Molecular basis of iron homeostasis

and its translation to the clinic

Tomas Ganz, PhD, MD Systemic iron homeostasis Liver (storage, recycling) Spleen (recycling, storage)

Liver Fe

20-25 mg/day Plasma Erythrocytes Fe-Tf 1-2 mg/day

1 ml of PRBC = 1 mg Fe (usage) Duodenum (absorption) Losses 1-2 mg/day During erythropoietic stimulation Liver (storage, recycling) Spleen (recycling, storage)

Liver Fe

Plasma Erythrocytes Fe-Tf

1 ml of PRBC = 1 mg Fe Bone marrow (usage) Duodenum (absorption) Losses During infection/inflammation Liver (storage, recycling) Spleen (recycling, storage)

Liver Fe Inflammation

Plasma Erythrocytes Fe-Tf

1 ml of PRBC = 1 mg Fe Bone marrow (usage) Duodenum (absorption) Losses —an iron-regulatory peptide hormone • Made in the liver as 84 aa preprohepcidin • Cleaved to 25 aa bioactive hepcidin by furin • Secreted by hepatocytes • Regulates intestinal iron absorption and the distribution of iron to tissues

DTHFPICIFCCGCCHRSKCGMCCKT

Jordan et al. JBC 2009 Tomas Ganz Hepcidin peptide lowers plasma iron

Synthetic hepcidin 50 µg IP/mouse

40

M) 30 

20 * * *

Serum IronSerum ( * 10 * *

0 0 1 2 3 4 5 6 24 48 72 96 Time (hours) Rivera et al. Blood 2005 • 12 transmembrane segment protein • Found in plants, invertebrates, vertebrates • In vertebrates, the receptor for hepcidin • The only cellular iron exporter known (Donovan et al.) • Present in the macrophages, duodenum, hepatocytes, and the placenta (Donovan et al., McKie et al., Abboud et al.)

45 61 115 127 203 206 324 343 393 450 512 518

23 80 96 152 186 228 307 362 374 471 493 537

N C Ferroportin is internalized by hepcidin and degraded in lysosomes

control + 1 µg/ml hepcidin

Nemeth et al. Science 2004 Regulation of intestinal iron absorption Dietary iron uptake Food Dietary iron uptake

ferritin ferritin Duodenal enterocytes Fpn

Fpn X Blood hepcidin Fe Fe Low hepcidin High hepcidin Regulation of erythrocyte iron recycling

Erythrocyte Erythrocyte uptake uptake Macrophages

ferritin ferritin

Fpn

Fpn X Iron release into plasma hepcidin Fe Fe Low hepcidin High hepcidin Hepcidin regulates dietary iron absorption and influx to plasma

Liver Spleen

Liver hepcidin Fe Fpn hepcidin Fpn

Plasma Fe-Tf Erythrocytes Fpn

Bone marrow Duodenum

Tomas Ganz Signals regulating hepcidin Inflammation Liver Spleen

Liver hepcidin Fe Fpn hepcidin Fpn

Plasma Erythrocytes Fe-Tf Fpn Iron signals

Bone marrow Duodenum

Erythropoietic signal Hepcidin excess in of inflammation • Hepcidin-dependent effects – Inflammatory cytokines increase hepcidin and cause Fe trapping in macrophages – Fe restricts synthesis and is inhibited • Hepcidin-independent effects – Shortened erythrocyte lifespan – Direct suppression of erythropoiesis by cytokines

Iron-restricted

Inflammation Liver Spleen

Plasma hepcidin clearance Fe-Tf RBC

Duodenum Bone Kidneys marrow Ablation of hepcidin is beneficial in anemia of inflammation induced by heat-killed Brucella abortus

