WO2018185341A1 - Régulateur de signalisation de bmp-smad et ses utilisations - Google Patents

Régulateur de signalisation de bmp-smad et ses utilisations Download PDF

Info

Publication number
WO2018185341A1
WO2018185341A1 PCT/EP2018/059040 EP2018059040W WO2018185341A1 WO 2018185341 A1 WO2018185341 A1 WO 2018185341A1 EP 2018059040 W EP2018059040 W EP 2018059040W WO 2018185341 A1 WO2018185341 A1 WO 2018185341A1
Authority
WO
WIPO (PCT)
Prior art keywords
bmp
hepcidin
tfr2
regulator
alk2
Prior art date
Application number
PCT/EP2018/059040
Other languages
English (en)
Inventor
Laura SILVESTRI
Clara Camaschella
Antonella NAI
Silvia COLUCCI
Alessia PAGANI
Original Assignee
Ospedale San Raffaele S.R.L.
Fondazione Centro San Raffaele
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ospedale San Raffaele S.R.L., Fondazione Centro San Raffaele filed Critical Ospedale San Raffaele S.R.L.
Publication of WO2018185341A1 publication Critical patent/WO2018185341A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • said regulator is an inhibitor of erythroid
  • Beta thalassemias are autosomal recessive disorders caused by mutations in the ⁇ -globin gene or its regulatory elements, resulting in reduced or even absent ⁇ -globin chain synthesis.
  • the unbalanced synthesis between normal a- and reduced/absent ⁇ -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 ⁇ -thalassemia.
  • Erythropoiesis is governed by erythropoietin (EPO), a hormone primarily produced by the kidney in adulthood and whose levels, stimulated by hypoxia, are high in ⁇ -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.
  • erythropoiesis 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).
  • HAMP hepcidin
  • ERFE contributes to iron loading in ⁇ -thalassemia (Kautz et al., 2015).
  • iron exerts a regulatory role on erythroid development: indeed iron-deficiency induces the production of EPO.
  • 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.
  • TFR2 Transferrin Receptor 2
  • TF-bound iron 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).
  • 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).
  • HAMP hepcidin
  • EPOR erythropoietin receptor
  • 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).
  • mice with specific genetic inactivation of Tfr2 in the bone marrow 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).
  • 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.
  • 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).
  • BMP Bone Morphogenetic Protein
  • SAD Stemmettal Decapentaplegic
  • 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.
  • 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).
  • HAMP hepcidin
  • 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.
  • 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).
  • beta thalassemia a disease characterized by anemia and splenomegaly due to ineffective erythropoiesis, and multi organ iron overload.
  • beta-thalassemia 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TFR2 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.
  • TFR1 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).
  • 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.
  • TFR2 systemic iron homeostasis is not altered whereas erythropoiesis is stimulated.
  • 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.
  • the present invention identifies a new level of BMP-SMAD and hepcidin regulation, based on the druggable target FKBP12.
  • tacrolimus a drug that complex with FKBP12 and displace it from the BMP receptor ALK2
  • 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.
  • 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.
  • 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.
  • the inhibitor of erythroid TFR2 alters EPO-EPOR signaling and/or inhibit
  • 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.
  • the regulator is a siRNA or tacrolimus.
  • the disease characterized by ineffective erythropoiesis is selected from the group consisting of: beta-thalassemia, congenital dyserythropoietic anemias, sideroblastic anemias, myelodysplastic syndromes.
  • anemia is selected from the group consisting of: sickle cell disease, malaria, chronic kidney disease, anemia of inflammation, hemolytic anemias.
  • 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.
  • tacrolimus is for use in the treatment and/or prevention of hematochromatosis or beta-thalassemia.
  • the regulator of the BMP-SMAD signaling is for use in combination with a therapeutic agent and/or intervention.
  • 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.
  • 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.
  • the pharmaceutical composition further comprises a therapeutic agent.
  • 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.
  • an activin ligand trap preferably sotatercept and luspatercept
  • an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor preferably an activin ligand trap, preferably sotatercept and luspatercept, an antisense oligonucleotide against Tmprss6, minihepcidin, transferrin and an antagonist of TFR1 receptor.
  • the pharmaceutical composition is for use in combination with gene therapy.
  • 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.
  • ineffective erythropoiesis means aberrant proliferation and decreased differentiation of erythroid cells.
  • low hepcidin means downregulation of hepcidin expression in the liver and/or decreased serum hepcidin levels.
  • 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.
  • 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.
  • 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.
  • 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.)
  • 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:
  • 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;
  • 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;
  • 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;
  • 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
  • SCA Single chain antibody
