation of the expression of the iron importer Transferrin receptor 1 (Levy et al., 1999) induces anemia, even severe.

In this context of mutual control of iron and erythropoiesis the inventors recently identified a role for Transferrin Receptor 2 (TFR2), a sensor of circulating TF-bound iron. TFR2 is a transmembrane protein, mainly expressed in the liver and in the erythroid compartment that is mutated in hemochromatosis type 3 (Camaschella et al., 2000). While hepatic TFR2 induces the transcription of hepcidin (HAMP) in response to increased transferrin saturation, through still unclear molecular mechanisms (Nemeth et al., 2005) (Wallace et al., 2009), erythroid TFR2 associates with the erythropoietin receptor (EPOR) in the endoplasmic reticulum and is required for the efficient transport of EPOR to the erythroid precursors surface (Forejnikova et al., 2010). In both cells TFR2 is stabilized on cell surface by diferric TF (Johnson et al., 2007) (Pagani et al., 2015), in this way sensing the amount of available iron and adjusting hepcidin levels and thus iron availability to erythropoiesis needs, through the induction of the erythroid regulator Erfe (Nai et al., 2015). Inventors' analysis of the role of TFR2 in erythropoiesis started from an intriguing finding that mice knock-out for Tfr2 when in iron deficiency develop erythrocytosis (Nai et al., 2014). The inventors hypothesized that this abnormality was accounted for by the loss of the erythroid Tfr2 in Tfr2 ^~'^~ animals. To test this hypothesis the inventors subsequently generated mice with specific genetic inactivation of Tfr2 in the bone marrow (BM, Tfr2^BMKO mice) showing that the lack of Tfr2 in erythroid cells induces erythrocytosis, increasing their EPO sensitivity, and mimics iron-deficiency in mice (Nai et al., 2015). In fact, in Tfr2^BMKO mice the proportion of bone marrow nucleated erythroid cells is higher and the apoptosis of late erythroid precursors is lower than in controls, despite comparable serum EPO levels. The inventors and others have shown that iron restriction partially corrects the β-thalassemia phenotype in the Hbb^th3/+ model of thalassemia intermedia (Nai et al., 2015) (Gardenghi et al., 2010) (Nai et al., 2012) (Schmidt et al., 2013) (Guo et al., 2013).

Hepcidin, the key regulator of iron homeostasis, is a short peptide mainly produced by the hepatocytes, which, secreted into the circulation, binds the sole cellular iron exporter ferroportin, triggering its internalization and degradation (Nemeth et al., 2004). Through this mechanism hepcidin reduces circulating iron by blocking dietary iron absorption in duodenal enterocytes and iron release from macrophages. Since iron is essential for several functions, as energy production, DNA synthesis, metabolic pathways and oxygen transport, hepcidin expression is tightly regulated in response to multiple stimuli as body iron concentration, erythropoiesis, inflammation, gluconeogenesis, hormones and drugs, including the mTOR inhibitor rapamycin (Mleczko-Sanecka et al., 2014).

Hepcidin synthesis is mainly regulated by the Bone Morphogenetic Protein (BMP)-Son of Mother Against Decapentaplegic (SMAD) pathway. In the presence of appropriate ligands, as BMP2 for basal activation (Koch et al., 2017) and BMP6 for the iron-dependent response (Andriopoulos et al., 2009; Canali et al., 2017; Kautz et al., 2008; Meynard et al., 2009), BMP type II receptors (BMPR-II), that are constitutively active, phosphorylate type I receptors (BMPR-I). Type I receptors activin receptor-like kinase-2 (ALK2) and activin receptor-like kinase-3 (ALK3) have a crucial role (Steinbicker et al., 201 1 ) in hepatocytes, while type II receptors (BMPR2 and ACVR2A) have a redundant function in hepcidin regulation (Mayeur et al., 2014). Activated type I receptors phosphorylate cytosolic SMAD1/5/8 that, after binding the cargo protein SMAD4, translocate to the nucleus as a multiprotein complex that interacts with the hepcidin promoter, inducing its expression. Decreased production of hepcidin leads to iron overload. In hereditary hemochromatosis defective hepcidin synthesis is caused by mutations in genes that regulate the liver BMP- SMAD pathway, as the BMP co-receptor hemojuvelin {HJV), hepcidin (HAMP) itself or its activators TFR2 and HFE (Camaschella, 2005). In β-thalassemia the expanded ineffective erythropoiesis, due to defective β-globin chain synthesis, downregulates hepcidin expression (Origa et al., 2007) through the erythroid regulator erythroferrone (Kautz et al., 2014).

Rapamycin, an immunosuppressive drug that inhibits mTOR in complex with the immunophilin FK506-binding protein 1A (FKBP12), activates hepcidin in vitro (Mleczko- Sanecka et al., 2014), but the underlying molecular mechanism is unknown. Interestingly, patients treated with rapamycin may develop mild anemia and microcytosis (Sofroniadou and Goldsmith, 201 1 ), conditions compatible with high hepcidin levels, suggesting that mTOR might modulate hepcidin in vivo.

Hjv KO mice recapitulate the most severe form of hereditary hemochromatosis. They are characterized by low hepcidin expression due to genetic inactivation of the BMP coreceptor Hjv. As a consequence, iron accumulates in several organs and tissues, as the liver, hearth and pancreas, but not in the spleen, in which iron concentration is even reduced compared to wild type mice because of ferroportin stabilization. Therapeutic approaches in iron loaded patients are currently based on phlebotomy. This approach is efficient to remove iron excess but not applicable to all patients. In addition, it does not correct the primary defect due to hepcidin deficiency. The inventors have demonstrated that tacrolimus (TAC) treatment of wild type mice with a single injection upregulated hepcidin expression and transiently increase serum hepcidin levels (Colucci et al., Blood 2017). Considering the short half-life of TAC the inventors hypothesized that a continuous administration of the drug might ameliorate iron overload redistributing iron to stores in Hjv KO mice. The inventors have demonstrated that this pharmacologic approach is effective in hepcidin upregulation in murine primary hepatocytes isolated form Hjv KO animals, demonstrating that Hjv is dispensable for TAC effect (Colucci et al., Blood 2017) providing the proof of concept for drug treatment of Hjv KO mice. Based on these data, the inventors treated hemochromatosis mice chronically with a suboptimal dose of TAC, unable to induce immunosuppression (Spiekerkoetter et al., JCI 2013).

There is a great interest in the development of targeted therapy for beta thalassemia, a disease characterized by anemia and splenomegaly due to ineffective erythropoiesis, and multi organ iron overload.

Traditional treatment of beta-thalassemia is based on regular blood transfusions and iron chelation life long, a costly, demanding and far from optimal treatment. The only definitive curative option for β-thalassemia is allogeneic bone marrow transplantation. However, this approach is applicable to only 30% of the patients with a HLA-matched sibling donor and limited by the risk of graft-versus-host disease. Gene therapy is an alternative experimental approach with in progress clinical trials that seem promising in reducing transfusional need. However, due to the complexity of the approach realistically only a minority of patients will be eligible for gene-therapy.

Both hemochromatosis and beta thalassemia are characterized by low hepcidin/iron overload. Current therapies based on phlebotomy in hemochromatosis, and on blood transfusions-iron chelation in beta thalassemia are symptomatic approaches, unsatisfactory or not applicable to all cases.

WO2012177921 relates to dsRNAs able to reduce expression levels of TFR2 to reduce hepcidin and increase body iron levels for the specific treatment of anemia of inflammation. In this document the dsRNA targets liver TFR2.

WO2014020140 disclose an antagonist of TFR1 receptor for the treatment of thalassemia. Therefore, there is still the need for alternative and/or combinatorial novel therapies of a disease characterized by ineffective erythropoiesis and/or of anemia and/or a disease characterized by iron overload and/or disease characterized by low hepcidin. SUMMARY OF THE INVENTION

The present invention is based on the finding that regulation of the BMP-SMAD signaling, in particular the deletion of TFR2 (including its haploinsufficiency) significantly ameliorates anemia and reduces ineffective erythropoiesis and iron overload in a mouse model of non- transfusion dependent beta-thalassemia. Specific erythroid TFR2 deletion by bone marrow transplantation of 77r2^~A hematopoietic stem cells in a wild type recipient provides the same positive results. All these findings define regulators of the BMP-SMAD signaling, such as TFR2 as a new therapeutic target for the treatment of beta-thalassemia.

Targeting TFR2, which in erythroid cells is a partner of EPO receptor, in order to reduce its expression is a novel original approach to be exploited alone or in combination with other treatment in beta-thalassemia.

The advantage of inhibiting TFR2 is that it improves erythropoiesis by inducing erythroblast proliferation and maturation and ameliorates anemia. This effect is achieved by a partial or a full inhibition of TFR2 in the bone marrow, without affecting systemic iron homeostasis. TFR2 is highly homologous to TFR1. However, at difference with TFR2, TFR1 is ubiquitously expressed and necessary for cellular iron intake. In the short term, inhibition of TFR1 has a positive effect because it reduces iron uptake by the thalassemia erythroid cells, but in the long term it could worsen erythropoiesis, as already demonstrated in thalassemia mice in which iron restriction was induced by administration of an iron deficient diet (Gardenghi et al., 2010).

On the contrary, TFR2 is expressed selectively in the liver (hepatocytes) and in erythroid cells It functions as an iron sensor and is not involved in iron uptake in vivo. Thus, by targeted inhibition of TFR2, systemic iron homeostasis is not altered whereas erythropoiesis is stimulated.

Further in the present invention regulation of BMP-SMAD signaling and subsequent molecular mechanism(s) of hepcidin activation by rapamycin was investigated, showing that it is mediated by FKBP12, which interacts with BMP type I receptors (Wang et al., 1996) to avoid leakage activation of the pathway (Song et al., 2010). In hepatocytes, FKBP12 preferentially binds the ALK2 receptor. ALK2 mutants with impaired binding to FKBP12 constitutionally activate the BMP-SMAD signaling and increase hepcidin expression in vitro in a ligand-independent manner. Tacrolimus (FK506), a drug that inhibits calcineurin in complex with FKBP12, activates hepcidin in vitro, ex vivo in primary hepatocytes, and in vivo in mice in the setting of acute treatment.

Interestingly, genetic or pharmacologic FKBP12 displacement renders ALK2 responsive to Activin A, a transforming-growth factor (TGF)-p ligand released in inflammation, suggesting a possible physiologic mechanism of BMP pathway activation and hepcidin increase in inflammation.

All together the present results clarify the hematologic effects of rapamycin, identify a novel mechanism of hepcidin regulation, indicate FKBP12 as a potential target for disorders with insufficient hepcidin production, as hemochromatosis and beta thalassemia, and suggest a role for Activin A in hepcidin control in inflammation.

The present invention identifies a new level of BMP-SMAD and hepcidin regulation, based on the druggable target FKBP12. The inventors have demonstrated that tacrolimus, a drug that complex with FKBP12 and displace it from the BMP receptor ALK2, upregulates hepcidin through BMP-SMAD pathway activation in primary hepatocytes, not only from wild type but also from Hjv KO mice, a genetic model of hemochromatosis, and in vivo in wild type mice (in an acute setting).

The present invention is also based on the fact that the inhibition of the erythroid form of the second transferrin receptor Tfr2 strongly and persistently ameliorates erythropoiesis in the thalassemia mice model and the targeting FKBP12 increases hepcidin, which on turn ameliorates both iron overload and anemia, a combination therapy of FKBP12 and TFR2 inhibition is proposed. This is tested in preclinical studies using the common beta thalassemia Hbb^th3/+ model (hbb: hemoglobin subunit beta; th3: third thalassemia model developed; +: heterozygous deletion) (Yang et al., 1995), the recognized model of iron loading anemias that include sideroblastic anemias, congenital dyserythropoietic anemias and some forms of hemolytic anemias.

Therefore, the present invention provides a regulator of the BMP-SMAD signaling for use in the treatment and/or prevention of a disease selected from the group consisting of: a disease characterized by ineffective erythropoiesis, anemia, a disease characterized by low hepcidin and by iron overload or a combination thereof.

The regulator may activate the BMP-SMAD signaling or may inhibit the BMP-SMAD signaling. The BMP-SMAD pathway activity is measured following the phosphorylation of SMAD1/5/8 by western blot analysis and the mRNA expression levels of BMP-SMAD target genes, as hepcidin, ID1 , SMAD7 and ATOH8.

Preferably the regulator of the BMP-SMAD signaling is selected from the group consisting of: an inhibitor of erythroid TFR2 or an inhibitor of hepatocyte FKBP12 or a combination thereof.

Still preferably the inhibitor of erythroid TFR2 alters EPO-EPOR signaling and/or inhibit

TFR2-EPOR binding orwherein the inhibitor of hepatocyte FKBP12 displaces FKBP12 from BMP type I receptor.

Still preferably the regulator is selected from the group consisting of: a small molecule, a peptide, a protein, an antisense oligonucleotide, a siRNA, an antisense expression vector or recombinant virus, an antibody or a fragment thereof.

More preferably the regulator is a siRNA or tacrolimus.

Preferably the disease characterized by ineffective erythropoiesis is selected from the group consisting of: beta-thalassemia, congenital dyserythropoietic anemias, sideroblastic anemias, myelodysplastic syndromes.

Preferably the anemia is selected from the group consisting of: sickle cell disease, malaria, chronic kidney disease, anemia of inflammation, hemolytic anemias.

Preferably the disease characterized by low hepcidin and by iron overload is selected from the group consisting of: hematochromatosis and ineffective erythropoiesis as beta- thalassemia, congenital dyserythropoietic anemias, sideroblastic anemias, myelodysplastic syndromes.

Still preferably tacrolimus is for use in the treatment and/or prevention of hematochromatosis or beta-thalassemia.

More preferably the regulator of the BMP-SMAD signaling is for use in combination with a therapeutic agent and/or intervention.

Preferably the therapeutic agent and/or intervention is selected from the group consisting of: a gene therapy, an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor.

It is a further object of the invention a pharmaceutical composition comprising a regulator of the BMP-SMAD signaling as defined above for use in the treatment and/or prevention of a disease selected from the group consisting of: a disease characterized by ineffective erythropoiesis, anemia, a disease characterized by low hepcidin and by iron overload or a combination thereof.

Preferably the pharmaceutical composition further comprises a therapeutic agent.

Preferably the therapeutic agent is selected from the group consisting of: an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor.

Preferably the pharmaceutical composition is for use in combination with gene therapy. In the present invention the regulator of BMP-SMAD, in particular by targeting ertyhroid TFR2 ameliorates erythropoiesis in thalassemia. The regulator of BMP-SMAD, in particular by targeting FKBP12 in the liver increases hepcidin and ameliorates ertyhropoiesis through iron restriction.

