Rein et Oxygène

Targeting the HIF Oxygen-Sensing Pathway for

Anemia Therapy

Volker H. Haase

The Krick-Brooks Chair in Nephrology Departments of Medicine, Cancer Biology,

Molecular Physiology and Biophysics Vanderbilt University, Nashville

Outline

Brief overview of the HIF pathway

Cellular and molecular regulation of erythropoietin

(EPO) production in the kidney

Application to renal anemia: Inhibition of HIF PHDs

stimulates EPO production in the kidney and liver

Application to renal anemia: HIF and iron metabolism

Application to renal anemia: HIF-prolyl hydroxylase

(HIF-PHIs) inhibitors in clinical trials

Physiologic Responses to Hypoxia

and Discovery of the HIF-PHD Pathway

Activation of hypoxic signaling occurs when tissues experience

a discrepancy between O2 demand and supply

Exercise Tumor

High altitude

Time Course of Selected Physiologic Responses to Hypoxia

Andrew M. Luks J Appl Physiol 2015;118:509-519

©2015 by American Physiological Society

Anglo-American Expedition to Pikes Peak, CO Aug. 1911, 14147 ft (JS Haldane, CG Douglas, Y Henderson, EC Schneider, and M Fitzgerald)

Pioneers of High Altitude Medicine

HIF controls the Hypoxic Induction of EPO

• Wang, G.L. and Semenza, G.L. (1995). Purification and characterization of hypoxia-inducible factor

1. J. Biol. Chem. 270, 1230-1237.

• Wang, G.L., Jiang, B.-H., Rue, E.A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a

basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci.

USA. 92, 5510-5514.

• Forsythe, J.A., Jiang, B.-H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., and Semenza, G.L. (1996).

Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

Mol. Cell. Biol. 16, 4604-4613.

• Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (1993). Inducible operation of the erythropoietin

3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc.

Natl. Acad. Sci. USA. 90, 2423-2427.

• Maxwell, P.H. et al. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors

for oxygen-dependent proteolysis. Nature. 399, 271-275.

• Jaakkola, P. et al. (2001). Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by

O2-regulated prolyl hydroxylation. Science. 292, 468-472

• Epstein, A.C.R. et al.. (2001). C. elegans EGL-9 and mammalian homologs define a family of

dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 107, 43-54.

• Iliopoulos, O., Levy, A.P., Jiang, C., Kaelin, W.G., Jr., and Goldberg, M.A. (1996). Negative regulation

of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc. Natl. Acad. Sci. USA. 93,

10595-10599.

• Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.-Y., Huang, L.E., Chau, V., Pavletich, N., and

Kaelin, W.G., Jr. (2000). Ubiquitination of hypoxia-inducible factor requires direct binding to the β-

domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423-427.

• Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S., and

Kaelin, W.G., Jr. (2001). HIF targeted for VHL-mediated destruction by proline hydroxylation:

implications for O2 sensing. Science. 292, 464-468

Discovery of the HIF-Oxygen Sensing Pathway

Gregg L. Semenza

Johns Hopkins

Peter J. Ratcliffe

Oxford

William G. Kaelin, Jr.

Harvard

Transcriptional Targets of HIF

HIF

Vasculogenesis/Angiogenesis Heme oxygenase-2 NOS-2, PAI - I VEGF, VEGF-R (FLT-1)

Iron metabolism /Erythropoiesis Ceruloplasmin EPO (HIF-2 > HIF-1) Transferrin Transferrin R

Metabolism: Glycolysis (HIF-1) Fat metabolism (HIF-2) Adenylate kinase-3 Carbonic anhydrase-9 Glut-1 and -3 Glycolytic enzymes (Hexokinase, LDH,PGK, etc.) Leptin

Proliferation/Cell survival Cyclin G2, EPO, Heme oxygenase 1 IGF-2, IGFBP1, -2, -3 NOS-2, NIP-3, p21 TGF-b3, VEGF, WT1 Indirect: c-Myc

HYPOXIA via

ECM production/Cell migration CxCR4, c-met, CTGF, PAI-1, Procollagen prolyl hydroxylase-a1

Epithelial Barrier Function ITF, MDR-1, CD73

Transcription factors Ets-1, DEC-1/Stra13

Regulation of HIF-α Stability

Angiotensin II

Non-hypoxic

stimuli:

Interleukin1,

TNF-a, Growth

factors

HIF: hypoxia-inducible factor

The Hydroxylation Reaction

2OG-dependent Dioxygenases

- at least 60 in mammals -

Loenarz and Schofield, Nature Chemical Biology, 2008 PHD1, PHD2, PHD3

FIH

Cellular and Molecular Regulation of

EPO Production in the Kidney

GATA-2/3 (5’-TGATAA-3’)

Hypoxic Regulation of EPO: HIF-2

EPO: erythropoietin; HIF: hypoxia-inducible factor.

The Number of Renal EPC

Determines EPO Output

EPC: erythropoietin-producing cell; EPO: erythropoietin; HIF: hypoxia-inducible factor; PHD: prolyl-4-hydroxylase domain.