  • 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 ⁇ -thalassemia in mice. JCI.
  • 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
  • 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.
  • transferrin (REF: Li H., Rybicki AC, Suzuka SM:, von Bondsdorff L., Breuer W., Hall CB., Cabantchik Zl., Bouhassira EE.,
  • 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.
  • 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.
  • ASO antisense oligonucleotide
  • 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.
  • 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.
  • the inhibitor may be an antisense oligonucleotide (ASO) directed against FKBP12; a compound such as tacrolimus (FK506) or rapamycin targeting liver FKBP12.
  • ASO antisense oligonucleotide
  • FKBP12 a compound such as tacrolimus (FK506) or rapamycin targeting liver FKBP12.
  • 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.
  • 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.
  • 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.
  • 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
  • Hepcidin NP_066998.1
  • unmodified ASO-TFR2 may be used to reduce, and not to completely suppress, liver and erythroid TFR2 mRNA levels.
  • hepcidin regulated by liver TFR2
  • EPO-EPOR signaling controlled by erythroid TFR2
  • modified ASO may be used.
  • modified ASO specifically target erythroid cells.
  • liver FKBP12 liver FKBP12
  • ASO hepatocyte-complexed drugs
  • Liposome-complexed drugs may also be used for liver targeting by molecules such as tacrolimus or rapamycin.
  • 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.
  • the concentration range of oligonucleotide can be from 0.1 nM to 500 ⁇ , preferably the in vitro range is between 0.2 nM and 300 ⁇ , preferably the in vitro range is between 0.5 nM to 200 ⁇ .
  • Preferred in vivo range is 0.1 -500 mg/ kg, preferably in vivo range is 1 -50 mg/kg.
  • 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.
  • cells of interest 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.
  • 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.
  • 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.
  • oligonucleotide refers 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.
  • the "oligonucleotides" of the invention as described herein have a phosphodiester backbone.
  • 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.
  • 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.
  • PNAs peptide nucleic acids
  • 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.
  • chimeric oligonucleotides are with one or more phosphodiester linkages at the 3' end.
  • chimeric oligonucleotides are with one or more phosphodiester linkages at the 5' end.
  • chimeric oligonucleotides are with one or more phosphodiester linkages at the 3' and 5' ends.
  • siRNA short interfering RNA
  • 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
  • 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
  • siRNAs are 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.
  • dsRNA double-stranded RNA
  • 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
  • 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.
  • RNA may serve as siRNA for use of the present invention
  • siRNA double stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • 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.
  • the inhibitor of TFR2 and the inhibitor of FKBP12 may be administered together or one before the other (subsequently) in any order.
  • FIG. 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.
  • 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).
  • RBC Red Blood Cells count
  • B Hemoglobin levels
  • MCV Mean Corpuscolar Volume
  • MH Mean Corpuscolar Hemoglobin
  • SIC spleen iron content
  • FIG. 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.
  • RBC Red Blood Cells number
  • Hb, B Hemoglobin levels
  • MCV Mean Corpuscular Volume
  • MH Mean Corpuscular Hemoglobin
  • 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.
  • FIG. 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.
  • Hbb th3/+ thalassemic
  • Tfr2- /Hbb th3/+ Tfr2- /Hbb th3/+
  • 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.
  • FIG. 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.
  • Hbb th3/+ thalassemic
  • Tfr2 BMKO /Hbb th3/+ Tfr2 BMKO /Hbb th3/+
  • 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).
  • LIC liver iron content
  • SIC spleen iron content
  • KIC kidney iron content
  • HIC heart iron content
  • HIC heart iron content
  • HOC heart iron content
  • E liver Hamp mRNA levels
  • E liver Hamp mRNA levels relative to Hypoxanthine Phosphoribosyltransferase 1 ⁇ Hprtl
  • TS serum transferrin saturation
  • 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG. 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.
  • 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).
  • LIC liver iron content
  • SIC spleen iron content
  • KIC kidney iron content
  • HIC heart iron content
  • HIC heart iron content
  • HOC heart iron content
  • E liver Hamp mRNA levels
  • E Hypoxanthine Phosphoribosyltransferase 1 ⁇ Hprtl
  • TS serum transferrin saturation
  • FIG. 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.
  • FIG. 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.
  • FIG. 11 Rapamycin upregulates hepcidin through activation of the BMP-SMAD pathway.
  • 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 .
  • FIG. 12 Tacrolimus upregulates hepcidin through BMP-SMAD pathway activation.
  • TAC 1 ⁇ g/ml
  • CA calcineurin inhibitor cyclosporine A
  • 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 ⁇ g/ml), CA (1 ⁇ g/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 .
  • FIG. 13 Displacement of FKBP12 form ALK2 increases hepcidin through BMP- SMAD pathway activation.
  • HuH7 cells were transiently transfected with FKBP12 FLAG and ALK2 wt"MYC and treated with tacrolimus (TAC, 1 ⁇ g/ml), rapamycin (RAPA, 100 nM), GPI-1046 (100 ⁇ g/ml) or vehicle for 15 hrs.