In the present invention ineffective erythropoiesis means aberrant proliferation and decreased differentiation of erythroid cells.

In the present invention low hepcidin means downregulation of hepcidin expression in the liver and/or decreased serum hepcidin levels.

In the present invention iron overload means an uncontrolled accumulation of iron in tissues and organs as liver, spleen, blood, heart, pancreas, kidney.

A further aspect of the present invention provides antibodies which are immunoreactive or bind to the peptides of the present invention. Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations, are provided. Monoclonal antibodies are made from antigen- containing peptides of the present invention or fragments by methods well known in the art (Kohler, et al., Nature, 256: 495,1975; Current Protocols in Molecular Biology, Ausubel et al., ed., 1989). Antibodies which bind to the peptides of the present invention or a region of TfR2 represented by the peptides of the present invention can be prepared using an intact polypeptide or fragments containing peptides of interest as the immunizing antigen. A polypeptide or a peptide, used to immunize an animal can be derived from translated cDNA or chemical synthesis and is purified and conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e. g., a mouse, a rat, or a rabbit). If desired, polyclonal antibodies can be further purified, for example, by binding to and eluting from a matrix to which a polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies. (See, for example, Coligan, et al., Unit 9, Current Protocols In Immunologv, Wiley Interscience, 1991 , incorporated by reference.)

The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, Fab'2 and Fv, which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor and are defined as follows:

1 ) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of a whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab'2, the fragment of an antibody molecule, can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) Fab'2, the fragment of the antibody that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction; Fab'2 is a dimmer of two Fab' fragments held together by two disulfide bonds;

4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference.)

Preferably the therapeutic agent and/or intervention is selected from the group consisting of: a gene therapy (for instance by reintroduction of the correct sequence of the beta-globin in hematopoietic stem cells), an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6 (NM_153609; REF: Guo S., Casu C, Gardenghi S., Booten S., Aghajan M., Peralta R., Watt A., Freier S., Monia BP. and Rivella S., Reducing TMPRSS6 ameliorates hemochromatosis and β-thalassemia in mice. JCI. 2013; 123(4): 1531 -41 ) , minihepcidin (REF: Ramos E., Ruchala P., Goodnough JB. Kautz L., Preza GC, Nemeth E. and Ganz T. Minihepcidin prevent iron overload in a hepcidin- deficient mouse model of severe hemochromatosis. Blood. 2012; 120(18):3829-36), transferrin (REF: Li H., Rybicki AC, Suzuka SM:, von Bondsdorff L., Breuer W., Hall CB., Cabantchik Zl., Bouhassira EE., Fabry ME. and Ginzburg YZ.. Transferrin therapy ameliorates disease in beta-thalassemic mice. Nat Med. 2010; 16(2): 177-82), an antagonist of TFR1.

An inhibitor of TFR2 is a compound or agent that specifically targets TFR2. The inhibitor may inhibit the function and/or the expression of TFR2. The inhibitor may also inhibit the binding TFR2-EPOR. In particular the inhibitor may be an antisense oligonucleotide (ASO) directed against TFR2 or a molecule, a peptide or an antibody of a functional fragment thereof that interferes with the TFR2-EPOR binding. "Interfering" means a molecule or peptide/ antibody that impedes or reduces the binding between TFR2 and EPOR.

TFR2 inhibition may be measured by measuring mRNA levels of TFR2 and/or mRNA levels of BCLXL and/or FASL and/or EPOR and/or erythroferrone (all EPO-EPOR target genes) and comparing them to a reference or reference value. The mRNA may be analysed by quantitative real time PCR in isolated reticulocytes. As reference or reference value, expression levels of the same genes may be analyzed in reticulocytes from the same individual before TFR2 inhibitor treatment. An inhibitor of FKBP12 is a compound that specifically targets FKBP12. The inhibitor may inhibit the function and/or the expression of TFR2. In particular the inhibitor may be an antisense oligonucleotide (ASO) directed against FKBP12; a compound such as tacrolimus (FK506) or rapamycin targeting liver FKBP12. In particular targeting FKBP12 may displace it from BMP type I receptor for hepcidin activation.

Inhibition of FKBP12 may be measured by measuring mRNA levels of FKBP12 (when applicable) and BMP-SMAD target genes (as Id1 and Atoh8), preferably in peripheral blood mononuclear cells (PBMCs), Serum hepcidin (a BMP-SMAD target gene) may also be measured as in index of FKBP12 inhibition. The measured mRNA levels may be compared to a reference or reference value. As reference or reference value, the expression levels of ID1 and ATOH8 in PBMCs from the same individual before FKBP12 inhibitor treatment may be analysed and serum hepcidin from the same individual before FKBP12 inhibitor treatment may be analysed.

As used herein, the term "TFR2" and "FKBP12" refers to substantially pure native TFR2 and FKBP12 proteins, respectively or recombinantly expressed/produced proteins, including variants thereof encoded by mRNA, and generated by alternative splicing of a primary transcript, and further including fragments thereof which retain native biological activity.

Sequences (NCBI Reference Sequence):

TFR2 sequence: NM_003227.3 NCBI Reference Sequence, PRI 06-OCT-2016 (https://www.ncbi.nlm.nih.gov/nuccore/NM 003227.3)

FKBP12 sequence: NM_000801.4 NCBI Reference Sequence, PRI 01 -DEC-2016

(https://www.ncbi.nlm.nih.gov/nuccore/NM 000801 .4)

EPOR: NM_000121.3

BCLXL (BAD): NM_004322.3

FASL: NM_000639.2

erythroferrone: NM_001291832.1

ID1 : NMJ 81353.2

ATOH8: NM_032827.6

Hepcidin: NP_066998.1 In the present invention, in order to target erythroid TFR2, unmodified ASO-TFR2 may be used to reduce, and not to completely suppress, liver and erythroid TFR2 mRNA levels. With this approach, hepcidin (regulated by liver TFR2) is left unmodified but EPO-EPOR signaling (controlled by erythroid TFR2) is modified. Alternatively, modified ASO may be used. Such modified ASO specifically target erythroid cells. When an inhibitor or TFR2- EPOR binding is used to inhibit TFR2 function, specific targeting is not needed since the TFR2-EPOR binding occurs exclusively in erythroid cells.

Specific targeting of liver (hepatocyte) FKBP12 may be achieved by using ASO properly modified to selectively target hepatocytes. Liposome-complexed drugs may also be used for liver targeting by molecules such as tacrolimus or rapamycin.

Disease characterized by ineffective erythropoiesis are diseases with expansion of immature erythroblast and defective erythroid maturation. Beta thalassemia (intermedia and major) may be the prototype. Other forms comprise congenital dyserythropoietic anemia, sideroblastic anemia and some forms of myelodysplasia.

Anemia is a condition characterized by low hemoglobin levels and defective tissue oxygenation, as sickle cell disease, malaria, chronic kidney disease, anemia of inflammation, hemolytic anemias.

A disease characterized by iron overload is a condition (hemochromatosis, ineffective erythropoiesis) characterized by iron accumulation in several organs and tissues, as liver, pancreas, gonads, heart. Increased serum iron concentration, high transferrin saturation and high serum ferritin are also present.

A disease characterized by low hepcidin is a condition characterized by low and inappropriate (considering the amount of serum and liver iron concentration) hepcidin expression, as in hereditary hemochromatosis and in ineffective erythropoiesis

DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides or dinucleotide dimers, siRNA ect ...may be systemically delivered and can be administered alone, or in combination with physiologically acceptable carriers, including solvents, perfumes or colorants, stabilizers, sunscreens or other ingredients, for medical or cosmetic use. They can be administered in a vehicle, such as water, saline, or in another appropriate delivery vehicle. The delivery vehicle can be any appropriate vehicle, which delivers the oligonucleotides, deoxynucleotides, dinucleotides, SiRNA or dinucleotide dimers. In one embodiment, the concentration range of oligonucleotide can be from 0.1 nM to 500 μΜ, preferably the in vitro range is between 0.2 nM and 300 μΜ, preferably the in vitro range is between 0.5 nM to 200 μΜ. Preferred in vivo range is 0.1 -500 mg/ kg, preferably in vivo range is 1 -50 mg/kg.

The oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, or composition comprising one or more of the foregoing, is administered to (introduced into or contacted with) the cells of interest in an appropriate manner.

The "cells of interest" as used herein are those cells which may become affected or are affected by a disease as defined in the present invention. Particularly preferred are hepatocytes and erythroid cells.

The oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, siRNA, agent that have the desired therapeutic use, or composition comprising one or more of the foregoing, is applied at an appropriate time, in an effective amount. The "appropriate time" will vary, depending on the type and molecular weight of the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, siRNA or other agent employed, the condition to be treated or prevented, the results sought, and the individual patient.

An "effective amount" as used herein, is a quantity or concentration sufficient to achieve a measurable desired result. The effective amount will depend on the type and molecular weight of the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, siRNA, or agent employed, the condition to be treated or prevented, the results sought, and the individual patient. For example, for the treatment and/or prevention of a disease characterized by ineffective erythropoiesis and/or of anemia and/or a disease characterized by iron overload, the effective amount is the amount necessary to reduce or relieve any one of the features of the disease, as low hemoglobin levels, low red blood cells number, increased reticulocytes, increased apoptosis of erythroid precursors, increased proliferation of early erythroid precursors, splenomegaly, high EPO and erythroferrone levels, low hepcidin, tissue iron overload, high serum iron concentration.

As demonstrated herein, the inhibitors of the present invention are active in vitro and in vivo in their unmodified form, e. g. sequences of unmodified oligonucleotides linked by phosphodiester bonds. As used herein, the terms "oligonucleotide", "dinucleotide," etc., refer to molecules having ribose and/or deoxyribose as the sugar, and having phosphodiester linkages ("phosphate backbone") as occur naturally, unless a different linkage or backbone is specified.

Oligonucleotides are relatively short polynucleotides. Polynucleotides are linear polymers of nucleotide monomers in which the nucleotides are linked by phosphodiester bonds between the 3' position of one nucleotide and the 5' position of the adjacent nucleotide. Unless otherwise indicated, the "oligonucleotides" of the invention as described herein have a phosphodiester backbone.

To enhance systemic delivery, the oligonucleotides of the invention may be modified so as to either mask or reduce their negative charges or otherwise alter their chemical characteristics. This may be accomplished, for example, by preparing ammonium salts of the oligonucleotides using readily available reagents and methods well known in the art. Preferred ammonium salts of the oligonucleotides include trimethyl-, triethyl-, tributyl-, tetramethyl-, tetraethyl-, and tetrabutyl-ammonium salts. Ammonium and other positively charged groups can be convalently bonded to the oligonucleotide to facilitate its transport accross the stratum comeum, using an enzymatically degradable linkage that releases the oligonucleotide upon arrival inside the cells of the viable layers of the epidermis.

Another method for reducing or masking the negative charge of the oligonucleotides includes adding a polyoxyethylene spacer to the 5'phosphate groups of the oligonucleotides and/or the internal phosphates of the oligonucleotides using methods and reagents well known in the art. This, in effect, adds a 6-or 12-carbon modifier (linker) to the phosphate that reduces the net negative charge by +1 and makes the oligonucleotides less hydrophilic. Further negative charge reduction is achieved by adding a phosphoroamidite to the end of the polyoxyethylene linker, thereby providing an additional neutralizing positive charge. The phosphodiester backbone of the oligonucleotides of the present invention can also be modified or synthesized to reduce the negative charge. A preferred method involves the use of methyl phosphonic acids (or chiral- methylphosphonates), whereby one of the negatively charged oxygen atoms in the phosphate is replaced with a methyl group. These oligonucleotides are similar to oligonucleotides having phosphorothioate linkages which comprise a sulfate instead of a methyl group and which are also within the scope of the present invention. The oligonucleotides of the present invention can also take the form of peptide nucleic acids (PNAs) in which the bases of the nucleotides are connected to each other via a peptide backbone.

Other modifications of the oligonucleotides such as those described, for example, in US Patent Nos. 6,537, 973 and 6,506, 735 as well as in (Stenvang et al., 2012) (all of which are incorporated herein by reference for all of the oligonucleotide modifications described therein) and others will be readily apparent to those skilled in the art.

The oligonucleotides can also be "chimeric" oligonucleotides which are synthesized to have a combination of two or more chemically distinct backbone linkages, one being phosphodiester. In one embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 3' end. In one embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 5' end. In another embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 3' and 5' ends.

As used herein, the term "short interfering RNA" or "siRNA" means double stranded ribonucleotide sequences of typically 15-50 base pairs and preferably 19-27 base pairs in length that are highly negatively charged and soluble predominantly in water siRNA may be composed of either two annealed ribonucleotide sequences or a single ribonucleotide sequence that forms a hairpin structure One of ordinary skill would understand that siRNA is responsible for RNA interference, the process of sequence-specific post-transcriptional gene silencing in animals and plants siRNAs are generated by ribonuclease Hi cleavage from longer double- stranded RNA (dsRNA) which are homologous to the silenced gene or by delivering synthetic RNAs to cells Techniques for the design of such molecules for use in targeted inhibition of gene expression are well known to one of skill in the art

The use of siRNAs is well known in the art. At least one siRNA is targeted to a specific gene sequence of interest leading to a down-regulation of the protein encoded by this Mrna. Any drugable or non-drugable gene of interest may be targeted by designing an siRNA sequence homologous to the mRNA of interest, With the entire human genome now sequenced, any portion thereof may serve as a target sequence in designing an siRNA for use in the present method of treatment of the invention. Thus, the sequence for an siRNA for use in the present invention may be obtained from plasma DNA or the human genome. siRNA may be generated by ribonuciease III cleavage from longer double-stranded RNA (dsRNA) which are homologous to the silenced gene or by delivering synthetic RNAs to cells. The siRNAs for use in the present invention may also be derived from known anti- sense sequences. The siRNA for use in the present invention are double stranded ribonucleotide sequences of typically 15 to 50 base pairs in length that are highly negatively charged and soluble predominantly in water Preferably, the siRNA sequence of the present invention ranges from about 19 to about 27 base pairs in length and is highly homologous or 100% homologous to the target sequence The siRNA may be blunt ended or else have base pair overhangs The siRNA further may include a repeating amino aod sequence consisting of serine-aspartic acid-threonine and/or phosphorothioate backbone modifications.

Various types of RNA may serve as siRNA for use of the present invention For example, double stranded RNA (dsRNA) micro-RNA (miRNA) short hairpin RNA (shRNA) or combinations thereof, may be used.

