Iron: a global issue in hematology

Clara Camaschella, MD

Università Vita Salute San Raffaele e

IRCCS San Raffaele - Milano

Firenze, 18-19 settembre 2015

Clara Camaschella

I have nothing to disclose

Heme

Iron is essential in Hematology

Hb,

cytochromes

Iron deficiency anemia

Anemia of chronic diseasesIron/Sulfur clusters

Enzymes, IRP proteins

Iron may be toxic in Hematology

Fenton reaction: Fe2+ + H2O2 HO. + HO- + Fe3+

Systemic iron overload

(blood transfusions, ineffective erythropoiesis)

Erythroid iron overload

(sideroblastic anemia)

Cellular iron handling

(Hentze, Galy, Muckenthaler & Camaschella, Cell 2010)

IRPs regulation

ferritinophagy

ALAS2

Iron distribution and regulation

(Nemeth et al, Science 2004)

Senescent

erythrocytesEnterocyte

FPN

FPN

Fe

IL-6

Macrophage

DMT1

HEPCIDIN

HEPCIDIN

Systemic iron regulation: physiology

(Hentze, Galy, Muckenthaler & Camaschella, Cell 2010)

Mechanisms of adaptation in iron deficiency anemia

(Camaschella C, N Engl J Med 2015)

Hemochromatosis: a disorder of hepcidin

skin

joints

pancreas

heart

Dietary iron

pituitary

liver

Liver: fibrosis, cirrhosis, HCC

Cardiac iron

Joint disease Endocrine damageSkin pigmentation

Hepcidin regulation and rare disorders

of the pathway

(Adapted from Hentze et al Cell, 2010)

Genetic

Iron overload

6

liver iron circulating iron

Genetic

iron deficiency

IRIDA

(Silvestri et al,

Cell Met 2008;

Finberg et al,

Nat Genet 2008)

erythropoiesis

EPO

?

erythroid regulator(s)

Hepcidin suppression: iron deficiency,

hypoxia and expanded erythropoiesis

Proposed erythroid regulators: GDF15, TWSG1, sHJV, HIF-1a, ERFE

EPO

NTD b-thalassemia,

CDA, Sideroblastic

anemia…….

A novel candidate erythroid regulator….

*a member of the C1q/TNF-related protein family (Kautz et al, Nat Genet 2014)

……. a therapeutic target?

*

The Hbbth3/+ mouse: a model of thal intermedia

RBC (106/μl) Hb (g/dl) Hct (%) Retics (%)

wt 9.5 ±0.4 16.5±0.5 45.7±0.8 1.4±0.4

Hbbth3/+ 6.6±0.1 10.9±0.3 30.5±0.8 30.3±0.9

Anemia and increased

reticulocyte count

(Yang et al, PNAS 1995)Abnormal red cell

morphology

(Yang et al, PNAS 1995)

Hbbth3/+wt

Ineffective erythropoiesis

(Gardenghi et al, JCI 2010)

Splenomegaly

(Libani et al, Blood 2008)

Iron overload

wt Hbbth3/+

(De Franceschi et al, Haematologica 2006)

Low hepcidin

Genetic loss of Tmprss6 ameliorates the

b-thalassemic phenotype

Tmprss6-/-Hbbth3/+

X

Double mutants followed up to 6 months:

Hepcidin increases and iron overload is prevented

Ineffective erythropoeisis and anemia improve(Nai et al Blood 2012)

TMPRSS6 gene silencing had similar effects in b-thal and

HFE-hemochromatosis preclinical models

(Schmidt et al, Blood 2013; Guo et al, JCO, 2013)

(Camaschella C, NEJM 2013)

Inhibiting the hepcidin inhibitor

Positive effect of iron restriction in b-thalassemia

• Transferrin infusions (Hbbth3/+, Hbbth1/th1)

(Li H et al, Nat Med 2010; Gelderman et al, Haematologica 2015)

• Use of “minihepcidin”

(Preza et al, JCI 2011; Ramos Blood 2012; Casu et al, ASH 2014)

• Inhibition of the hepcidin inhibitor Tmpss6

(Schmidt et al, Blood 2013; Guo et al, JCO, 2013)

Tmprss6-/- and Erfe-/- phenotype comparison

IDA with high hepcidin

Tmprss6-/- Erfe-/- mouse

Mild anemia (young mice)

(Nai et al, Blood 2012)

Hb increase (1.5 g/dl)

Tmprss6-/-, Hbbthal3/+ Erfe-/-, Hbbthal3/+

Hb unchanged

(Kautz et al, Blood 2015)

Erythropoiesis and iron absorption in thalassemia

EpoR

pJak2

pStat5 Cell Replication

Protection from

apoptosis

Epo

ß-Thalassemia: ROS &

Apoptosis

pJak2

Increased

number of

progenitor

erythroid cells

GDF11

Decreased cell

differentiation

Erythroferrone

GDF15……

courtesy of Stefano Rivella

Hepcidin

Increased iron absorption

Implications

Iron overload exacerbates ineffective erythropoiesis in β-

thalassemia)

Iron restriction (or manipulation of hepcidin pathway by

“hepcidin agonists”) may be exploited as a therapeutic

tool in β-thalassemia

Is iron a modifier of normal, effective erythropoiesis?

Why two transferrin receptors?