  • TAC tacrolimus
  • RAPA rapamycin
  • GPI-1046 100 ⁇ g/ml
  • Vehicle 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.
  • 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).
  • 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).
  • 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.
  • HAMP Hepcidin
  • ID1 E expression levels were quantified by qRT-PCR and normalized to the housekeeping gene
  • 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 ⁇ ). Cells were lysed and analyzed for the luciferase activity that was normalized to an untreated mean value of 1.
  • DM dorsomorphin
  • HAMP-Luc hepcidin promoter firefly luciferase reporter
  • 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.
  • FIG. 14 The BMP coreceptor hemojuvelin (HJV) is dispensable for FKBP12- dependent hepcidin activation.
  • HJV The BMP coreceptor hemojuvelin
  • 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) 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.
  • DMSO vehicle
  • TAC tacrolimus
  • 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.
  • SMAD1/5/8 phosphorylation was analyzed in HuH7 transfected with the Smad1 FLAG expressing vector and 5 ⁇ g or 10 ⁇ g of ALK2 wt_MYC , ALK2 R206H_MYC or ALK2 R258"MYC .
  • cells were treated for 15 hrs with 10 ng/ml Activin A.
  • RQ relative quantification. Error bars indicate SD.
  • FIG. 17 Pharmacologic displacement of FKBP12 from ALK2 leads to Activin A- dependent SMAD1/5/8 activation and hepcidin upregulation.
  • HAMP-Luc hepcidin promoter luciferase reporter vector
  • ALK2 wt ⁇ MYC expressing vector were pre-treated with 1 ⁇ g/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.
  • Transfected cells were incubated with TAC (1 ⁇ g/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 .
  • 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).
  • 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
  • FIG. 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 ⁇ g/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.
  • 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 .
  • FIG 19 The ALK2 inhibitor DMH1 abrogates the FKBP12-dependent hepcidin regulation.
  • Hep3B cells were pre-treated for 3 hrs with DMH 1 (0,5 ⁇ g/ml) and then incubated for 15 hrs with rapamycin (RAPA, 100 nM), Torinl (T1 , 100 nM) (A), tacrolimus (TAC, 1 ⁇ ) or Cyclosporin A (CA, 1 ⁇ 9/ ⁇ ) (B).
  • RAPA rapamycin
  • T1 Torinl
  • T1 tacrolimus
  • CA Cyclosporin A
  • B Cyclosporin A
  • FIG. 20 ALK2-FKBP12 binding capacity influences hepcidin activation through BMP-SMAD pathway.
  • HAMP-Luc hepcidin promoter luciferase reporter vector
  • ALK2 R206H-MYC-FLAG (red
  • _ K2 R258S-MYC-FLAG (g reen line) expressing vectors.
  • Figure 21 Hepcidin expression and liver iron content in mice treated with tacrolimus.
  • Hamp Liver hepcidin
  • LIC Liver iron content
  • Figure 22 The drug-competitive inhibition of ALK2-FKBP12 interaction renders ALK2 responsive to Activin A .
  • TAC Tacrolimus
  • HAMP-Luc hepcidin promoter reporter vector
  • BRE-Luc BRE-Luc
  • 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.
  • FIG. 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.
  • Hbb th3/+ mice (with heterozygous deletion of both betal and beta2 genes, a model of non transfusion-dependent ⁇ -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.
  • TS transferrin saturation
  • 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).
  • BMT bone marrow transplantation
  • 5 * 10 6 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.
  • mice 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.
  • 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 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).
  • 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.
  • rat- anti-mouse CD16/CD32 BD Pharmingen
  • PE rat anti-mouse Ter1 19 BD Biosciences
  • APC rat anti- mouse CD44 BD Biosciences
  • 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 CantoTM II.
  • 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.
  • 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.
  • 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.
  • REV ccaacagtgtaatctggtgagccactgttcttgt SEQ ID NO: 12
  • pCMV6-ALK2 Q207E - MYC - FLAG FW: aaagaacagtggctcgcgagattacactgttggag SEQ ID NO: 12
  • pCMV6-ALK2 FW ctcatctcagaagaggatctgtgagcaaatgatatcctggatta
  • pCMV6-FKBP12 FLAG FW ggatctggcagcaaatgatctcgaggattacaaggatgacg
  • pCMV6-FKBP12 FW gtggagcttctaaaactggaatagcgtacgcggcc SEQ ID NO:
  • DMEM Dulbecco's modified Eagle's medium
  • FBS heat-inactivated fetal bovine serum
  • Hep3B cells (BS TCL 78, Istituto Zooprofilatticosperimentale 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.
  • MEM Minimal essential medium
  • 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.
  • 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 ⁇ m cell strainer.
  • HCs Hepatocytes
  • NPCs non-parenchymal cells
  • HCs were resuspended in Williams-E medium (4% FBS, 1 % P/S, Glutamax) (Thermo Fisher Scientific) and plated into collagen-coated 12-well (3 * 10 5 cells/well).
  • FBS 4% FBS, 1 % P/S, Glutamax
  • Another group of hepatocytes was serum starved for 3 hrs.
  • Serum supplemented cells were incubated for 3 hrs with tacrolimus (1 ⁇ 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 ⁇ g/ml), Cyclosporin A (1 M g/ml) and GPI-1046 (100 ⁇ g/ml). Cells were lyzed and RNA isolated for gene expression analysis.
  • 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.
  • BRE BMP Responsive Element
  • 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).
  • 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.
  • SybrGreen GAPDH FW ccccggtttctataaattgagc
  • HAMP FW ctgttttcccacaacagacg
  • ID1 FW tccagcacgtcatcgactac
  • 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 ⁇ g) 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).
  • ECL chemiluminescence detection kit