The siRNAs may be chemically modified. Several chemistries such as phosphorothioates" or boranophosphates, 2 -O-Methyl, 2'-0-allyl, 2'-methoxyethyl (MOE) and 2'- deoxyfluoconucleotides, or Locked Nucleic Acids (LNA) have been described and form part of the present invention. Modified siRNA with improved pharmacological properties may be obtained by minimally modifying siRNAs on the 3'-end of each strand in order to prevent 3'- exonuclease digestion: the 3'-dideoxynucleotide overhang of 21 -nt siRNA may be replaced by a universal 3'-hydroxypropyl phosphodiester moiety and the modification of the two first base-pairing nucleotides on 3'-end of each strand further enhances serum stability.

In the present invention the inhibitor of TFR2 and the inhibitor of FKBP12 may be administered together or one before the other (subsequently) in any order.

The invention will be illustrated by means of non limiting examples in reference to the following figures.

Figure 1 : Hematological and iron parameters of Hbb^th3/+ mice with germ-line deletion of Tfr2. The hematological parameters of both male and female Hbb^th3/+ mice with germ- line deletion of none (Tfr2^+/+), a single (Tfr2^+/~) or both (Tfr2^~f~) allele of Tfr2 were determined at different time points (4, 10, 15, 20, 25, 29, 33 and 36 weeks of age); while iron parameters were determined in a pool of 10-week-old Hbb^th3/+ and T1r2^~'^~/Hbb^m,+ mice. In the figure are graphed: Red Blood Cells count (RBC, A), Hemoglobin levels (B), Mean Corpuscolar Volume (MCV, C), Mean Corpuscolar Hemoglobin (MCH, D), liver iron content (LIC, E) and spleen iron content (SIC, F). The dotted red line indicates mean value in wild-type mice. Error bars indicate standard error. Unpaired 2-tailed Student t-test was performed and asterisks refer to statistically significant differences. *p<0.05; **p<0.01 ; ***p<0.005

Figure 2: Hematological parameters of Hbb^th3/+ mice with erythroid specific deletion of Tfr2. The hematological parameters of mice were analyzed monthly from 9 to 22 weeks after transplantation with a thalassemic (Hbb^th3/+) or a Tfr2- /Hbb^th3/+ (Tfr2^BMKO/Hbb^th3/+) bone marrow. Mice were fed a standard diet. In the figure are graphed: Red Blood Cells number (RBC, A); Hemoglobin levels (Hb, B); Mean Corpuscular Volume (MCV, C) and Mean Corpuscular Hemoglobin (MCH; D). Mean values of 6-8 animals for genotype are graphed. The dotted red line indicates mean value in wild-type mice. Error bars indicate standard error. Asterisks refer to statistically significant differences. *P<0.05; **P<0.01 ; ***P<0.005.

Figure 3: Analysis of erythropoiesis and serum EPO levels of Tfr2^BMKO/Hbb^th3/+ mice 9 weeks after BMT. Mice were analyzed 9 weeks after transplantation with a thalassemic (Hbb^th3/+) or a Tfr2- /Hbb^th3/+ (Tfr2^BMKO/Hbb^th3/+) bone marrow. Mice were fed a standard diet. In the figure are graphed: body weight (A), spleen weight normalized to body weight (B); percentage of Ter1 19⁺ cells on alive cells and subpopulation composition [Gated Clusters: proerythroblasts (I), basophilic erythroblasts (II), polychromatic erythroblasts (III), orthochromatic erythroblasts and immature reticulocytes (IV) and mature red cells (V)] based on Ter1 19/CD44 expression and forward scatter (reflecting cell size) both in the BM and in the spleen (C); percentage of reticulocytes in peripheral blood (D) and serum erythropoietin (EPO) levels (E). The dotted red line indicates mean value in wild-type mice. Bars indicate standard error. Asterisks refer to statistically significant differences. **P<0.01 ; ***P<0.005.

Figure 4: Analysis of iron parameters of Tfr2^BMKO/Hbb^th3/+ mice 9 weeks after BMT. Iron parameters and hepatic hepcidin (Hamp) expression were determined in mice 9 weeks after transplantion with a thalassemic (Hbb^th3/+) or a Tfr2 -/Hbb^th3/+ (Tfr2^BMKO/Hbb^th3/+) bone marrow. Mice were fed a standard diet. In the figure are graphed: liver iron content (LIC, A); spleen iron content (SIC, B); kidney iron content (KIC, C); heart iron content (HIC, D); liver Hamp mRNA levels (E) relative to Hypoxanthine Phosphoribosyltransferase 1 {Hprtl) and serum transferrin saturation (TS, F). The dotted red line indicates mean value in wild-type mice. Bars indicate standard error. Figure 5: Analysis of EPO target genes in the BM of Tfr2^BMKO/Hbb^th3/* mice 9 weeks after BMT. Bone marrow cells were extracted from the femurs of Hbb^th3/+ and Tfr2^BMKO/Hbb^th3/+ mice 9 weeks after BMT and the expression levels of EPO target genes were determined. In the figure are graphed: mRNA expression of Erythroferrone {Erfe, A), B-cell lymphoma-extra large {BCI-XL, B), Fas ligand {Fasl, C) and Erythropoietin Receptor (Epor, D) relative to Glyceraldehyde 3-Phosphate dehydrogenase (Gapdh). Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05.

Figure 6: Analysis of EPO target genes in the spleen of Tfr2^BMKO/Hbb^th3/+ mice 9 weeks after BMT. The expression of EPO target genes was analyzed in the spleen Hbb^th3/+ and Tfr2^BMKO/Hbb^{t 3/+} mice 9 weeks after BMT. In the figure are graphed: mRNA expression of Erythroferrone {Erfe, A), B-cell lymphoma-extra large {BCI-XL, B), Fas ligand {Fasl, C) and Erythropoietin Receptor {Epor, D) relative to Glyceraldehyde 3-Phosphate dehydrogenase {Gapdh). Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05; ^**P<0.01 ; ^***P<0.005.

Figure 7: Analysis of erythropoiesis and serum EPO levels of Tfr2^BMKO/Hbb^th3/+ mice 22 weeks after BMT. Mice were analyzed 22 weeks after transplantation with a thalassemic {Hbb^th3/+) or a Tfr2- /Hbb^th3/+ {Tfr2^BMKO/Hbb^th3/+) bone marrow. Mice were fed a standard diet. In the figure are graphed: body weight (A); percentage of reticulocytes in peripheral blood (B); percentage of Ter1 19⁺ cells on alive cells and subpopulation composition [Gated Clusters: proerythroblasts (I), basophilic erythroblasts (II), polychromatic erythroblasts (III), orthochromatic erythroblasts and immature reticulocytes (IV) and mature red cells (V)] based on Ter1 19/CD44 expression and forward scatter (reflecting cell size) in the BM (C) and in the spleen (D); serum erythropoietin (EPO) levels (E) and spleen weight normalized to body weight (F). The dotted red line indicates mean value in wild-type mice. Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05; ^**P<0.01.

Figure 8: Analysis of iron parameters of Tfr2^BMKO/Hbb^th3/+ mice 22 weeks after BMT. Iron parameters and hepatic hepcidin {Hamp) expression were determined in mice 22 weeks after transplantion with a thalassemic {Hbb^th3/+) or a Tfr2-^f-/Hbb^th3/+ {Tfr2^BMKO/Hbb^th3/+) bone marrow. Mice were fed a standard diet. In the figure are graphed: liver iron content (LIC, A); spleen iron content (SIC, B); kidney iron content (KIC, C); heart iron content (HIC, D); liver Hamp mRNA levels (E) relative to Hypoxanthine Phosphoribosyltransferase 1 {Hprtl) and serum transferrin saturation (TS, F). The dotted red line indicates mean value in wild-type mice. Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05; ^***P<0.005.

Figure 9: Analysis of EPO target genes in the BM Ter119⁺ cells of Tfr2^BMKO/Hbb^th3/+ mice 22 weeks after BMT. Ter1 19⁺ cells were isolated from the BM extracted from the femurs of Hbb^th3/+ and Tfr2^BMKO/Hbb^th3/+ mice 22 weeks after BMT and the expression levels of EPO target genes were determined. In the figure are graphed: mRNA expression of Erythroferrone {Erfe, A), B-cell lymphoma-extra large {BCI-XL, B), Erythropoietin Receptor (Epor, C) and Fas ligand (Fasl, D) relative to Glyceraldehyde 3-Phosphate dehydrogenase {Gapdh). Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05.

Figure 10: Analysis of EPO target genes in the spleen of Tfr2^BMK°/Hbb^th3/+ mice 22 weeks after BMT. The expression of EPO target genes was analyzed in the spleen Hbb^th3/+ and Tfr2^BMKO/Hbb^th3/+ mice 22 weeks after BMT. In the figure are graphed: mRNA expression of Erythroferrone {Erfe, A), B-cell lymphoma-extra large {BCI-XL, B), Erythropoietin Receptor {Epor, C) and Fas ligand {Fasl, D) relative to Glyceraldehyde 3- Phosphate dehydrogenase {Gapdh). Bars indicate standard error. Asterisks refer to statistically significant differences. ^*P<0.05; ^***P<0.005.

Figure 11 : Rapamycin upregulates hepcidin through activation of the BMP-SMAD pathway. A) Hep3B cells, treated with rapamycin (RAPA, 100 nM) or Torinl (T1 , 100 nM) for 15 hrs, were lysed and whole cell extract loaded onto a 10% SDS-PAGE for Western Blot analysis. mTOR activation was detected by following Ser240/244 and Ser235/236 phosphorylation of the mTOR target protein S6RP. Total S6RP and actin were analyzed for normalization of gel loading. A representative Western Blot, made in triplicate, is shown. B, E) Hep3B cells were treated with RAPA or T1 as described in A. Total RNA was isolated and analyzed by qRT-PCR for hepcidin {HAMP) (B) and ID1 (E) expression and normalized to an untreated mean value of 1. GAPDH was used as housekeeping gene. A representative experiment, made in triplicate, is shown. C) Primary murine hepatocytes were treated with RAPA (100 nM) or T1 (100 nM) for 5 hours. RNA was isolated and qRT- PCR was performed to analyze hepcidin {Hamp) expression. Hrptl was used as housekeeping gene. mRNA expression ratio was normalized to an untreated mean value of 1. A representative experiment, made in triplicate, is shown. D). Hep3B cells were transfected with the BRE-Luc reporter vector and treated with RAPA (100 nM) or T1 (100 nM) as described in A. Cells were lysed and analyzed for the luciferase activity, that was normalized to an untreated mean value of 1 . A representative experiment, made in triplicate, is shown. Error bars indicate SD. RQ: relative quantification, ns: non significant; *p< .05; **p< .01 ; ***p< .001 ; ****p< .0001 .

Figure 12: Tacrolimus upregulates hepcidin through BMP-SMAD pathway activation. A) Luciferase activity was analyzed in Hep3B cells transfected with the BRE-Luc reporter vector and treated with Tacrolimus (TAC, 1 μg/ml) or vehicle for 15 hrs. The luciferase activity of treated cells was normalized to an untreated mean value of 1 . A representative experiment, made in triplicate, is shown. B, C) Hep3B cells, treated with TAC (1 μg/ml), the calcineurin inhibitor cyclosporine A (CA, 1 μg/ml) or vehicle for 15 hrs, were processed for RNA purification. Hepcidin (HAMP) and ID1 expression were quantified by qRT-PCR and normalized to the housekeeping gene GAPDH. A representative experiment, made in triplicate, is shown. D, E) Murine primary hepatocytes were isolated and treated with TAC (1 μg/ml), CA (1 μg/ml) or vehicle for 18 hrs. Hepcidin (Hamp) and Id1 expression were evaluated by qRT-PCR and normalized to the housekeeping gene Hprtl. A representative experiment, made in triplicate, is shown. mRNA expression ratio was normalized to an untreated mean value of 1 . Error bars indicate SD. RQ: relative quantification, ns: non significant; **p< .01 ; ***p< .001 ; ****p< .0001 .

Figure 13: Displacement of FKBP12 form ALK2 increases hepcidin through BMP- SMAD pathway activation. A) HuH7 cells were transiently transfected with FKBP12^FLAG and ALK2^wt"MYC and treated with tacrolimus (TAC, 1 μg/ml), rapamycin (RAPA, 100 nM), GPI-1046 (100 μg/ml) or vehicle for 15 hrs. Protein extracts were immunoprecipitated with the anti-FLAG M2 affinity gel (Sigma-Aldrich). Total extract and immunoprecipitated proteins were loaded onto a 12% SDS-PAGE and analyzed by WB. ALK2 and FKBP12 were detected by using the anti-MYC and anti-FLAG antibodies, respectively. B) HuH7 cells were transfected with FKBP12^FLAG in the presence of ALK2^wt_MYC or ALK2^R206H"MYC or ALK2^Q207E"MYC or ALK2^R258S"MYC, or empty vector. When indicated, cells were treated for 15 hrs with BMP6 (100 ng/ml). Whole cell extract was immunoprecipitated and analyzed as described in A). C) HuH7 cells were transfected with the Smad1 ^FLAG expressing vector in the presence of ALK2^wt_MYC or ALK2^R206H"MYC or ALK2^Q207E"MYC or ALK2^R258S"MYC or empty vector (mock). When indicated, transfected cells were treated with BMP6 (50 ng/ml) for 30 min or 1 .5 hrs. Cells were lysed, loaded onto a 10% SDS-PAGE and analyzed by Western Blot. Activation of the BMP-SMAD pathway was detected by using an antibody recognizing the phospho-S MAD 1/5/8 and total SMAD1. ALK2 was detected by using the anti-MYC antibody. D, E) RNA was isolated from HuH7 cells transfected with ALK2^wt"MYC, ALK2^R206H~

^MYC, ALK2^Q207E"MYC or ALK2^R258S"MYC expressing vector. Hepcidin (HAMP) (D) and ID1 (E) expression levels were quantified by qRT-PCR and normalized to the housekeeping gene

GAPDH. mRNA expression ratio was normalized to an untreated mean value of 1. F) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter (HAMP-Luc) and increasing concentration of ALK2^wt"MYC"FLAG, ALK2^{R206 "MYC"FLAG}, ALK2^Q207F-^myc-^flag or

ALK2^{R258S"MYC"FLAG} expressing vector. Cells transfected with the highest concentration of ALK2 cDNA were treated with dorsomorphin (DM, 10 μΜ). Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated mean value of 1. G) Hep3B cells were transfected with hepcidin promoter firefly luciferase reporter (HAMP-Luc), _ALK2wt-MYc _ALK2R206H-MYC _A|__K2R258S-MYC _{or empty vector (mock}) _and increasing concentration of FKBP12. Luciferase activity was normalized to an untreated mean value of 1. A, B, C, D, E, F are representative results of experiments performed in triplicate. Error bars indicate SD. RQ: relative quantification. The ANOVA two-way analysis was used in F (ALK2 wt vs ALK2 mutants). ^* p< .05; ^**p< .01 ; ^***p< .001 ; ^****p< .0001. WB results are representative of three independent experiments.