TFR1 TFR2

Family member 1 Family member 2

Transmembrane glycoprotein Transmembrane glycoprotein:

homologus to TFR1 extracellular domain

IRE elements 3’UTR No IRE elements

Binds diferric-TF at high affinity Binds diferric-TF at lower affinity than TFR1

Inactivation in mouse: embryolethal Inactivation: in mice and humans

(severe iron deficiency) iron overload

Ubiquitous expression Restricted expression to hepatocytes,

erythroid cells - EpoR interaction

(Foretnikova et al, Blood 2010)

Sequential markers of erythroid development

BFU-E CFU-E

Erythroid Progenitors

Terminal erythropoiesis

GlyAEPOR TFR1 (CD71)

CD36TFR2

Perls’ 250x

Mutations of TFR2Type 3 Hemocromatosis

Autosomal recessive, rare

Elevated TF saturation

Hepatic iron load – low hepcidin

Early onset of iron overload

Phlebotomy-responsive

Camaschella et al, Nat Genet 2000

Girelli et al, Gastroenterol 2002

Tfr2 KO

Wild type

Fleming et al, PNAS 2002

Girelli et al, Haematologica 2011

Oral iron

Murine models

Type Phenotype Reference

Tfr2Y245X/ Y245X

iron overload - low Hamp Fleming et al, 2002

Tfr2 KO iron overload - low Hamp Wallace et al, 2007Roetto et al, 2010

Tfr2LCKO iron overload - low Hamp Wallace et al 2007Roetto et al, 2010

Tfr2 KO/Hfe KO severe - low Hampiron overload

Wallace et al, 2009

Tmprss6 KO/Tfr2 KO iron deficiency - high Hamperythrocytosis

Nai et al, 2014

Tmprss6 KO/Tfr2LCKO iron deficiency - high Hampno erythrocytosis

Nai et al, 2014

Tfr2 KO

(Nai et al Haematologica 2014)

Generation of a BM-specific Tfr2-/- model

Tfr2-/- or wt Tfr2-/- or wt BMwt

BM

(Nai, Lidonnici et al, Blood 2015)

Iron parameters and hepatic gene expression

in Tfr2BMKO mice

(Nai, Lidonnici et al, Blood 2015)

(Nai, Lidonnici et al, Blood 2015)

Erythroid cells are increased and apoptosis decreased

in the bone marrow of Tfr2 BMKO mice

II III IV V

Serum EPO

I

B

I

D

Increased EPO sensitivity in Tfr2BMKO mice

TFR2 TFR2

ERFE

Tfr2BMKO mice phenotype

Erythrocytosis, increased erythroid precursors and

decreased apoptosis.

Normal Epo levels. Increased expression of Epo

target genes in isolated erythroblasts

1. Tfr2BMKO erythroblasts have increased Epo

sensitivity

2. Deletion of erythroid Tfr2 mimics iron-deficiency

TFR2 links erythropoiesis and hepcidin

TFR2 is stabilized on plasma membrane by the diferric transferrin ligand(Johnson et al, Mol Biol Cell 2007; Pagani et al, Haematologica 2015)

Does TFR2 modulate the Epo sensitivity in ID?

Lack of membrane TFR2 stabilization in ID. Degradation?

TFR2 releases a soluble form in iron deficiency in vitro(Pagani et al Haematologica 2015)

Conclusion

TFR2 is a component of a novel iron-sensing mechanism

that adjusts erythropoiesis according to iron availability,

by modulating the erythroblast Epo sensitivity

Loss of TFR2 in iron deficiency (likely by sTFR2 shedding) optimizes eryhtropoiesis and increases ERFE, leading to hepcidin suppression and iron acquisition

TFR2 links hepcidin and iron homeostasis to erythropoiesis

TFR2 = liver sensor of circulating iron (excess)

TFR2 = erythroid sensor of circulating iron (deficiency)

PP

P

IL-6

IL-6R

JAK2

STAT3

PP

SMAD1/5/8

SMAD4

BMPRHJV

TMPRSS6

HAMPBRE SREBRE

Activin-b

Inflammation

Hepcidin upregulation by inflammatory cytokines....

……decreases iron available for

pathogens

……explains iron maldistribution,

macrophage sequestration and

iron-restricted erythropoiesis in

anemia of inflammation (ACD)

Hepcidin

antagonists

(modified from Fung & Nemeth

Haematologica 2013)

Class 1

Inhibitors of hepcidinproduction

BMP binders (sHJV, heparin)BMPR inhibitors (LDN-193189)

HJV antisense oligonucleotidesHepcidin antisense oligonucleotides

Anticytokines (IL6, IL6R, TNFalpha)

ESA

Class 2

Hepcidin neutralizing peptides

Antihepcidin monoclonal antibodiesAnticalinSpiegelmers

Class 3

Interfering withhepcidin/FPN binding

Anti-FPN monoclonal antibodiesThiol modifiers

Iron handling by tumor cells

(Torti & Torti. Nat Rev Cancer 2013)

A)Normal cell Cancer cell

Role of hepcidin/ferroportin axis in cancer

Breast cancer: Pinnix et al. Sci Transl Med. 2010

Prostate cancer: Tesfay et al. Cancer Res. 2015

Multiple Myeloma: Gu et al. Cancer Res. 2015

Campanella et al Haematologica 2013

Bordini et al, Haematologica 2015

Acknowledgments

(PRIN) - Rome

Vita-Salute University and

San Raffaele Scientific Institute

Tiget, HSR

Giuliana Ferrari

M. Rosa Lidonnici

Giacomo Mandelli

Antonella Roetto, TorinoDomenico Girelli, Verona

Paolo Arosio, Brescia

Institut Cochin, Université Paris

Descartes

Frédérique Verdier

Patrick Mayeux

Catherine Lacombe

Carlos Lopez Otin, Oviedo