  • HuH7 cells transfected with FKBP12 FLAG and ALK2 MYC (wild type or mutants) expressing vectors were treated with tacrolimus (1 ⁇ g/ml), rapamycin (100 nM) or GPI-1046 (100 ⁇ g/ml), when indicated.
  • tacrolimus 1 ⁇ g/ml
  • rapamycin 100 nM
  • GPI-1046 100 ⁇ g/ml
  • 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 ⁇ of Laemmli sample buffer (without ⁇ -mercaptoethanol) and incubated at 95°C for 5 minutes. After centrifugation, ⁇ -mercaptoethanol was added to supernatants. Samples were then subjected to 12% SDS-PAGE and immunodetection was performed as described in Western Blot Analysis.
  • 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.
  • 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 ⁇ capacity, 0.1 1 ⁇ /hrs drug delivery).
  • the drug was prepared as followed.
  • Tacrolimus FK506, Cayman Chemicals
  • DMSO 100% 5 mg/ml
  • 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 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.
  • Tfr2 is expressed in the liver and in erythroid cells.
  • Tfr2 BMKO /Hbb th3/+ young mice have a more effective erythropoiesis than Hbb th3/+ , but comparable splenomegaly and iron-overload
  • 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.
  • LIC spleen
  • KIC kidney
  • HIC heart
  • HIC heart
  • TS transferrin saturation
  • the EPO-EPOR signaling pathway is overactive in erythroid tissues from young Tfr2 BMKO /Hbb th3/+ mice
  • 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).
  • 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).
  • Tfr2 BMKO /Hbb th3/+ old mice further improve erythropoiesis and reduce iron accumulation
  • 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.
  • 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
  • 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).
  • FKBP12 tacrolimus
  • the inventors modulated FKBP12 binding by using tacrolimus (FK506), that interacts with the same FKBP12-binding pocket that binds rapamycin (Wilson et al., 1995).
  • 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).
  • 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.
  • 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).
  • GPI- 1046 treatment upregulates endogenous hepcidin ( Figure 18C) and Id1 ( Figure 18D) expression in murine primary hepatocytes.
  • 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 ).
  • DMH1 4-(6-(4-isopropoxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl) quinolone
  • DMH1 4-(6-(4-isopropoxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl) quinolone
  • 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.
  • the human hepatoma cell line HuH7 transiently transfected with ALK2 wt and FKBP12
  • the different efficiency shown by the three drugs on FKBP12 displacement is probably related to their different half-life or FKBP12 affinity.
  • 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.
  • 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).
  • 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
  • HJV BMP coreceptor hemojuvelin
  • 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.
  • 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.
  • the inventors analyzed both the TGF- ⁇ and the BMP-SMAD pathway by measuring the luciferase activity under the control of the CAGA and the BRE responsive elements, respectively.
  • TGF- ⁇ 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).
  • the BRE- Luc activity increases in cells transfected with mutants ALK2 in the presence of Activin A ( Figure 16E).
  • 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).
  • the TGF- ⁇ signaling measured by CAGA-Luc activity, is increased by Activin A and unaffected by tacrolimus (Figure 17D).
  • 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 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.
  • mice 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.
  • 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 ⁇ -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 ⁇ -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 ⁇ -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.
  • 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 ).
  • erythroid TFR2 is a candidate therapeutic target for ⁇ -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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • IRIDA Iron Refractory Iron Deficiency Anemia
  • TMPRSS6 the IRIDA causative gene
  • 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).
  • BMP6 binds both ALK2 and ALK3, whereas BMP2 preferentially interacts with ALK3 (Hurst et al., 2017; Lavery et al., 2008; Upton et al., 2007).
  • 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).
  • 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.
  • inflammatory cytokines as interleukin 6 (IL6)
  • JNK Janus Kinase
  • STAT Signal Transducer and Activator of Transcription
  • 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).
  • 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).
  • 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.
  • 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.
  • 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 ⁇ -thalassemia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un régulateur de la signalisation de BMP-SMAD destiné à être utilisé dans le traitement et/ou la prévention d'une maladie choisie dans le groupe constitué par: une maladie caractérisée par une érythropoïèse inefficace, une anémie, une maladie caractérisée par une faible hepcidine et par une surcharge en fer ou une combinaison de celles-ci. De préférence, ledit régulateur est un inhibiteur de l'érythroïde TFR2 ou un inhibiteur de l'hépatocyte FKBP12 ou une combinaison de ceux-ci. La présente invention concerne également des compositions pharmaceutiques relatives et leurs utilisations.
PCT/EP2018/059040 2017-04-07 2018-04-09 Régulateur de signalisation de bmp-smad et ses utilisations WO2018185341A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762483172P 2017-04-07 2017-04-07
US62483172 2017-04-07