Figure 14: The BMP coreceptor hemojuvelin (HJV) is dispensable for FKBP12- dependent hepcidin activation. A) Primary hepatocytes from Hjv KO mice were isolated and treated for 18 hrs with rapamycin (RAPA, 100 nM), Torinl (T1 , 100 nM), Tacrolimus (TAC, 1 μg/ml), Cyclosporin A (CA, 1 μg/ml) and GPI-1046 (100 μg/ml). RNA was isolated and hepcidin (Hamp) levels measured by qRT-PCR. Hprtl was used as housekeeping gene. mRNA expression ratio was normalized to an untreated mean value of 1. A representative experiment, made in triplicate, is shown. B) TAC dependent Hamp fold change increase was evaluated in wild type and Hjv KO hepatocytes. Data are plotted as fold change relative to untreated cells. C) Primary hepatocytes from wild type and Hjv KO mice were isolated and treated with increasing concentrations of TAC. Hamp expression was measured by qRT-PCR as described in A). Error bars indicate SD. RQ: relative quantification. FC: fold change, ns: non significant;^* p< .05; ^**p< .01 ; ^***p< .001 ; ^****p< .0001. Figure 15: Tacrolimus increases hepcidin expression in vivo. A) Schematic representation of the experimental design: C57BL/6 wild type male mice (n= 3-6 mice/group) were treated with vehicle (DMSO) or 10 mg/kg tacrolimus (TAC) via subcutaneous (sc) injection and sacrificed at different time points. B) Liver hepcidin (Hamp) expression was quantified by qRT-PCR and normalized to the housekeeping gene Hprtl. Hepcidin was expressed as a ratio compared to total liver iron content (LIC). C) Spleen iron content (SIC). Error bars indicate SD. ^*p< .05; ^***p< .001.

Figure 16: ALK2-FKBP12 resistant mutants activate hepcidin through Activin A.

Hep3B cells were transfected with the hepcidin promoter luciferase reporter vector (HAMP- Luc) and ALK2^wt_MYC (black line), ALK2^R206H"MYC (red line) or ALK2^R258S"MYC (green line) and treated for 15 hrs with increasing concentrations of BMP2 (A), BMP6 (B) and Activin A (C). Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated-ALK2^wt"MYC mean value of 1. Hep3B cells were transfected with the SMAD2/3 reporter vector (CAGA-Luc) (D) or the SMAD1/5/8 reporter vector (BRE-Luc) (E) in the presence of ALK2^wt"MYCi ALK2^R206H"MYC or ALK2^R258S"MYC When indicated, cells were incubated for 15 hrs with BMP6 (1 ng/ml) or Activin A (10 ng/ml). Luciferase activity was normalized to an untreated-ALK2^wt"MYC mean value of 1. F) SMAD1/5/8 phosphorylation was analyzed in HuH7 transfected with the Smad1 ^FLAG expressing vector and 5 μg or 10 μg of ALK2^wt_MYC, ALK2^R206H_MYC or ALK2^R258"MYC. When indicated, cells were treated for 15 hrs with 10 ng/ml Activin A. Whole cell extract, loaded onto a 10% SDS-PAGE, was analyzed by Western Blot. Anti-FLAG antibody was used to detect SMAD1. A representative Western Blot, made in triplicate, is shown. RQ: relative quantification. Error bars indicate SD. The ANOVA two-way analysis was used in A, B, C (ALK2 wt vs ALK2 mutants), ns: non significant; ^* p< .05; ^***p< .001 ; ^****p< .0001.

Figure 17: Pharmacologic displacement of FKBP12 from ALK2 leads to Activin A- dependent SMAD1/5/8 activation and hepcidin upregulation. A) Hep3B cells, transfected with the hepcidin promoter luciferase reporter vector (HAMP-Luc) and ALK2^wt~ ^MYC expressing vector, were pre-treated with 1 μg/ml tacrolimus (TAC) or vehicle for 3 hrs and then treated with increasing concentrations of Activin A, in presence or absence of TAC for 15 hrs. Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated mean value of 1. B) Primary murine hepatocytes from wild type mice were pre-treated for 3 hrs with 1 μg/ml TAC and incubated for 5 hrs with Activin A (10 ng/ml) in presence or absence of TAC. Hepcidin (Hamp) mRNA expression was quantified by qRT- PCR and normalized to the housekeeping gene Hprtl. mRNA expression ratio was normalized to an untreated mean value of 1. A representative experiment, made in triplicate, is shown. SMAD1/5/8 and SMAD2/3 signaling pathways were analyses in Hep3B cells transfected with the BRE-Luc (C) and the CAGA-Luc (D) reporter vectors. Transfected cells were incubated with TAC (1 μg/ml) ± Activin A (10 ng/ml) and then lysed for analysis of the luciferase activity that was normalized to an untreated mean value of 1 . A representative experiment, made in triplicate, is shown. E) Murine primary hepatocytes were pre-treated with 100 nM rapamycin (RAPA) for 3 hrs and treated with Activin A (10 ng/ml) for 5 hrs, in the presence or absence of RAPA. As control, cells were treated with Torinl (T1 ). Cells were processed and analyzed as described in B). F) Hep3B cells, transfected with the hepcidin promoter luciferase reporter vector (HAMP-Luc) were treated for 15 hrs with increasing concentration of BMP6 in presence or absence of 10 ng/ml of Activin A. Luciferase activity was analyzed and normalized to an untreated mean value of 1 . A representative experiment, made in triplicate, is shown. The ANOVA two-way analysis was used in A, B, F (ALK2 wt vs ALK2 mutants). RQ: relative quantification, ns: non significant; **p< .01 ; ****p< .0001

Figure 18: The non-immunosuppressive drug GPI-1046 activates SMAD1/5/8 signaling and hepcidin expression. Hep3B cells were transfected with the SMAD1/5/8 (BRE-Luc) (A) or the hepcidin promoter (HAMP-Luc) (B) luciferase reporter vectors and treated with GPI-1046 (100 μg/ml) or vehicle for 20 hrs. Cells were lysed and luciferase activity was analyzed and normalized to an untreated mean value of 1 . A representative experiment, made in triplicate, is shown. C, D) Total RNA was purified from primary murine hepatocytes treated with GPI-1046 (100 μθ/ιηΙ) for 18 hrs. Hepcidin {Hamp) (C) and Id1 (D) expression were quantified by qRT-PCR and normalized to an untreated mean value of 1 . Hprtl was used as housekeeping gene. RQ: relative quantification. Error bars indicate SD. ***p< .001 ; ****p< .0001 .

Figure 19: The ALK2 inhibitor DMH1 abrogates the FKBP12-dependent hepcidin regulation. Hep3B cells were pre-treated for 3 hrs with DMH 1 (0,5 μg/ml) and then incubated for 15 hrs with rapamycin (RAPA, 100 nM), Torinl (T1 , 100 nM) (A), tacrolimus (TAC, 1 μ^νη\) or Cyclosporin A (CA, 1 μ9/ιτιΙ) (B). Total RNA was isolated and Hepcidin (HAMP) expression quantified by qRT-PCR and normalized to the housekeeping gene GAPDH. mRNA expression ratio was normalized to an untreated mean value of 1. RQ: relative quantification. Error bars indicate SD. ^* p< .05.

Figure 20: ALK2-FKBP12 binding capacity influences hepcidin activation through BMP-SMAD pathway. A) Hep3B cells were transfected with the hepcidin promoter luciferase reporter vector (HAMP-Luc) in the presence of ALK2^wt"MYC"FLAG (black line),

_ALK2R206H-MYC-FLAG _(red |j_{ne)> A}|__K2Q207E-MYC-FLAG _(bkje |j_{ne) Qr A}|__K2R258S-MYC-FLAG _{(g reen} line) expressing vectors. Cells, treated with increasing concentrations of tacrolimus, were lysed and analyzed for luciferase activity. Luciferase activity was normalized to an untreated-ALK2 (wt or mutants) mean value of 1. A representative experiment, made in triplicate, is shown. The ANOVA two-way analysis was applied (ALK2 wt vs ALK2 mutants). B) Hep3B cells, transfected with the HAMP-Luc reporter vector in the presence of ALK2^wt~ ^MYC, ALK2^R206H"MYC or ALK2^R258S"MYC, were treated for 6 hrs with DMH1 (0,5 pg/ml) and then incubated for 15 hrs with Activin A (10 ng/ml) ± DMH1. Cells were lysed and luciferase activity was quantified and normalized to an untreated mean value of 1. RQ: relative quantification. A t-test analysis was applied. Asterisks in black refer to: ActA or DMH1 vs ut; Asterisks in blue refer to: ActA+DMM vs ActA. Error bars indicate SD. ^**p< .01 ; ^***p< .001 ; ^****p< .0001.

Figure 21 : Hepcidin expression and liver iron content in mice treated with tacrolimus.

A) Liver hepcidin (Hamp) expression was quantified by qRT-PCR and normalized to the housekeeping gene Hprtl. B) Liver iron content (LIC). Error bars indicate SD. ^**p< .01.

Figure 22: The drug-competitive inhibition of ALK2-FKBP12 interaction renders ALK2 responsive to Activin A . A) Primary murine hepatocytes were treated for 3 hrs with Tacrolimus (TAC, 1 g/rnl) or vehicle and then incubated for 5 hrs with Activin A (0-10 ng/ml) in the presence or absence of TAC. Total RNA was isolated and Id1 expression was quantified by qRT-PCR. mRNA expression ratio was normalized to an untreated mean value of 1. Hprtl was used as housekeeping gene. B) RNA was purified from primary murine hepatocytes treated with Torinl (T1 , 100nM), rapamycin (RAPA, 100 nM) or a combination of RAPA plus Activin A (10 ng/ml). Id1 expression was measured by qRT-PCR as described in A. C, D) Hep3B cells, transfected with the hepcidin promoter reporter vector (HAMP-Luc) (C) or the BRE luciferase reporter vector (BRE-Luc) (D), were treated with GPI-1046 (100 μg/ml) or vehicle for 5 hrs and then incubated or not with Activin A (10 ng/ml) ± GPI-1046 for 15 hrs. Luciferase activity was normalized to an untreated mean value of 1. Representative experiments, made in triplicate, are shown. RQ: relative quantification. Error bars indicate SD. A t-test analysis was applied. Asterisks in black refer to: -GPI-1046 vs +GPI-1046. Asterisks in blue refer to: GPI-1046/-ActA vs GPI-1046/+ActA. ^* p< .05; ^**p< .01 ; ^***p< .001 ; ^****p< .0001 .

Figure 23: Modulation of hepcidin by FKBP12-ALK2 interaction. A) Normal iron concentration: hepcidin levels are maintained preferentially through BMP2-ALK3-HJV signaling. ALK2 is inactive since bound to FKBP12. B) In inflammation, Activin A is increased and FKBP12 is released form ALK2 through unknown mechanism/s. Activin A signals through ALK4 to activate SMAD2/3 phosphorylation, and also through ALK2 thanks to FKBP12 displacement. ALK2-mediated SMAD1/5/8 phosphorylation upregulates hepcidin. C) In iron overload conditions, BMP6 is upregulated and signals preferentially through ALK2 thanks to FKBP12 displacement.

Figure 24: Hepcidin and iron changes by TAC treatment in Hjv KO mice. Hjv KO mice were treated with TAC or vehicle for 28 days. Liver expression of hepcidin (A), the BMP- SMAD target gene Id1 (B) and the ligand Bmp6 (C were evaluated by qRT-PCR using Hprtl as housekeeping gene. Spleen Iron Content (SIC) was evaluated as well. ^*: p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Material and methods

Materials and Methods referred to figures 1-10

Mouse models and bone marrow transplantation (BMT)

Hbb^th3/+ mice (with heterozygous deletion of both betal and beta2 genes, a model of non transfusion-dependent β-thalassemia intermedia) on a pure C57BL/6N background (Jackson Laboratories, Bar Harbor, ME, USA) and Tfr2^~f~ mice on a pure 129S2 background (Roetto et al., 2010) were crossed obtaining the Tfr2^+/~ and Tfr2^+/VHbb^{t 3/+} progeny on a mixed C57/129S2 background; then these animals were back-crossed generating Tfr2^~'^~ /Hbb^th3/+, Tfr2^+/VHbb^th3/+ and Hbb^th3/+ mice. Mice were given a standard diet and blood was collected by tail vein puncture for hematological analyses at 4, 10, 15, 20, 25, 29, 33 and 36 weeks of age. A cohort of animals was sacrificed at 10 weeks of age, while the others will be sacrificed at 36 weeks of age. At sacrifice blood was collected for transferrin saturation (TS) determination and serum EPO quantification. Liver, spleen, kidneys and heart are weighed, dissected and snap-frozen immediately for RNA analysis or dried for tissue iron quantification or processed for FACS or histological analysis. Blood marrow (BM) cells were harvested and processed for flow cytometry analysis or for Ter1 19+ cells purification and RNA analysis.

BM cells isolated from some animals sacrificed at 10 weeks of age were used for bone marrow transplantation (BMT) procedure as previously described (Nai et al., 2015). In brief, 5^*10⁶ BM cells recovered from the femur of Tir2^''^'l bb^m double mutants and Hbib^f/,3/+ rriice (expressing the CD45.2 B-cell surface antigen) were transplanted into lethally irradiated C57BL/6-Ly-5.1 male mice (expressing the CD45.1 B-cell surface antigen), generating thalassemic mice with (Hbb^th3/+) or without Tfr2 (Tfr2^BMKO/Hbb^th3/+) in the BM. Mice were given a standard diet and blood was collected by tail vein puncture for hematological analyses at 9, 13, 17, 21 and 22 weeks after BMT. A cohort of animals was sacrificed 9 weeks after BMT, while the other at 22 weeks. At sacrifice animals were analyzed as previously described and donor/host chimerism was evaluated.

All mice were maintained in the animal facility of San Raffaele Scientific Institute (Milano, Italy) in accordance with the European Union guidelines. The study was approved by the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute.