18

16 hepcidin KO WT 14 saline

12 Brucella 10

8 Hemoglobin (g/dL)

6 0 7d 14d 21d 28d Time

Kim et al. Blood 2014 Hepcidin deficiency in β-

secreted suppressors less hepcidin Fe overload  Worse anemia

 • Failure of red cell precursors to mature into functional RBCs • Anemia, increased erythropoietin • Expansion of immature RBC precursors • Hepcidin-suppressive signal causes hepcidin deficiency Anemia • Increased Fe absorption, Fe overload Rational design of peptide agonists • Strategy for development: structure-function study of hepcidin and ferroportin

C

N 45 61 115 127 203 206 324 343 393 450 512 518

23 80 96 152 186 228 307 362 374 471 493 537

N C

Hepcidin Ferroportin 25 aa peptide hormone 571 aa protein 4 disulfide bonds 12 transmembrane segments

Preza at al. JCI 2011, Ramos et al. Blood 2012 Minihepcidin PR73

Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-Ahx- Ida(NHPal)-CONH2 iminodiacetic acid, L-threonine, L-histidine, L-3,3-diphenylalanine, L-β-homoproline, L- arginine, L-cysteine, L-arginine, L-β-homophenylalanine, 6-aminohexanoic acid, iminodiacetic acid palmitylamide (PEG-free) Mouse model of β-thalassemia: minihepcidin improves anemia and decreases

1600 16 1400 14 p = 0.001 p = 0.0003 1200 12

g/g) 1000 10  800 8

6 600 Liver iron ( iron Liver Hemoglobin (g/dl) Hemoglobin 4 400

2 200

0 0 WT TH3/solvent TH3/MH WT TH3/solvent TH3/MH Mouse/treatment Mouse/treatment

M004 (2.65 mg/kg) or solvent was administered 2x weekly SQ x 6 wks

C Casu, S. Rivella et al. and Merganser Biotech A search for erythroid factor suppressing hepcidin • Stimulated erythropoiesis in mice by phlebotomy (0.5 ml) or EPO injection (200 U) 4

phlebotomy 2 * p<0.05 EPO ** p<0.01 1 * ***p<0.001 * ** 0.5

mRNA expression *** *** 0.25 *** *** Hamp 0.125 Liver 0.062 CTRL 4h 9h 12h 15h 24h

• Gene chip expression profiling of the bone marrow to identify erythroid transcripts that 1) change before the hepcidin suppression and 2) encode secreted proteins Erythroferrone (Erfe) Erythroferrone (Fam132b, CTRP15)

TNFα-like

Polybasic cleavage sites?

Signal sequence

Collagen stalk GPP-rich

• 50 kD glycoprotein highly expressed in erythropoietin-stimulated erythroblasts Erfe knockout mice do not suppress hepcidin in response to bleeding • Erfe1+/+, +/- and -/- mice were phlebotomized (0.5 ml) Hepcidin mRNA Hemoglobin recovery

* p<0.05 ** p<0.01 *** p<0.001 Dose-response for mouse erythroferrone on hepatocytes

1.0 EC50 = 7 ng/ml ~ 140 pM 0.8

0.6

0.4

Fractional response Fractional 0.2

0.0

0 1 3 10 30 100 300 1000 Conc ng/ml mErfe (24-340) Mouse model of β-thalassemia: Ablation of Erfe reverses hepcidin suppression and improves iron overload

Hepcidin Liver iron 200 *** 300 *** *** *** * 150

200 100

50 100 serum hepcidin (ng/mL)

0 g non-heme iron / g liver wet weight 0  WT WT Th3/+ Th3/+ Erfe-/-Th3/+ Erfe-/-Th3/+ Development of new treatments

• Prevent iron overload and improve erythropoiesis in β-thalassemia – minihepcidins (hepcidin agonists) – erythroferrone antagonists • Release sequestered iron in anemia of inflammation and anemia of chronic kidney disease – hepcidin antagonists – erythroferrone agonists • UCLA licensed the intellectual property related to minihepcidin and erythroferrone to two startup companies for drug development Key contributors

Ella Nemeth: Piotr Ruchala: Everything Minihepcidin design

Airie Kim: León Kautz: Mouse models Erythroferrone of anemia of inflammation