Publications (1)

Publication Number Publication Date
WO2018185341A1 true WO2018185341A1 (fr) 2018-10-11

Family

ID=62046868

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/059040 WO2018185341A1 (fr) 2017-04-07 2018-04-09 Régulateur de signalisation de bmp-smad et ses utilisations

Country Status (1)

Country Link
WO (1) WO2018185341A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021180840A1 (fr) * 2020-03-12 2021-09-16 Heinrich-Heine-Universität Düsseldorf Inhibition de fkbp1a pour la thérapie du carcinome mammaire triple négatif
WO2021231851A1 (fr) * 2020-05-15 2021-11-18 Celgene Corporation Méthodes et compositions pour traiter l'anémie à l'aide de pièges à ligands actriib et d'inhibiteurs de mtor
WO2022149113A1 (fr) * 2021-01-11 2022-07-14 Università Degli Studi Di Torino Activateurs de l'axe bmp/smad destinés à être utilisés dans le traitement et/ou la prévention de l'atrophie musculaire

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6506735B1 (en) 1998-02-03 2003-01-14 Methyl Gene, Inc. Optimized antisense oligonucleotides complementary to DNA methyltransferase sequences
US6537973B1 (en) 1992-03-16 2003-03-25 Isis Pharmaceuticals, Inc. Oligonucleotide inhibition of protein kinase C
WO2012177921A2 (fr) 2011-06-21 2012-12-27 Alnylam Pharmaceuticals, Inc Compositions et procédés d'inhibition de l'expression du peptide antimicrobien hepcidine (hamp) ou du gène lié à hamp
WO2014020140A1 (fr) 2012-08-02 2014-02-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation d'un antagoniste du récepteur de transferrine pour le traitement de la thalassémie

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6537973B1 (en) 1992-03-16 2003-03-25 Isis Pharmaceuticals, Inc. Oligonucleotide inhibition of protein kinase C
US6506735B1 (en) 1998-02-03 2003-01-14 Methyl Gene, Inc. Optimized antisense oligonucleotides complementary to DNA methyltransferase sequences
WO2012177921A2 (fr) 2011-06-21 2012-12-27 Alnylam Pharmaceuticals, Inc Compositions et procédés d'inhibition de l'expression du peptide antimicrobien hepcidine (hamp) ou du gène lié à hamp
WO2014020140A1 (fr) 2012-08-02 2014-02-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation d'un antagoniste du récepteur de transferrine pour le traitement de la thalassémie