Hematological Analysis

Hemoglobin (Hb) concentration, red blood cell (RBC) count and erythrocyte indexes (MCV, MCH) were measured on a Sysmex KX-21 automated blood cell analyzer (Sysmex America).

Transferrin saturation was calculated as the ratio between serum iron and total iron binding capacity, using The Total Iron Binding Capacity Kit (Randox Laboratories Ltd.), according to the manufacturer's instructions. Serum EPO levels were measured using mouse EPO quantikine set (R&D System), according to the manufacturer's instructions.

Tissue iron content

To measure iron concentration, tissue samples were dried at 1 10°C overnight, weighed, and digested in 1 mL of acid solution (3M HCI, 0.6M trichloroacetic acid) for 20 hours at 65°C. The clear acid extract was added to 1 mL of working chromogen reagent (1 volume of 0.1 % bathophenanthroline sulfate and 1 % thioglycolic acid solution, 5 volumes of water, and 5 volumes of saturated sodium acetate). The solutions were then incubated for 30 minutes at room temperature until color development and the absorbance measured at 535 nm. A standard curve was plotted, using an acid solution containing increasing amounts of iron diluted from a stock solution of Titrisol iron standard (Merck). Flow cytometry

Percentage of reticulocytes was determined by flow cytometry after staining with thiazole orange dye (Sigma-Aldrich).

For phenotypic analysis by flow cytometry, BM and spleen cells were pre-treated with rat- anti-mouse CD16/CD32 (BD Pharmingen) in order to block unspecific Ig binding, and subsequently stained with PE rat anti-mouse Ter1 19 (BD Biosciences) and APC rat anti- mouse CD44 (BD Biosciences) for 30 min in the dark at 4°C.

Donor/host chimerism was evaluated on mouse peripheral blood and BM cells from transplanted mice by using FITC-conjugated anti-mouse CD45.1 (BD Biosciences) and APC-conjugated anti-mouse CD45.2 antibodies (BD Biosciences). Cells were analyzed at FACS Canto™ II.

Isolation of Ter1 19+ cells

BM cells were incubated with MACS Ter1 19 MicroBeads (Myltenyi Biotec) and separation was performed following manufacturer's instructions. Both the positive and the negative fractions were recovered for RNA analysis.

Quantitative RT-PCR

Total Liver and Spleen: RNA was extracted from murine liver and spleen using the guanidinium thiocyanate-phenol-chloroform method (Trizol Reagent) (Invitrogen), following manufacturer's recommendations. RNA (2 μg) was used for quantitative polymerase chain reaction (qPCR) analysis for first-strand synthesis of cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to manufacturer's instructions. For real-time PCR analysis, specific murine Assays-on- Demand products (20x) and TaqMan Master Mix (2x) (Applied Biosystems) or specific murine oligos (designed using the Universal ProbeLibrary Assay Design Center by Roche and generated by PRIMM) and SYBRgreen Master Mix (2x) (Applied Biosystems) were used. Each cDNA sample was amplified in triplicate and the RNA level was normalized to the corresponding level of Hprtl or Gapdh mRNA. Primers used for qRT-PCR are in Tables 1 and 2.

Table 1. Oligonucleotide primers used for qRT-PCR by TaqMan

Table 2. Oligonucleotide primers used for qRT-PCR by SybrGreen

Isolated BM cells: RNA was extracted from BM cells using the mini RNeasy kit (Qiagen), following manufacturer's instructions. RNA (500 ng) was used for cDNA synthesis with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer's instructions. For real-time PCR analysis, specific murine oligos (designed using the Universal ProbeLibrary Assay Design Center by Roche and generated by PRIMM) and SYBRgreen Master Mix (2x) (Applied Biosystems) were used; the reactions were run on 7900HT Fast Real-Time PCR System (Applied Biosystems) in a final volume of 15 μΙ_. Each cDNA sample was amplified in duplicate and the RNA level was normalized to the corresponding level of Gapdh mRNA. Primers used for qRT-PCR are in Table 2 (see above).

Statistics

Data are presented as mean ± SD. Unpaired 2-tailed Student's t-test was performed using using GraphPad Prism 5.0 (GraphPad). P<0.05 was considered statistically significant. Materials and Methods referred to figures 11-23

Expressing Vectors

The pCMV6-ALK2^MYC-^FLAG and the pCMV6-FKBP12^MYC-^FLAG expressing vectors were from OriGene (Rockwille, MD, USA). The pcDEF/FLAG-mSmad1 was kindly provided by Prof. Takenobu Katagiri (Fukuda T et al., 2009) Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Japan). The pGL2-HAMP-Luc was generated as described (Pagani et al., 2008). The pGL3-BRE-Luc and the pGL3-CAGA- Luc reporter vectors were a kind gift of Prof. Stefano Piccolo (Inui M. et al., 201 1 ) (University of Padua, Italy). The ALK2 mutants R206H, Q207E and R258S were generated by mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol. The MYC-tagged ALK2 variants were obtained by mutagenesis through insertion of a STOP codon after the MYC sequence. The pCMV6-FKBP12-FLAG was generated through mutagenesis and in-frame excision of the MYC-tag and the pCMV6-FKBP12 by the insertion of a STOP codon after the cDNA sequence through mutagenesis. Primers used for mutagenesis are in Table 3. Mutagenesis were verified by DNA sequencing.

Table 3 - Oligonucleotides for mutagenesis

Plasmid mutated Sequences (5'→ 3')

pCMV6-ALK2^R206H-^MYC-^FLAG FW: acaaagaacagtggctcaccagattacactgttgg SEQ ID

NO: 1 1

REV: ccaacagtgtaatctggtgagccactgttctttgt SEQ ID NO: 12 pCMV6-ALK2^Q207E-^MYC-^FLAG FW: aaagaacagtggctcgcgagattacactgttggag SEQ ID

NO: 13

REV: ctccaacagtgtaatctcgcgagccactgttcttt SEQ ID NO: 14

pCMV6-ALK2^R258S"MYC-^FLAG FW: gtacaacactgtgatgctgagcaatgaaaatatcttaggtttc

SEQ ID NO: 15

REV: gaaacctaagatattttcatggctcagcatcacagtgttgtac SEQ ID NO: 16

pCMV6-ALK2 FW: ctcatctcagaagaggatctgtgagcaaatgatatcctggatta

R206H/Q207E/R258S-MYC SEQ ID NO: 17

REV: taatccaggatatcatttgctcacagatcctcttctgagatgag SEQ ID NO: 18

pCMV6-FKBP12^FLAG FW: ggatctggcagcaaatgatctcgaggattacaaggatgacg

SEQ ID NO: 19

REV: eg tcatccttg taatcctcg ag atcatttg ctg ccag atcc

SEQ ID NO: 20

pCMV6-FKBP12 FW: gtggagcttctaaaactggaatagcgtacgcggcc SEQ ID

NO: 21

REV: ggccgcgtacgctattccagttttagaagctccac SEQ ID NO: 22

Cell Culture, reagents and treatments

Cell culture media were from Thermo Fisher Scientific (Waltham, MA, USA). Rapamycin, Torinl and Cyclosporin A were from Tocris (Tocris Bioscience, Bristol, UK). Tacrolimus/FK506 and GPI-1046 were from Cayman Chemicals (Michigan, USA). DMH1 and β-cyclodextrin were from Sigma-Aldrich (Milan, Italy). BMP2, BMP6 and Activin A were from R&D Systems (Minneapolis, MN, USA). HuH7 cells (Verga-Falzacappa M.V. et al., 2008) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 200 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). Hep3B cells (BS TCL 78, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, Italy) were cultured in Minimal essential medium (MEM), supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 200 mg/ml streptomycin, 1 mM sodium pyruvate, and 10% heat-inactivated FBS.

Primary hepatocytes were isolated following the standard two-step perfusion method described in the literature (Goncalves et al., 2007; Klaunig et al., 1981 ; Shen et al., 2012) with minor modifications. Mice were anesthetized with Avertin. For in situ perfusion, Vena Cava Inferior was cannulated and perfused (pump flux: 5ml/min) with Liver Perfusion Medium (Thermo Fisher Scientific) and Liver Digest Medium (Thermo Fisher Scientific). After digestion, the liver capsule was mechanically disrupted to release cells into the medium. Debris and membranes were removed by a 100 μ m cell strainer. Hepatocytes (HCs) were separated from non-parenchymal cells (NPCs) through low speed centrifugation (50 g for 3 minutes). HCs were resuspended in Williams-E medium (4% FBS, 1 % P/S, Glutamax) (Thermo Fisher Scientific) and plated into collagen-coated 12-well (3^*10⁵ cells/well). Four hours after isolation, in a group of hepatocytes cell culture media was replaced with 4% FBS Williams-E medium (1 % P/S, Glutamax) for 18 hrs. Another group of hepatocytes was serum starved for 3 hrs. Serum supplemented cells were incubated for 3 hrs with tacrolimus (1 μ g/ml) in serum free media and then Activin A (10 ng/ml) was added for additional 5 hrs. Serum starved hepatocytes were incubated for 18 hours in serum free media with Torinl (100 nM), rapamycin (100 nM), tacrolimus (1 μ g/ml), Cyclosporin A (1 M g/ml) and GPI-1046 (100 μ g/ml). Cells were lyzed and RNA isolated for gene expression analysis.

Luciferase Assay

Hepcidin promoter activation was studied by using the pGL2-Hamp-Luc in which the firefly luciferase cDNA is under the control of the 2.9 Kb human hepcidin promoter region (Pagani et al., 2008). SMAD1/5/8 activation was measured by using the pGL3-BRE-Luc plasmid, in which the firefly luciferase cDNA was under the control of BMP Responsive Element (BRE) sequences obtained from human ID1. SMAD2/3 activation was assayed by using the pGL3- CAGA-Luc vector, in which the luciferase cDNA was under the control of the CAGA sequences. The pGL3-BRE and pGL3-CAGA vectors were kindly provided by Stefano Piccolo (Inui M. et al., 201 1 ), University of Padua, Padua, Italy.

Hep3B cells, seeded at 70%-80% of confluence in a 48-well plate, were transiently transfected using Lipotectamine, according to manufacturer's instructions (Invitrogen, Carlsbad, CA), with 250 ng Hamp-Luc, 200 ng BRE-Luc, 200 ng CAGA-Luc in combination with 15 ng of pRL-TK Renilla luciferase vector (Promega) as a control of transfection efficiency, and with or without expressing vectors encoding wild type or mutant ALK2 (10- 100 ng). Eighteen hours after transfection cells were serum-starved (MEM + 2% FBS), and, when indicated, incubated with tacrolimus, rapamycin, Torinl , GPI-1046, DMH1 or dorsomorphin in low-serum media.

After 24 hrs, cells were lysed and luciferase activity was determined according to manufacture's protocol (Dual Luciferase Reporter Assay, Promega). Relative luciferase activity was calculated as the ratio of Firefly (reporter) to Renilla luciferase activity and expressed as a multiple of the activity of cells transfected with the reporter alone.

Quantitative Real Time PCR

Total RNA was extracted from cells and murine tissues with the UPzol reagent (Biotechrabbit, Dusseldorf, Germany) and cDNA was synthetized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Massachusetts, USA). Gene expression levels were measured by quantitative real-time PCR using the SybrGreen PCR Master Mix (Applied Biosystems) or the TaqMan Gene Expression Master Mix (Applied Biosystems). HPRT1 or GAPDH were used as housekeeping genes. Primers used for qRT- PCR are listed in Table 4.

Table 4 - Oligonucleotides for qRT-PCR

Gene Sequence (5'→ 3') or Id

SybrGreen GAPDH FW: ccccggtttctataaattgagc

SEQ ID NO: 23

REV: caccttccccatggtgtct

SEQ ID NO: 24

HAMP FW: ctgttttcccacaacagacg

SEQ ID NO: 25

REV: agatggggaagtgggtgtct

SEQ ID NO: 26

ID1 FW: tccagcacgtcatcgactac

SEQ ID NO: 27

REV: tcagcgacacaagatgcg

SEQ ID NO: 28

TaqMan Hprtl Mm01318743_m1 Hamp Mm00519025_m1

Id1 Mm00775963_g1

Western Blot Analysis of phospho-SMAD proteins

HuH7 cells were transfected with wild type or mutants ALK2. When indicated, cells were transfected also with Smad1 ^FLAG expressing vector. After 40 hrs, cells were lysed in NET/Triton buffer plus protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). Protein extracts (20-50 μg) were diluted in Laemmli sample buffer, incubated 5 minutes at 95°C, subjected to 10% or 12% SDS-PAGE and then transferred to Hybond C membrane (Amersham Bioscience Europe GmbH, Freiburg im Breisgau, Germany) by standard Western blot techniques. Blots were incubated with anti-PhosphoSMAD1/5/8 (1 :1000, Cell Signaling, Danvers, MA), anti-SMAD1 (1 :1000, Cell Signaling), anti-FLAG (1 :1000, Sigma- Aldrich), anti-MYC (1 :1000, Cell Signaling), according to standard procedures. Blots were incubated with relevant HRP-conjugated antisera and developed using a chemiluminescence detection kit (ECL; Amersham Biosciences Europe GmbH).

Immunoprecipitation

HuH7 cells transfected with FKBP12^FLAG and ALK2^MYC (wild type or mutants) expressing vectors were treated with tacrolimus (1 μg/ml), rapamycin (100 nM) or GPI-1046 (100 μg/ml), when indicated. One mg of cell lysates was incubated with the anti-FLAG M2 affinity gel (Sigma Aldrich) at 4°C for 2 hrs. After gel washing, samples were eluted with 20 μΙ of Laemmli sample buffer (without β-mercaptoethanol) and incubated at 95°C for 5 minutes. After centrifugation, β-mercaptoethanol was added to supernatants. Samples were then subjected to 12% SDS-PAGE and immunodetection was performed as described in Western Blot Analysis.

Mice treatment

Wild type C57BL/6 male mice aged 7 weeks were purchased from Charles River. Mice were housed under a standard 12-hour light/dark cycle with water and chow ad libitum in a pathogen-free animal facility of San Raffaele Scientific Institute, in accordance with the European Union guidelines. The study was approved by the Institutional Animal Care and Use. A single dose of tacrolimus (10 mg/kg in DMSO) was administered by subcutaneous injections. Mice were sacrificed at 3, 6, 9 and 18 hrs post-injection. Control mice were injected with DMSO and sacrificed at 3 and 18 hrs post-injection. Mice were anesthetized and then sacrificed by cervical dislocation. Liver was dissected and immediately snap- frozen for RNA analysis. Liver and spleen were dried for tissue iron quantification according to standard procedures.