Kidney at baseline:

small EPC pool size

Erythrocytosis Syndromes Resulting from Genetic Defects in HIF-2 Oxygen Sensing

HIF-2 and PHD2 mutations confer protection from high altitude-associated illnesses

Hemoglobin concentration of high-altitude

Tibetans and Bolivian Aymara. Cynthia Beall et

al. Am J Phys Anthropol 1998, 106: 385-400.

Genetic Evidence for High-Altitude Adaptation

in Tibet. Tatum S. Simonson et al. Science 2010,

329: 72-75.

Sequencing of 50 human exomes reveals

adaptation to high altitude. Xin Yi et al. Science.

2010, 329: 75-8.

Application to Renal Anemia I:

Inhibition of HIF-PHDs Stimulates EPO

Synthesis in Kidney and Liver

200 X

EPO Neg. Ctl

400 X 200 X

EPO

Phd2-/- Phd2-/- Phd2-/-

Cre-/- Cre-/- Cre-/-

Targeting Phd2 in Peritubular Interstitial Cells

EPO: erythropoietin; PHD: prolyl-4-hydroxylase domain.

Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

Targeting Phd2 in Renal Interstitial Cells

Co: control; EPO: erythropoietin; Hb: hemoglobin; Hct: hematocrit; PHD: prolyl-4-hydroxylase domain; RBC: red blood cells;

Retic: reticulocyte; VEGF: vascular endothelial growth factor.

Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

Distinct Interstitial Cell Populations

Regulate Renal EPO Output

EPO: erythropoietin; PHD: prolyl-4-hydroxylase domain; REPC: renal EPO-producing cell.

Kobayashi H, et al. J Clin Invest. 2016;126(5):1926-1938.

The Liver as a Source of EPO:

Role of Individual PHDs

EPO: erythropoietin; PHD: prolyl-4-hydroxylase domain.

Tojo Y, et al. Mol Cell Biol. 2015;35(15):2658-2672.

The hypoxic induction of EPO in the kidney and liver is HIF-

2-dependent

Inactivation of PHD2 alone is sufficient to stimulate the

production of renal EPO

There are at least two distinct populations of EPO-

producing cells in the kidney that differ in their regulation

of HIF-2 activity and EPO production

Inactivation of at least 2 PHD enzymes in the liver is

required to stimulate erythropoiesis

Key Points

EPO: erythropoietin; HIF: hypoxia-inducible factor; PHD: prolyl-4-hydroxylase domain.

Application to Renal Anemia II:

HIF and iron metabolism

Pathogenesis of Renal Anemia

EPO: erythropoietin.

Koury MJ, Haase VH. Nat Rev Nephrol. 2015;11(7):394-410.

REPC in Renal Injury

CKD: chronic kidney disease; EPO: erythropoietin; REPC: renal EPO-producing cell.

HIF in Iron Metabolism

PHD inhibition stimulates both renal and hepatic EPO

production

Myofibroblast differentiation reduces EPO producing capacity

in the kidney

PHD therapy in CKD: maintenance of EPO-producing

capacity in interstitial cells that already produce EPO versus

conversion of non-EPO producing interstitial cells into EPO-

producing cells

HIF activation enhances iron uptake and utilization. HIF

effects on hepcidin production appear to be indirect

Key Points

CKD: chronic kidney disease; EPO: erythropoietin; HIF: hypoxia-inducible factor; PHD: prolyl-4-hydroxylase domain.

Application to Renal Anemia III:

HIF-PHD Inhibitors: Clinical trials

The Hydroxylation Reaction

HIF-Prolyl Hydroxylase Inhibition Beyond erythropoiesis: what to look for

VEGF

Metabolic effects (glucose, cholesterol, fat

metabolism, uric acid, FGF 23)

Blood pressure, hemodynamics

effects on kidney disease progression

PHD2 vs. PHD1 vs. PHD3 - IC50 profiles

HIF PHDs have other hydroxylation targets

effects on other dioxygenases (FIH, Jmjds, others)

tissue distribution

Biological effects in kidney vs. liver vs. other tissues

(bone marrow, gastrointestinal tract)

Genetic variations

HIF-Prolyl Hydroxylase Inhibition How to evaluate compound actions

pharmacodynamic and pharmacokinetic considerations

Haase V.H.; Hemodialysis International, in press

HIF-Prolyl Hydroxylase Inhibition

Overview of compounds

HIF-PHIs

Chemical structure

Haase V.H.; Hemodialysis International, in press

HIF-Prolyl Hydroxylase Inhibition

Clinical experience from phase 2 trials

HIF-PHIs have been effective in stimulating erythropoiesis

To date HIF-PHIs have been well tolerated in phase 2 trials

Some of the compounds have clinical effects beyond

erythropoeisis

Long term safety evaluations are pending

The effects on CKD progression are not known

HIF-Prolyl Hydroxylase Inhibition

Summary