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1989
AHEARN, I.M. ET AL., MOL. CELL, vol. 41, 2011, pages 173 - 185
ANDRIOPOULOS, B. ET AL., NAT. GENET., vol. 41, 2009, pages 482 - 487
AREZES, J. ET AL., CELL HOST MICROBE, vol. 17, 2015, pages 47 - 57
ASSHOFF, M. ET AL., BLOOD BLOOD, September 2016 (2016-09-01), pages 740092
BESSON-FOURNIER, C. ET AL., BLOOD, vol. 120, 2012, pages 431 - 439
BESSON-FOURNIER, C. ET AL., BLOOD, vol. 129, 2017, pages 533 - 536
CAMASCHELLA C; ROETTO A; CALI A ET AL., NAT GENET, vol. 25, no. 1, 2000, pages 14 - 15
CAMASCHELLA, C., BLOOD, vol. 106, 2005, pages 3710 - 3717
CAMERON, A.M. ET AL., CELL, vol. 83, 1995, pages 463 - 472
CANALI, S. ET AL., BLOOD, vol. 129, 2017, pages 405 - 414
CANALI, S. ET AL., ENDOCRINOLOGY, vol. 157, 2016, pages 1146 - 1162
CASU C; OIKONOMIDOU PR; LO PRESTI V ET AL., BLOOD, vol. 128, 2016, pages 1012
CHAIKUAD, A. ET AL., J. BIOL. CHEM., vol. 287, 2012, pages 36990 - 36998
COLIGAN ET AL.: "Current Protocols In Immunologv", 1991, WILEY INTERSCIENCE
COLUCCI S ET AL., BLOOD, vol. 130, no. 19, 9 November 2017 (2017-11-09), pages 2111 - 2120
DANIEL W COYNE: "Hepcidin: clinical utility as a diagnostic tool and therapeutic target", KIDNEY INTERNATIONAL, vol. 80, no. 3, 15 June 2011 (2011-06-15), pages 240 - 244, XP055170102, ISSN: 0085-2538, DOI: 10.1038/ki.2011.141 *
DARGAN, S.L. ET AL., BIOCHEM. J., vol. 361, 2002, pages 401 - 407
DENG, Z. ET AL., AM. J. HUM. GENET., vol. 67, 2000, pages 737 - 744
DIGICAYLIOGLU M; LIPTON SA, NATURE, vol. 412, no. 6847, 2001, pages 641 - 647
FINBERG, K.E. ET AL., NAT. GENET., vol. 40, 2008, pages 569 - 571
FOREJTNIKOVA H ET AL., BLOOD, vol. 116, no. 24, 2010, pages 5357 - 5367
FUKUDA T. ET AL., J BIOL CHEM, vol. 240, 2009, pages 7149 - 7156
GARDENGHI S; RAMOS P; MARONGIU MF ET AL., J CLIN INVEST, vol. 120, no. 12, 2010, pages 4466 - 4477
GKOUVATSOS, K. ET AL., PLOS ONE, vol. 9, 2014, pages e85530
GONGALVES, L.A.; VIGARIO, A.M.; PENHA-GONGALVES, C., MALAR. J., vol. 6, 2007, pages 169
GUO S.; CASU C.; GARDENGHI S.; BOOTEN S.; AGHAJAN M.; PERALTA R.; WATT A.; FREIER S.; MONIA BP.; RIVELLA S.: "Reducing TMPRSS6 ameliorates hemochromatosis and P-thalassemia in mice", JCI, vol. 123, no. 4, 2013, pages 1531 - 41, XP055135245, DOI: doi:10.1172/JCI66969
GUO S; CASU C; GARDENGHI S ET AL., J CLIN INVEST, vol. 123, no. 4, 2013, pages 1531 - 1541
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
HARRISON, C.A. ET AL., J. BIOL. CHEM., vol. 279, 2004, pages 28036 - 28044
HATSELL, S.J. ET AL., SCI. TRANSL. MED., vol. 7, 2015, pages 303ra137 - ra303ra137
HAUPT, J. ET AL., HUM. MOL. GENET., vol. 23, 2014, pages 5364 - 5377
HEALEY, E.G. ET AL., NAT. STRUCT. MOL. BIOL., vol. 22, 2015, pages 458 - 465
HINO, K. ET AL., PROC. NATL. ACAD. SCI., vol. 112, 2015, pages 15438 - 15443
HUANG F.W. ET AL., J CLIN INVEST, vol. 115, 2005, pages 2187 - 2191
HURST, L.A. ET AL., NAT. COMMUN., vol. 8, 2017, pages 14079
INUI M. ET AL., NAT CELL BIOL, vol. 13, 2011, pages 1368 - 1375
IRIE-SASAKI J; SASAKI T; MATSUMOTO W ET AL., NATURE, vol. 409, no. 6818, 2001, pages 349 - 354
JOHNSON MB ET AL., MOL BIOL CELL, vol. 18, no. 3, 2007, pages 743 - 754
KANAMORI, Y. ET AL., SCI. REP., vol. 6, 2016, pages 38702
KANG, C.B., NEUROSIGNALS, vol. 16, 2008, pages 318 - 325
KASHII Y; UCHIDA M; KIRITO K ET AL., BLOOD, vol. 96, no. 3, 2000, pages 941 - 949
KAUTZ L ET AL., NAT GENET, vol. 46, no. 7, 2014, pages 678 - 684
KAUTZ L; JUNG G; DU X ET AL., BLOOD, vol. 126, no. 17, 2015, pages 2031 - 2037
KAUTZ, L. ET AL., BLOOD, vol. 112, 2008, pages 1503 - 1509
KAUTZ, L. ET AL., NAT. GENET., vol. 46, 2014, pages 678 - 684
KLAUNIG, J.E. ET AL., IN VITRO, vol. 17, 1981, pages 913 - 925
KOCH, P.-S. ET AL., BLOOD, vol. 129, 2017, pages 415 - 419
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
LATOUR, C. ET AL., HEPATOLOGY, vol. 63, 2016, pages 126 - 137
LAVERY, K. ET AL., J. BIOL. CHEM., vol. 283, 2008, pages 20948 - 20958
LEVY JE ET AL., NAT GENET, vol. 21, no. 4, 1999, pages 396 - 399
LI H.; RYBICKI AC.; SUZUKA SM; BONDSDORFF L.; BREUER W.; HALL CB.; CABANTCHIK ZI.; BOUHASSIRA EE.; FABRY ME.; GINZBURG YZ.: "Transferrin therapy ameliorates disease in beta-thalassemic mice", NAT MED, vol. 16, no. 2, 2010, pages 177 - 82, XP055046897, DOI: doi:10.1038/nm.2073
LIBANI IV; GUY EC; MELCHIORI L ET AL., BLOOD, vol. 112, no. 3, 2008, pages 875 - 885
LIU Y ET AL., BLOOD, vol. 108, no. 1, 2006, pages 123 - 133