Materials and Methods referred to figure 24

Hjv KO male mice (Huang FW et al., JCI 2005), 9-1 1 weeks of age, were anesthetized and subcunaneosly implanted with miniosmotic pumps (Alzet, 100 μΙ capacity, 0.1 1 μΙ/hrs drug delivery). The drug was prepared as followed. Tacrolimus (FK506, Cayman Chemicals) were resuspended in DMSO 100% (5 mg/ml) and then diluited 1 :1 with EtOH 15%. The final concentration of the drug in the pump was 2.5 mg/ml. The amount of the drug delived in 24 hrs is 8.9 The pumps were filled with FK506 or vehicle (DMSO 100%, EtOH 15%; 1 :1 ). Mice were sacrificed 28 days later and analyzed for iron and hematological parameters according to standard procedures (Colucci et al., Blood 2017). Briefly, total RNA was isolated from the liver, retrotranscribed and analyzed for hepcidin, Id1 and Bmp6 expression. Liver and spleen were processed for quantification of total iron content. EXAMPLES

Loss of total Tfr2 ameliorates anemia of Hbb^th3/+ mice with a gene-dosage effect

To generate thalassemic mice with germ-line homozygous or heterozygous deletion of Tfr2, Hbb^{t 3/+} males and Tfr2^~f~ females were crossed and heterozygous animals intercrossed to generate mice with the desired genotype. Mice, maintained at a standard diet, were analyzed for hematological parameters from 4 to 25 weeks of age. Red blood cells number (RBC, Figure 1A) and hemoglobin levels (Hb, Figure 1 B) are both increased in mice lacking Tfr2 and this effect is maintained during time. This is not accompanied by alterations of erythrocyte indexes: MCV (Figure 1 C) and MCH (Figure 1 D) are comparable in thalassemic mice with or without one or two copies of Tfr2. Interestingly, a gene dosage effect of Tfr2 deletion was observed on Hb and RBCs improvement. The amelioration of hematologic phenotype occurs despite a more severe iron burden in Tfr2^~'^~lHbb^m,+ mice compared to Hbb^th3/+ mice, as shown by higher liver iron content (LIC, Figure 1 C), whereas spleen iron content (SIC) is comparable between thalassemic mice with or without Tfr2 (Figure 1 D).

Deletion of erythroid Tfr2 induces a long lasting amelioration of anemia in Hbb^th3/+ mice

Tfr2 is expressed in the liver and in erythroid cells. The inventors hypothesize that the improvement of the anemia phenotype in thalassemic animals lacking total Tfr2 is due to the effect of erythroid TFR2 rather than hepatic TFR2.

To demonstrate this hypothesis, lethally irradiated wild type male mice were transplanted with BM derived cells from Hbb^th3/+ and T 2^~'^~lHbb^m,+ donor mice. Nine weeks after bone marrow transplantation (BMT) engraftment of donor cells, evaluated by FACS analysis, was nearly complete (about 99%). Mice were fully analyzed at 9 and 22 weeks after BMT, whereas hematological parameters were followed monthly. As shown in Figure 2, a consistent and persistent amelioration of RBCs count (Figure 2A) and Hb levels (Figure 2B) was observed in thalassemic mice lacking erythroid Tfr2. At difference with what observed in Tfr2^'f'IHbb^m/+, deletion of BM Tfr2 in thalassemic mice causes a decrease in both MCV (Figure 2C) and MCH (Figure 2D), suggesting iron restricted erythropoiesis, likely related to the lower iron burden of these animals in which thalassemia is "acquired" through BMT. Although Hb and RBCs are stable during time in Hbb^{t 3/+} mice, at 22 weeks after BMT a trend towards phenotype worsening is observed in Tfr2^BMKO/Hbb^th3/+ animals, with decreasing Hb levels that reach a value comparable to Hbb^th3/+ animals. RBCs count persits significantly higher in thalassemic mice lacking erythroid Tfr2.

Tfr2^BMKO/Hbb^th3/+ young mice have a more effective erythropoiesis than Hbb^th3/+, but comparable splenomegaly and iron-overload

At 9 weeks after BMT both Tfr2^BMKO/Hbb^th3/+ and Hbb^th3/+ animals appear viable and indistinguishable from one another, with comparable body weight (Figure 3A). As expected, the erythropoiesis of Hbb^{t 3/+} mice is highly compromised, with all the hallmarks of the disease: splenomegaly (Figure 3B), high proportion of immature erythroid progenitor cells both in the BM and in the spleen (Fig. 3C), high percentage of reticulocytes (Fig. 3D), and remarkably high serum EPO levels (Fig. 3E). The absence of Tfr2 in the erythroid compartment moderately improves erythropoiesis in Hbb^{t 3/+} mice, reducing reticulocytes percentage (Figure 3D) and inducing a trend towards decreased accumulation of erythroid precursors stage lll-IV (polychromatic-orthochromatic erythroblasts and reticulocytes) and increase in stage V cells (mature RBCs) both in BM and spleen (Figure 3C). The amelioration of anemia in double mutant mice decreases serum EPO levels (Figure 3E), as expected, but surprisingly spleen sizes are not reduced (Figure 3B).

Iron burden in liver (LIC, Figure 4A), spleen (SIC, Figure 4B), kidney (KIC; Figure 4C) and heart (HIC, Figure 4D) is comparable in thalassemic mice with or without erythroid Tfr2. Compatible with these results, hepatic hepcidin levels {Hamp, Figure 4E), and transferrin saturation (TS, Figure 4F) are unaffected by the lack of erythroid Tfr2. Overall these data suggest that anemia improvement is not associated with decreased iron accumulation.

The EPO-EPOR signaling pathway is overactive in erythroid tissues from young Tfr2^BMKO/Hbb^th3/+ mice

In order to elucidate the mechanisms of the observed amelioration of the thalassemic phenotype induced by the absence of Tfr2, the inventors investigated the EPO-EPOR signaling pathway to assess whether it was overactive as the inventors demonstrated in wt erythroid cells (Nai et al., 2015). Relative to serum EPO levels, BM cells from Tfr2^BMKO/Hbb^th3/+ mice express higher levels of the E PO-E PO R- J AK2-STAT5 target gene Erfe (Kautz et al., 2014) (Figure 5A), but not of Bcl-xl, another target of the same pathway (Silva et al., 1999; Socolovsky et al., 1999; Socolovsky et al., 2001 ) (Figure 5B). In the same cells the expression of Fasl, a pro-apoptotic target inhibited by the EPO-EPOR-PI3K-AKT- FOXO3a pathway (Kashii et al., 2000) was significantly reduced (Figure 5C), while the expression of Epor itself, a target of the PI3K-AKT-GATA1 pathway (Palani et al., 2008), was unaltered (Figure 5D). On the contrary in the more homogeneous erythroid tissue of the spleen of thalassemic mice, both the JAK2-STAT5 and PI3K-AKT signaling pathways are upregulated in Tfr2^BMKO/Hbb^{t 3/+} mice relative to serum EPO. Indeed, the expression levels of Erfe (Figure 6A), Bcl-xl (Figure 6B) and Epor (Figure 6D) are higher in double mutant mice relative to thalassemic controls, while Fasl mRNA levels are reduced (Figure 6C).

Tfr2^BMKO/Hbb^th3/+ old mice further improve erythropoiesis and reduce iron accumulation

At 22 weeks after BMT both Tfr2^BMKO/Hbb^th3/+ and Hbb^th3/+ animals appear viable and indistinguishable from one another, with comparable body weight (Figure 7A). The erythropoiesis of Hbb^th3/+ mice is similar to that observed at 9 weeks after BMT. However, despite the reduction of Hb levels and RBCs count, the absence of Tfr2 in the erythroid compartment further improves erythropoiesis in Hbb^{t 3/+} mice as shown by the reduced percentage of circulating reticulocytes (Figure 7B) and a significant reduction of stage IV erythroid precursors and increased stage V cells both in BM (Figure 7C) and spleen (Figure 7D). Also serum EPO levels are reduced in double mutant mice as compared to Hbb^{t 3/+} animals (Figure 7E), but, as observed at 9 weeks after BMT, spleen size is unmodified (Figure 7F).

At this time point iron accumulation in liver (LIC, Figure 8A), spleen (SIC, Figure 8B), kidney (KIC; Figure 8C) and heart (HIC, Figure 8D) is strongly reduced in Tfr2^BMKO/Hbb^th3/+ mice compared to thalassemic control animals, although still inappropriately high compared to wild type mice. The only exception is iron in the spleen that appears to be strongly decreased in Tfr2^BMKO/Hbb^{t 3/+} animals compared to Hbb^{t 3/+} mice. In accordance with reduction of LIC, Hamp is decreased (Figure 8E), suggesting that, on a long-term, the lack of Tfr2 in the thalassemic bone marrow induces changes in iron homeostasis. On the contrary, transferrin saturation is comparable between Tfr2^BMKO/Hbb^{t 3/+} and Hbb^{t 3/+} mice (Figure 8F). The spleen EPO-EPOR signaling pathway is overactive in Tfr2^BMKO/Hbb^th3/+ old mice

Given the unclear results obtained on total BM in animals analyzed 9 weeks after BMT, the inventors decided to isolate Ter1 19+ (erythroid) cells from the BM in this cohort of mice. However, the inventors observed no differences in the expression levels of Erfe (Figure 9A), Bcl-xl (Figure 9B) and Epor (Figure 9C) between Tfr2^BMKO/Hbb^th3/+ and Hbb^th3/+ mice relative to serum EPO levels. Only the mRNA level of Fasl was significantly reduced in Tfr2^BMKO/Hbb^th3/+ animals (Figure 9D), confirming the overactivation of the PI3K-AKT- FOX03a signaling pathway. On the contrary in the spleen, as occurred at 9 weeks after BMT, both the JAK2-STAT5 and PI3K-AKT signaling pathways appear overactive in Tfr2^BMKO/Hbb^th3/+ mice. Indeed, the expression of Erfe (Figure 10A), Bcl-xl (trend toward increase, Figure 10B) and Epor (Figure 10C) relative to serum EPO levels is higher in double mutant mice than in thalassemic controls, while the mRNA levels of Fasl are reduced (Figure 10D). The mTOR inhibitor rapamycin increases hepcidin expression activating the BMP- SMAD pathway

To investigate whether hepcidin activation by rapamycin was mTOR dependent, human hepatoma derived cells were incubated with either the mTOR complex 1 (mTORCI ) inhibitor rapamycin, or Torinl , an ATP-competitive inhibitor of mTORCI and mTORC2. Both drugs efficiently inhibit mTOR signaling, as demonstrated by decreased phosphorylation of the mTOR target S6 ribosomal protein (S6RP) (Figure 1 1A). Rapamycin upregulates endogenous hepcidin expression in Hep3B cells (Figure 1 1 B) and, as reported (Mleczko-Sanecka et al., 2014), in murine primary hepatocytes (Figure 1 1 C), On the contrary, Torinl is ineffective, suggesting that hepcidin modulation is not dependent on mTOR inhibition but, rather, a rapamycin-specific effect.

Next, the inventors asked whether rapamycin modulates the BMP-SMAD pathway. Rapamycin treatment upregulates the luciferase activity in hepatoma cells transfected with the BMP responsive element (BRE)-Luc reporter vector (Figure 1 1 D) that expresses the luciferase cDNA under the control of an element exclusively activated by the SMAD1/5/8- SMAD4 complex. In addition, rapamycin, but not Torinl , increases the endogenous expression of the BMP-SMAD target gene Inhibitor of DNA Binding 1 (ID1 ), both in Hep3B cells (Figure 1 1 E) and in murine primary hepatocytes (data not shown). Overall these data demonstrate that rapamycin increases hepcidin expression through the activation of the BMP-SMAD signaling pathway.

Drugs targeting FKBP12 activate hepcidin through the BMP-SMAD pathway

To inhibit mTOR, rapamycin complexes with FKBP12, an immunophilin reported to interact with BMP type I receptors, to avoid ligand-independent activation of the pathway (Chaikuad et al., 2012; Spiekerkoetter et al., 2013; Wang et al., 1996). To explore the potential role of FKBP12 in hepcidin activation the inventors modulated FKBP12 binding by using tacrolimus (FK506), that interacts with the same FKBP12-binding pocket that binds rapamycin (Wilson et al., 1995). Since tacrolimus exerts its immunosuppressive effect inhibiting calcineurin, as control cells were treated with cyclosporine A that inhibits calcineurin through a different mechanism. Tacrolimus upregulates the luciferase activity of Hep3B cells transfected with the BRE-Luc reporter vector, indicating a SMAD1/5/8-SMAD4-dependent signaling (Figure 12A). In agreement, both endogenous hepcidin (Figure 12B) and ID1 (Figure 12C) are upregulated in Hep3B cells treated with tacrolimus, but not in cells treated with cyclosporine A. A similar effect on hepcidin (Figure 12D) and Id1 (Figure 12E) is observed in murine primary hepatocytes, where the effect of cyclosporine A on hepcidin is even suppressive. To further exclude any potential contribution of the immunosuppression on hepcidin regulation, the inventors used GPI-1046. This synthetic compound, designed based on tacrolimus-FKBP12 complex structure, interacts with FKBP12, but lacks immunosuppressive activity (Sich et al., 2000; Sun et al., 2003). GPI-1046 treatment increases the luciferase activity in Hep3B cells transfected with both BRE-Luc (Figure 18A) and HAMP-Luc (Figure 18B) reporter vectors, the latter expressing the luciferase cDNA under the control of the hepcidin promoter region (Pagani et al., 2008). In addition, GPI- 1046 treatment upregulates endogenous hepcidin (Figure 18C) and Id1 (Figure 18D) expression in murine primary hepatocytes. Overall these data indicate that FKBP12 sequestration is sufficient to activate the BMP-SMAD signaling pathway and to increase hepcidin expression.

FKBP12 functionally interacts with the BMP type I receptor ALK2

Hepcidin activation is dependent on BMP type I receptors ALK2 and ALK3. ALK3 is crucial for basal activation of the pathway while both ALK2 and ALK3 regulate hepcidin in response to iron and BMP ligand (Steinbicker et al., 201 1 ). To discriminate which of the two receptors binds FKBP12 to suppress hepcidin expression, the inventors took advantage of the inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl) quinolone), which specifically blocks BMP signaling by targeting the intracellular kinase domain of ALK2 (Mohedas et al., 2013). If FKBP12 binds preferentially ALK2, its pharmacologic displacement in the presence of DMH1 will not activate hepcidin. On the contrary, if FKBP12 binds ALK3, DMH1 treatment will not interfere with the hepcidin activation by rapamycin/tacrolimus. As shown in Supplemental Figure 2, DMH1 strongly inhibits endogenous hepcidin upregulation by rapamycin (Figure 19A) and tacrolimus (Figure 19B), indicating that the effect is mediated by FKBP12 displacement from ALK2.

To further confirm the interaction of FKBP12 with ALK2, the human hepatoma cell line HuH7, transiently transfected with ALK2^wt and FKBP12, was treated with rapamycin, or tacrolimus, or GPI-1046 and the interaction between ALK2 and FKBP12 was assessed by immunoprecipitation. The inventors observed a strong interaction between FKBP12 and ALK2 that is completely abrogated by tacrolimus and significantly reduced by rapamycin and GPI-1046 (Figure 13A). The different efficiency shown by the three drugs on FKBP12 displacement is probably related to their different half-life or FKBP12 affinity.

To unequivocally demonstrate that FKBP12 binds preferentially ALK2, the inventors replaced amino acid residues within (R206, Q207) or closed to (R258) the glycine-serine- rich (GS) intracellular domain of the receptor, essential for FKBP12 binding (Hatsell et al., 2015; Hino et al., 2015). The inventors introduced pathological mutations reported in patients. Indeed R206H, Q207E and R258S ALK2 substitutions are responsible of the rare genetic dominant disorder Fibrodysplasia Ossificans Progressiva (FOP, OMIM #135100), characterized by heterotopic ossification of soft tissues secondary to high and uncontrolled activity of the BMP-SMAD pathway (Chaikuad et al., 2012). These mutations have been reported to be partially resistant to the suppressive effect of FKBP12 (Haupt et al., 2014). The inventors confirm by immunoprecipitation that both R206H and Q207E have defective binding to FKBP12, and that R258S fails to interact with the immunophilin (Figure 13B). To further investigate the FKBP12 binding ability of ALK2, Hep3B cells, transfected with the HAMP-Luc reporter vector and wild type or mutants ALK2, were treated with increasing concentration of tacrolimus to displace FKBP12. The inventors expected a reduced effect of tacrolimus in mutants ALK2 because of the low amount of associated FKBP12. As shown in Figure 20A, the fold change of hepcidin activation by tacrolimus is lower in cells transfected with mutants than with wild type ALK2, suggesting that the former have a reduced FKBP12 binding capacity. Consistent with this finding, phosphorylation of SMAD1/5/8 is higher in cells transfected with mutants than with wild type ALK2 (Figure 13C). This experiment confirms that defective FKBP12-ALK2 interaction increases BMP- SMAD signaling, causing hepcidin (Figure 13D) and ID1 (Figure 13E) upregulation. Activation of hepcidin by ALK2 mutants is dose-dependent and is blunted by the inhibitor of the BMP-SMAD pathway dorsomorphin (Figure 13F), as assessed by the HAMP-Luc assay. Interestingly, overexpression of wild type ALK2 does not activate the signaling pathway in the absence of the ligand.

To further confirm the role of FKBP12 as modulator of ALK2 activity and hepcidin expression, Hep3B cells were transfected with the HAMP-Luc reporter vector, wild type or mutants ALK2 and increasing concentrations of FKBP12. As shown in Figure 13G, overexpression of FKBP12 is able to partially inhibit the activity of ALK2 mutants on hepcidin promoter, whereas has no effect in wild type ALK2 transfected cells, suggesting that in the absence of the ligand ALK2 is inactive, likely blocked by endogenous FKBP12. Since Hep3B cells express endogenous ALK2 and ALK3, these results indirectly confirm that FKBP12 acts preferentially through ALK2 binding.

Hemojuvelin is dispensable for FKBP12-dependent hepcidin regulation

The main activator of hepcidin, with an in vivo role, is the BMP coreceptor hemojuvelin (HJV). Inactivation of HJV causes juvenile hemochromatosis in humans and severe iron overload in mice, due to extremely low hepcidin levels. To investigate whether HJV is essential for FKBP12-dependent hepcidin regulation murine hepatocytes isolated from Hjv KO mice were treated with rapamycin or tacrolims or GPI-1046. All drugs upregulate hepcidin, demonstrating that the BMP coreceptor Hjv is dispensable for hepcidin activation mediated by loss of FKBP12-ALK2 interaction (Figure 14A). Irrespective of the different basal hepcidin levels the fold change of hepcidin activation by tacrolimus is the same in wild type and Hjv KO hepatocytes (Figure 14B). Activation of hepcidin by tacrolimus decreases at the highest drug concentrations (Figure 14C), likely because calcineurin inhibits hepcidin in both wild type (Figure 12D) and Hjv KO hepatocytes (Figure 14A). Acute tacrolimus treatment upregulates hepcidin in vivo

To explore whether FKBP12 is functional in vivo, adult C57BL/6 male wild type mice were treated with a single subcutaneous injection of tacrolimus (10 mg/kg) or vehicle (DMSO) for different time points (from 3 to 18 hrs) (Figure 15A). To normalize for variation of liver iron the inventors expressed hepcidin as the hepcidin/LIC (liver iron concentration) ratio (hepcidin mRNA levels and LIC are shown in Figure 21 A and 21 B, respectively). The ratio was significantly increased (2x) by tacrolimus at 6 hrs post-injection (Figure 15B). This effect was accompanied by increased spleen iron content (Figure 15C) pointing out that the drug may modulate hepcidin in vivo, at least in an acute setting. FKBP12 regulates the ALK2 ligand responsiveness to Activin A

Since ALK2 is responsible for the ligand-dependent hepcidin activation (Steinbicker et al., 201 1 ), the inventors investigated the response of wild type and mutants ALK2 to different ligands in hepatoma cells. The inventors tested BMP2, which is highly expressed in mouse liver endothelial cells and involved in hepcidin regulation (Koch et al., 2017), and BMP6, which is increased by liver iron content.

Mutants ALK2 activate hepcidin in a dose-dependent manner and with the same efficiency of wild type ALK2 when stimulated with both BMP2 (Figure 16A) and BMP6 (Figure 16B), suggesting that mutations do not affect the hepcidin response to the iron-related ligands. The inventors also tested Activin A that has been recently proposed to be a ligand of ALK2 mutants in Fibrodysplasia Ossificans Progressiva (Hatsell et al., 2015; Hino et al., 2015). As shown in Figure 16C, only mutants ALK2 upregulate hepcidin in response to Activin A, while wild type ALK2 is unresponsive.

To investigate the signaling pathway of hepcidin regulation induced by Activin A, the inventors analyzed both the TGF-β and the BMP-SMAD pathway by measuring the luciferase activity under the control of the CAGA and the BRE responsive elements, respectively.

In basal conditions the TGF-β pathway is comparable among ALK2^wt, ALK2^R206H and ALK2^R258S-transfected cells and is increased by Activin A with the same efficiency in both wild type and mutants ALK2 (Figure 16D). The BMP-SMAD signaling increased in cells transfected with mutants ALK2 and following BMP6 treatment, remains unchanged in wild type ALK2 transfected cells treated with Activin A (Figure 16E). On the contrary the BRE- Luc activity increases in cells transfected with mutants ALK2 in the presence of Activin A (Figure 16E). As a further confirmation of this finding the phosphorylation level of SMAD1/5/8, high in cells overexpressing mutants ALK2, is further increased by Activin A (Figure 16F), and the effect on hepcidin activation is strongly decreased by the ALK2- inhibitor DMH1 (Figure 20B). Thus Activin A upregulates the BMP-SMAD pathway only in the presence of mutants ALK2 that show impaired binding and are resistant to the effect of FKBP12. To investigate whether modulation of the FKBP12-ALK2 interaction may affect the receptor responsiveness, Hep3B cells, transfected with ALK2^wt, were treated with tacrolimus to displace FKBP12 and with increasing concentrations of Activin A. In this case ALK2^wt becomes responsive to Activin A, increasing the HAMP-Luc activity (Figure 17A). The effect of tacrolimus on Activin A response is maintained in primary murine hepatocytes (Figure 17B), mediated by the BMP-SMAD pathway, as shown by increased Id1 expression in primary hepatocytes (Figure 22A) and by upregulation of the BRE-Luc activity in Hep3B cells (Figure 17C). As expected, the TGF-β signaling, measured by CAGA-Luc activity, is increased by Activin A and unaffected by tacrolimus (Figure 17D).

The change of the ligand responsiveness is due to FKBP12 sequestration, since endogenous hepcidin (Figure 17E) and Id 1 (Figure 22B) expression are further enhanced through the BMP-SMAD pathway in murine primary hepatocytes treated with rapamycin and Activin A. In addition, the acquisition of Activin A responsiveness is independent from mTOR and calcineurin, since it occurs also in cells treated with GPI-1046 (Figure 22C), through the BMP-SMAD pathway activation (Figure 22D). Overall these results indicate that FKBP12 binding to ALK2 modulates the Activin A responsiveness of the receptor.

BMP6 synergizes with Activin A for hepcidin activation

Since BMP6 is upregulated by high iron levels, the inventors asked how the ALK2-FKBP12 interaction is regulated in this context. HuH7 cells, transfected with ALK2 and FKBP12, were treated with BMP6 increasing concentration and their interaction studied by immunoprecipitation. Less FKBP12 is bound to ALK2 at the highest BMP6 concentrations (Figure 13B), suggesting that phosphorylation of ALK2 induced by the ligand negatively interferes with FKBP12 binding. In agreement with this finding, Activin A synergizes with high BMP6 and further increases hepcidin activation in the HAMP-Luc assay (Figure 17F). Overall these results suggest that high levels of BMP6 in iron overload favor Activin A responsiveness of ALK2.

Chronic administration of TAC does not affect hematological parameters of Hjv KO mice

Hjv KO mice implanted with vehicle-filled or TAC-filled pumps were sacrificed after 28 days of treatment and hematological parameters were evaluated.

Table 5. Hematological parameters of Hjv KO mice treated with TAC or vehicle

RBCs Hb HCT MCV MCH PTL WBCs

(10⁶/μΙ) (g/dL) (%) (fL) (pg) (10³/μΙ) (10³/μΙ) vehicle 1 1 .7±1 19.4±1 .5 60.9±5.6 52.1 ±0.9 14.9±0.1 548.7±179.9 4.9±3.8

TAC 1 1 .4±1 .5 16.9±1 .8 58.8±9 51 .3±2.2 14.9±0.4 642.6±164.2 4.4±2.4 As shown in Table 5, TAC treated mice do not show any variation of RBCs count and Hb content, platelets and WBCs, with a differential comparable to that of vehicle treated mice.

Chronic administration of TAC upregulates hepcidin in Hjv KO mice through BMP- SMAD pathway activation

After 28 days of treatment, mice were sacrificed, and RNA was isolated from the liver. Hepcidin and Id1 were measured by qRT-PCR using Hprtl as housekeeping gene. Due to the strong correlation between mRNA and protein levels, hepcidin expression is considered an indirect measure of serum hepcidin (Colucci et al., Blood 2017). As shown in Figure 24A, hecpidin expression is increased in TAC-treated Hjv KO mice. This is paralleled by upregulation of the BMP-SMAD target gene Id1 (Figure 24B), thus suggesting that the BMP- SMAD pathway in upregulated by TAC administration. The hepcidin activator Bmp6, whose expression is upregulated by liver iron concentration, is unchanged by TAC treatment (Figure 24C). Spleen iron content was measured in vehicle and treated mice. Due to stabilization of the iron exporter ferroportin mediated by the lack of hepcidin, spleen iron (SIC) of Hjv KO mice is lower than wt animals. If hepcidin is upregulated in TAC-treated KO mice, the inventors would expect an increase of SIC. As shown in Figure 24D, a trend towards SIC increase was observed in TAC treated compared to vehicle treated animals. Discussion

Beta-thalassemias are disorders caused by mutations in the β-globin gene characterized by ineffective erythropoiesis, severe anemia, splenomegaly and iron overload. It has been estimated that about 1.5% of the global population (80 to 90 million people) are carriers of β-thalassemia, with about 60 thousand anemic and symptomatic individuals born annually, the great majority in the developing world. Traditional treatment is based on life long blood transfusions and iron chelation, a costly, demanding and far from optimal treatment. The only established and definitive cure for β-thalassemia is allogeneic bone marrow transplantation. However, this approach is limited by the scarcity of HLA-matched sibling donors and the risk of graft-versus-host disease after transplantation. Gene therapy will in the future offer an alternative approach to solve these criticisms since in progress clinical trials seem promising in decreasing transfusion needs (Negre et al., 2016). However, realistically only a minority of patients will be eligible for a gene-therapy based approach. For these reasons the search for alternative and/or combinatorial novel therapies is a real clinical need. Here the inventors show that the genetic loss of Tfr2 results in a long lasting impressive improvement of anemia and in amelioration of ineffective erythropoiesis in Hbb^{t 3/+} mice. This benefit is present early in life and persists up to 5 months in animals lacking both Tfr2 in the whole organism and selectively in the bone marrow, making TFR2 (and especially erythroid TFR2) a novel candidate therapeutic target to be tested in β- thalassemia. Data obtained in mice suggest that this treatment redistribute iron in the different organs but may deplete the spleen suggesting the need to monitor the degree of TFR2 inactivation. Results from animals with germ-line deletion of Tfr2 reveal a gene- dosage effect: this finding opens the possibility of a partial inhibition of Tfr2 to ameliorate the thalassemic phenotype without altering systemic iron homeostasis. Surprisingly splenomegaly is not reduced in the present model, despite the improvement of anemia and erythropoiesis: the overactivation of the EPO-EPOR-JAK2-STAT5 signaling pathway induced in the spleen by the absence of Tfr2 is likely responsible for this finding. Indeed, recent results demonstrated that the inhibition of this pathway induces an important reduction of splenomegaly in thalassemic mice, accompanied by only negligible amelioration of anemia (Casu et al., 2016).

Inventors' results prove that, as occurs in normal BM (Nai et al., 2015), Tfr2 deletion increases EPO sensitivity of thalassemic erythroid cells especially in splenic cells since EPO target genes are more expressed in Tfr2^BMKO/Hbb^th3/+ mice than in thalassemic control. However, at difference with results obtained in wt mice (Nai et al., 2015), in a thalassemic context the PI3K-AKT-FOXO3a pathway is overactive both in BM and spleen and is long- lasting, while the JAK2-STAT5 signaling is hyperactive mainly in the spleen and its activity decreases with time. It has been reported that PTPRC (CD45) is one of the most significantly upregulated genes by the EPO mediated PI3K-AKT pathway in CD34+ cells (Sivertsen et a., 2006). PTPRC is a suppressor of JAK kinase phosphorylation and, when absent in CD34+ cells, increases the number of EPO-dependent erythroid colonies (Irie- Sasaki et al., 2001 ).