MATHEA, S. ET AL., BIOCHEMISTRY, vol. 50, 2011, pages 10844 - 10850
MATHIAS LA; FISHER TC; ZENG L ET AL., EXP HEMATOL, vol. 28, no. 12, 2000, pages 1343 - 1353
MAURA POLI ET AL: "Hepcidin antagonists for potential treatments of disorders with hepcidin excess", FRONTIERS IN PHARMACOLOGY, vol. 5, 28 April 2014 (2014-04-28), pages 1 - 13, XP055486712, DOI: 10.3389/fphar.2014.00086 *
MAYEUR, C. ET AL., BLOOD, vol. 124, 2014, pages 2116 - 2123
MEYNARD, D., NAT. GENET., vol. 41, 2009, pages 478 - 481
MLECZKO-SANECKA, K. ET AL., BLOOD, vol. 123, 2014, pages 1574 - 1585
MOHEDAS, A.H. ET AL., ACS CHEM. BIOL., vol. 8, 2013, pages 1291 - 1302
NAI A; LIDONNICI MR; RAUSA M ET AL., BLOOD, vol. 125, no. 7, 2015, pages 1170 - 1179
NAI A; PAGANI A; MANDELLI G ET AL., BLOOD, vol. 119, no. 21, 2012, pages 5021 - 5029
NAI A; PELLEGRINO RM; RAUSA M ET AL., HAEMATOLOGICA, vol. 99, no. 6, 2014, pages 1016 - 1021
NEGRE O; EGGIMANN AV; BEUZARD Y ET AL., HUM GENE THER., vol. 27, no. 2, 2016, pages 148 - 165
NEMETH E ET AL., BLOOD, vol. 105, no. 4, 2005, pages 1803 - 1806
NEMETH, E. ET AL., SCIENCE, vol. 306, 2004, pages 2090 - 2093
ORIGA, R. ET AL., HAEMATOLOGICA, vol. 92, 2007, pages 583 - 588
PAGANI A ET AL., HAEMATOLOGICA, vol. 100, no. 4, 2015, pages 458 - 465
PAGANI, A. ET AL., BLOOD, vol. 118, 2011, pages 736 - 746
PAGANI, A. ET AL., BLOOD, vol. 129, 2017, pages 3392 - 3395
PAGANI, A. ET AL., HAEMATOLOGICA, vol. 93, 2008, pages 1466 - 1472
PALANI S; SARKAR CA, BIOPHYS J., vol. 95, no. 4, 2008, pages 1575 - 1589
PIGA ET AL.: "Luspatercept (ACE-536) Reduces Disease Burden, Including Anemia, Iron Overload, and Leg Ulcers, in Adults with Beta-Thalassemia: Results from a Phase 2 Study", 1 January 2015 (2015-01-01), XP002782304, Retrieved from the Internet <URL:http://www.bloodjournal.org/content/126/23/752> [retrieved on 20180622] *
RAJA KB ET AL., BLOOD, vol. 94, no. 9, 1999, pages 3185 - 3192
RAMOS E.; RUCHALA P.; GOODNOUGH JB.; KAUTZ L.; PREZA GC.; NEMETH E.; GANZ T.: "Minihepcidin prevent iron overload in a hepcidin-deficient mouse model of severe hemochromatosis", BLOOD, vol. 120, no. 18, 2012, pages 3829 - 36
ROETTO A; DI CUNTO F; PELLEGRINO RM ET AL., BLOOD, vol. 115, no. 16, 2010, pages 3382 - 3389
SCHMIDT PJ; TOUDJARSKA I; SENDAMARAI AK ET AL., BLOOD, vol. 121, no. 7, 2013, pages 1200 - 1208
SHEN, L. ET AL., J. VIS. EXP., vol. 64, 2012, pages e3917
SICH, C. ET AL., EUR. J. BIOCHEM., vol. 267, 2000, pages 5342 - 5355
SILVA M; BENITO A; SANZ C ET AL., J BIOL CHEM., vol. 274, no. 32, 1999, pages 22165 - 22169
SILVESTRI, L. ET AL., CELL METAB., vol. 8, 2008, pages 502 - 511
SINGH S; DEV A; VERMA R ET AL., PLOS ONE, vol. 7, no. 7, 2012, pages e38530
SIVERTSEN EA; HYSTAD ME; GUTZKOW KB ET AL., BR J HAEMATOL., vol. 135, no. 1, 2006, pages 117 - 128
SOCOLOVSKY M ET AL., BLOOD, vol. 98, no. 12, 2001, pages 3261 - 3273
SOCOLOVSKY M ET AL., CELL, vol. 98, no. 2, 1999, pages 181 - 191
SOFRONIADOU, S.; GOLDSMITH, D., DRUG SAF., vol. 34, 2011, pages 97 - 115
SONG, G.-A. ET AL., J. BIOL. CHEM., vol. 285, 2010, pages 22542 - 22553
SPIEKERKOETTER E; TIAN X; CAI J ET AL., J CLIN INVEST, vol. 123, no. 8, 2013, pages 3600 - 3613
SPIEKERKOETTER ET AL., J. CLIN. INVEST., vol. 123, 2013, pages 3600 - 3613
SPIEKERKOETTER, E. ET AL., AM. J. RESPIR. CRIT. CARE MED., vol. 192, 2015, pages 254 - 257
STEINBICKER, A.U. ET AL., BLOOD, vol. 118, 2011, pages 4224 - 4230
SUN, F. ET AL., BIOPHYS. J., vol. 85, 2003, pages 3194 - 3201
THEURL, I. ET AL., BLOOD, vol. 118, 2011, pages 4977 - 4984
THEURL, I. ET AL., HAEMATOLOGICA, vol. 96, 2011, pages 1761 - 1769
UPTON, P.D. ET AL., MOL. PHARMACOL., vol. 73, 2007, pages 539 - 552
V. ALBINANA ET AL: "Immunosuppressor FK506 Increases Endoglin and Activin Receptor-Like Kinase 1 Expression and Modulates Transforming Growth Factor-?1 Signaling in Endothelial Cells", MOLECULAR PHARMACOLOGY, vol. 79, no. 5, 10 February 2011 (2011-02-10), pages 833 - 843, XP055140980, ISSN: 0026-895X, DOI: 10.1124/mol.110.067447 *
VERGA FALZACAPPA M.V. ET AL., J MOL MED, vol. 86, 2008, pages 531 - 540
WALLACE DF ET AL., GASTROENTEROLOGY, vol. 137, no. 3, 2009, pages 1173 - 1175
WANG, T. ET AL., CELL, vol. 86, 1996, pages 435 - 444
WILSON, K.P. ET AL., ACTA CRYSTALLOGR. SECT. D BIOL. CRYSTALLOGR., vol. 51, 1995, pages 511 - 521
XIA, Y.; BABITT, J.L.; SIDIS, Y.; CHUNG, R.T.; LIN, H.Y., BLOOD, vol. 111, 2008, pages 5195 - 5204

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021180840A1 (fr) * 2020-03-12 2021-09-16 Heinrich-Heine-Universität Düsseldorf Inhibition de fkbp1a pour la thérapie du carcinome mammaire triple négatif
WO2021231851A1 (fr) * 2020-05-15 2021-11-18 Celgene Corporation Méthodes et compositions pour traiter l'anémie à l'aide de pièges à ligands actriib et d'inhibiteurs de mtor
WO2022149113A1 (fr) * 2021-01-11 2022-07-14 Università Degli Studi Di Torino Activateurs de l'axe bmp/smad destinés à être utilisés dans le traitement et/ou la prévention de l'atrophie musculaire

Similar Documents

Publication Publication Date Title
Nai et al. Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice
Mejias et al. CPEB4 increases expression of PFKFB3 to induce glycolysis and activate mouse and human hepatic stellate cells, promoting liver fibrosis
Mukherjee et al. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration
Chen et al. EGFR signaling promotes TGFβ-dependent renal fibrosis
Liu et al. Mesenchymal stem cells alleviate the early brain injury of subarachnoid hemorrhage partly by suppression of Notch1-dependent neuroinflammation: involvement of Botch
Gong et al. Claudin-14 underlies Ca++-sensing receptor–mediated Ca++ metabolism via NFAT-microRNA–based mechanisms
Zhou et al. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival
Besson-Fournier et al. Induction of activin B by inflammatory stimuli up-regulates expression of the iron-regulatory peptide hepcidin through Smad1/5/8 signaling
US11559522B2 (en) Methods for enhancing liver regeneration
Olerud et al. Thrombospondin-1: an islet endothelial cell signal of importance for β-cell function
Chen et al. Transforming growth factor β1 (TGF-β1) activates hepcidin mRNA expression in hepatocytes
WO2018185341A1 (fr) Régulateur de signalisation de bmp-smad et ses utilisations
Nordquist et al. Proinsulin C‐peptide prevents type‐1 diabetes‐induced decrease of renal Na+‐K+‐ATPase α1‐subunit in rats
Gui et al. LncRNAH19 improves insulin resistance in skeletal muscle by regulating heterogeneous nuclear ribonucleoprotein A1
Shao et al. Angiotensin type 2 receptor in pancreatic islets of adult rats: a novel insulinotropic mediator
KR20150131260A (ko) 결장직장암의 치료 방법
Sharma et al. Mechanisms of acquired resistance to anti-VEGF therapy for neovascular eye diseases
Bryson et al. The deleterious role of the prostaglandin E2 EP3 receptor in angiotensin II hypertension
Reardon et al. Safety and pharmacokinetics of dose-intensive imatinib mesylate plus temozolomide: phase 1 trial in adults with malignant glioma
Chen et al. RNA interference targeting the platelet‐derived growth factor receptor β subunit ameliorates experimental hepatic fibrosis in rats
US9539323B2 (en) Methods and compositions for malic enzyme 2 (ME2) as a target for cancer therapy
Farias Quipildor et al. Modulation of glucose production by central insulin requires IGF-1 receptors in AgRP neurons
Ahn et al. Substance P reduces infarct size and mortality after ischemic stroke, possibly through the M2 polarization of microglia/macrophages and neuroprotection in the ischemic rat brain
Efthymiou et al. Inhibition of AXL receptor tyrosine kinase enhances brown adipose tissue functionality in mice
US20100239596A1 (en) Grp78 and tumor angiogenesis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18719770

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18719770

Country of ref document: EP

Kind code of ref document: A1