At 9 weeks after BMT iron accumulation is not prevented by the loss of erythroid Tfr2 in all the tissues analyzed in thalassemic mice. The results suggest that it is also the case for 10- week-old animals with germ-line deletion of Tfr2. However, further iron accumulation is precluded in double mutant mice: indeed, at 22 weeks after BMT the iron burden of different organs is increased in thalassemic mice compared to 9 weeks after BMT, while this does not occur in Tfr2^BMKO/Hbb^th3/+ mice that have liver, kidney and heart iron comparable to values measured at 9 weeks while spleen iron is even lower. These results suggest that the persistently increased erythropoiesis consequent to the loss of erythroid Tfr2 strongly mobilizes iron from tissues and especially consumes spleen iron, a major site of erythropoiesis in these animals. This strong reduction of spleen iron is likely responsible for the worsening of the hematological phenotype observed in older Tfr2^BMKO/Hbb^{t 3/+} mice, despite the significant improvement of erythroid cells maturation. The inventors propose that in long-term spleen iron content (SIC) reduction may impair the compensatory spleen erythropoiesis, causing a final failure. To test this hypothesis Tfr2^BMKO/Hbb^th3/+ mice were challenged with iron-dextran injections in order to avoid this worsening of the hematological phenotype.

All together the present results prove that erythroid TFR2 is a candidate therapeutic target for β-thalassemia. This novel therapeutic approach can be applied to other disorders characterized by ineffective erythropoiesis (i.e. some hemolytic anemias and myelodysplastic syndromes) or to ameliorate other types of anemia by potentiating the erythroid response (sickle cell disease, malaria, chronic kidney disease, anemia of inflammation).

Here the inventors also characterize a new level of hepcidin regulation contributed by FKBP12 binding to the BMP type I receptor ALK2 in hepatocytes, a finding that provides novel insights into the control of systemic iron homeostasis and a potential pharmacological target for treatment of iron overload-low hepcidin disorders, as hemochromatosis and beta- thalassemia.

Inventors' study started from the analysis of the effect of rapamycin on hepcidin expression. To be functional, rapamycin interacts with FKBP12, a peptidyl-prolyl cis-trans cytosolic isomerase that belongs to the immunophilin superfamily. FKBP12 targets mTOR in complex with rapamycin and calcineurin in complex with tacrolimus, with immunosuppressive effect in both cases. It also modulates the H-Ras retrograde trafficking (Ahearn et al., 201 1 ), counteracts the phosphorylation status of epidermal growth factor (Mathea et al., 201 1 ), inositol trisphosphate receptors (Cameron et al., 1995; Dargan et al., 2002; Kang et al., 2008) and TGF-beta type I receptors (Wang et al., 1996), including BMP type-l receptors (Chaikuad et al., 2012; Spiekerkoetter et al., 2013). FKBP12 binding to the glycine-serine- rich domain of the latter receptors provides a safeguard against leaky constitutive BMP signaling.

Here the inventors demonstrate that hepcidin is activated by rapamycin and other compounds able to bind and sequester the immunophilin, independently from mTOR or calcineurin inhibition. The inventors show that the up-regulation of hepcidin expression requires the loss of FKBP12-ALK2 interaction. In agreement, the overexpression of ALK2 mutants with defective binding affinity to FKBP12, that are responsible of the rare genetic disease Fibrodysplasia Ossificans Progressiva, mirrors the drug effect on hepcidin up- regulation. The mechanism, characterized in vitro in hepatoma cells, is conserved in vivo, as shown by the transient increase of hepcidin expression observed in wild type mice treated with a single dose of tacrolimus. The mechanism is active also in humans as shown by a patient the inventors have identified affected by Iron Refractory Iron Deficiency Anemia (IRIDA), a condition characterized by high hepcidin levels, who was heterozygous compound for TMPRSS6 (the IRIDA causative gene) and ALK2 mutations (Pagani et al., 2017). Overall the present results indicate that ALK2 is the BMP type-l receptor targeted by FKBP12 in hepatocytes and that it activates hepcidin only when not bound to the immunophilin.

Then the inventors asked which the physiologic significance of ALK2-FKBP12 interaction is. Both BMP2 and BMP6 are expressed in liver endothelial cells and regulate hepcidin expression in hepatocyte in a paracrine manner. Only BMP6 is up-regulated in response to iron increase (Kautz et al., 2008), whereas BMP2 maintains the hepcidin basal activation (Koch et al., 2017). In several cell types, as mesenchymal stem cells and endothelial cells, BMP6 binds both ALK2 and ALK3, whereas BMP2 preferentially interacts with ALK3 (Hurst et al., 2017; Lavery et al., 2008; Upton et al., 2007). In agreement, silencing of ALK3 in hepatoma cells impairs both BMP2- and BMP6-dependent hepcidin activation, whereas ALK2 downregulation affects only the BMP6 response (Xia et al., 2008). In accordance with the in vitro data, Alk3 liver conditional inactivation in mice causes a stronger repression of hepcidin and severe iron overload than conditional inactivation of Alk2 (Steinbicker et al., 201 1 ), suggesting that ALK2 has a marginal role in basal hepcidin activation that may be explained by its interaction with FKBP12.

The inventors showed that, as reported for other tissues (Hatsell et al., 2015; Hino et al., 2015), ALK2 mutants signal through the non-canonical ligand Activin A in hepatocytes. The mechanism is activated by loss of FKBP12 binding, since it occurs also in wild type ALK2 after treatment with FKBP12 sequestering drugs. This result opens a new perspective on the hepcidin regulation in inflammation. It is well known that inflammatory cytokines, as interleukin 6 (IL6), increase hepcidin expression through the Janus Kinase (JAK) 2 - Signal Transducer and Activator of Transcription (STAT) 3 signaling and that, for a full hepcidin expression, a concomitant activation of the BMP-SMAD1/5/8 pathway is required (Pagani et al., 201 1 ; Theurl et al., 201 1 a). In accordance, suppression of the BMP pathway by the LDN-193189 inhibitor decreases hepcidin levels and has been proposed as a potential treatment of anemia of chronic disease (ACD) (Theurl et al., 201 1 b). However, the ligand that activates the BMP pathway in inflammation remained unknown. Interestingly, in mouse models of both acute (LPS) and chronic {Brucella Abortus) inflammation, treatment with follistatin, an activin(s) inhibitor that binds at high efficiency both Activin A and Activin B, reverts the hepcidin activation (Canali et al., 2016). Activin B, a member of the TGF-β superfamily released in inflammation, initially proposed as a potential ligand (Besson- Fournier et al., 2012) recently was dismissed as hepcidin regulator (Besson-Fournier et al., 2017). Activin A, a critical component of the inflammatory response, secreted in the circulation by immune and non-immune cell types, was not previously reported to take part in hepcidin activation (Canali et al., 2016; Kanamori et al., 2016). Activin A binds BMP type II receptors, ACVR2A and ACVR2B, and the type I receptor ALK4 (Harrison et al., 2004; Hino et al., 2015). The complex activates target genes through phosphorylation and nuclear translocation of the transcription factors SMAD2 and SMAD3 (Figure 23).

The inventors show that when ALK2-FKBP12 interaction is impaired, as in case of ALK2 mutants or in the presence of FKBP12 binding drugs, the receptor becomes responsive to Activin A and triggers hepcidin activation through SMAD1/5/8.

The inventors propose that a mechanism that reduces FKBP12 or interferes with its ALK2 binding may facilitate SMAD1/5/8 activation by Activin A to sustain hepcidin expression in the first phase of inflammation (Figure 23B), before IL6 effect becomes most relevant. Consistent with this interpretation the administration of momelotinib, developed as a Jak1/2 inhibitor that was shown to inhibit ALK2 strongly suppresses hepcidin activation in rats with inflammation-related anemia, but not in basal condition (Asshoff et al., 2017). These results suggest that in inflammation ALK2 is functional and not bound to FKBP12. The FKBP12-ALK2 interaction might be relevant also in conditions of iron overload in which increased BMP6 activates hepcidin through SMAD1/5/8. High concentrations of BMP6 reduce the FKBP12-ALK2 interaction (Figure 23C). Thus ALK2, inactive in basal condition, becomes functional in iron overload, and also sensitive to Activin A. The inventors speculate that the observed synergism between Activin A and high concentration of BMP6 in hepcidin activation might be relevant when inflammation occurs in severe iron loading, by further enhancing hepcidin expression in the attempts of achieving a protective condition of hypoferremia (Arezes et al., 2015).

Inventors' results suggest that hepcidin production undergo multiple control levels to coordinate systemic iron trafficking and homeostasis and are in agreement with the recently proposed model of two BMP-regulated pathways that additively contribute to hepcidin activation (Latour et al., 2016). In the first (that the inventors speculate involved ALK3) BMP2 and BMP6 interact with the coreceptor HJV and other hemochromatosis-proteins as HFE and TfR2, to allow endocytosis and signaling according to the proposed model (Healey et al., 2015). The second (ALK2 -dependent) would signal after BMP6 binding to type 1/type 2 preformed receptor complexes without requiring the HJV coreceptor. In agreement with the latter model the inventors showed that the BMP co-receptor HJV is dispensable for the ALK2 effect in Hjv KO primary hepatocytes treated with tacrolimus, although, because of the extremely low basal levels, the maximal hepcidin expression achieved is about half that of wild type hepatocytes.

As expected, HJV further potentiates the hepcidin activation induced by mutant ALK2 in vitro (not shown). HJV undergoes a posttranslational control by the serine protease matriptase-2, encoded by the TMPRSS6 gene, that cleaves the co-receptor from the cell membrane (Silvestri et al., 2008)(Figure 23). High hepcidin levels due to TMPRSS6 recessive mutations cause iron-refractory iron-deficiency anemia (IRIDA)(Finberg et al., 2008). The inventors recently reported a rare informative patient affected by both IRIDA and FOP, carrier of both ALK2^R258S and TMPRSS6 ^2J heterozygous mutations. This case indicates that in humans constitutively active ALK2 mutants do not upregulate hepcidin at a level able to induce an iron refractory anemia (Pagani et al., submitted), unless the activity of the inhibitor TMPRSS6 is impaired and some BMP co-receptor (HJV) function is maintained. Digenic inheritance in this patient is also compatible with the two BMP pathways model (Figure 23). Inventors' results indicate that FKBP12 is a novel target to treat conditions of iron overload due to low hepcidin. Rescue of the BMP-SMAD activity by tacrolimus has been achieved in pulmonary artery hypertension, a disease due to reduction of the BMP type II receptor BMPR2 (Deng et al., 2000). Chronic treatment with tacrolimus in low dose that do not trigger immunosuppression, rescues endothelial dysfunction of pulmonary hypertension in mice with conditional deletion of Bmpr2 in endothelial cells (Spiekerkoetter et al., 2013). More recently a pilot study with the same drug ameliorated the clinical condition in 3 patients with endstage pulmonary hypertension (Spiekerkoetter et al., 2015). Considering that in the present invention tacrolimus treatment upregulates hepcidin in Hjv KO hepatocytes and that in vivo Hjv is dispensable for hepcidin upregulation by iron (Gkouvatsos et al., 2014), tacrolimus-like drugs might be proposed for disorders due to impaired hepcidin production caused by low BMP-SMAD activation as hemochromatosis and β-thalassemia.

Chronic TAC administration increases hepcidin in Hjv KO mice through BMP-SMAD pathway activation. This upregulation is followed by a trend towards an increase in spleen iron content. Overall these data demonstrate that FKBP12 is a promising pharmacological target for amelioration of iron overload through hepcidin upregulation in a severe model of hemochromatosis.

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Claims

1. A regulator of the BMP-SMAD signaling for use in the treatment and/or prevention of a disease selected from the group consisting of: a disease characterized by ineffective erythropoiesis, anemia, a disease characterized by low hepcidin and by iron overload or a combination thereof.

2. The regulator of the BMP-SMAD signaling for use according to claim 1 wherein said regulator is selectyed from the group consisting of: an inhibitor of erythroid TFR2 or an inhibitor of hepatocyte FKBP12 or a combination thereof.

3. The regulator of the BMP-SMAD signaling for use according to claim 2 wherein the inhibitor of erythroid TFR2 alters EPO-EPOR signaling and/or inhibit TFR2-EPOR binding or wherein the inhibitor of hepatocyte FKBP12 displaces FKBP12 from BMP type I receptor.

4. The regulator of the BMP-SMAD signaling for use according to any one of previous claim wherein said regulator is selected from the group consisting of: a small molecule, a peptide, a protein, an antisense oligonucleotide, a siRNA, an antisense expression vector or recombinant virus, an antibody or a fragment thereof.

5. The regulator of the BMP-SMAD signaling for use according to any one of previous claim wherein said regulator is a siRNA.

6. The regulator of the BMP-SMAD signaling for use according to any one of claim 1 to 4 wherein said regulator is tacrolimus.

7. The regulator of the BMP-SMAD signaling for use according to any one of previous claim wherein the disease characterized by ineffective erythropoiesis is selected from the group consisting of: beta-thalassemia, congenital dyserythropoietic anemias, sideroblastic anemias, myelodysplastic syndromes.

8. The regulator of the BMP-SMAD signaling for use according to any one of previous claim wherein the anemia is selected from the group consisting of: sickle cell disease, malaria, chronic kidney disease, anemia of inflammation, hemolytic anemias.

9. The regulator of the BMP-SMAD signaling for use according to any one of previous claim wherein the disease characterized by low hepcidin and by iron overload is selected from the group consisting of: hematochromatosis and ineffective erythropoiesis as beta-thalassemia, congenital dyserythropoietic anemias, sideroblastic anemias, myelodysplastic syndromes.

10. The regulator of the BMP-SMAD signaling for use according to any one of previous claim in combination with a therapeutic agent and/or intervention.

1 1 . The regulator of the BMP-SMAD signaling for use according to claim 10 wherein the therapeutic agent and/or intervention is selected from the group consisting of: a gene therapy, an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor.

12. A pharmaceutical composition comprising a regulator of the BMP-SMAD signaling as defined in any one of claiml to 6 for use in the treatment and/or prevention of a disease selected from the group consisting of: a disease characterized by ineffective erythropoiesis, anemia, a disease characterized by low hepcidin and by iron overload or a combination thereof.

13. The pharmaceutical composition according to claim 12 further comprising a therapeutic agent.

14. The pharmaceutical composition according to claim 13 wherein the therapeutic agent is selected from the group consisting of: an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor.

15. The pharmaceutical composition according to any one of claim 12 to 14 in combination with gene therapy.