New Insights in the Understanding of Cholesterol Metabolism:

The Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in the Regulation of Low-Density Lipoprotein Cholesterol (LDL-C) and LDL Receptors (LDLRs) USA-145-100024(1) © 2014 Amgen Inc. All rights reserved. Not for Reproduction.

Table of Contents

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Pathway

Genetic Variants of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

2

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Pathway

© 2014 Amgen Inc. All rights reserved. Not for Reproduction.

Hepatic LDLRs Play a Central Role in Cholesterol Homeostasis

LDL LDLR Clathrin-coated vesicle Hepatocyte

LDL = low-density lipoprotein; LDLR = low-density lipoprotein receptor

1.

Steinberg D, et al.

Proc Natl Acad Sci

. 2009;106:9546-9547.

2.

Brown MS, et al.

J Lipid Res

. 2009;50:S15-S27.

4

Recycling of LDLRs Enables Efficient Clearance of LDL-C Particles

Increased LDLR surface concentration Lysosomal degradation LDLR recycling

LDL-C = low-density lipoprotein cholesterol

1.

Steinberg D, et al.

Proc Natl Acad Sci Biol

. 2009;29:431-438.

3.

. 2009;106:9546-9547.

2.

Goldstein JL, et al.

Arterioscler Thromb Vasc

Brown MS, et al.

Proc Natl Acad Sci

. 1979;76:3330-3337.

5

PCSK9 Regulates the Surface Expression of LDLRs by Targeting for Lysosomal Degradation Decreased LDLR surface concentration PCSK9 secretion Lysosomal degradation LDLR/PCSK9 routed to lysosome

PCSK9 = proprotein convertase subtilisin/kexin type 9

1.

3.

Qian YW, et al.

J Lipid Res

Brown MS, et al. . 2007;48:1488-1498.

Proc Natl Acad Sci

2.

Horton JD, et al. . 1979;76:3330-3337.

4.

9547.

5.

Goldstein JL, et al.

Arterioscler Thromb Vasc Biol

6 2007;282:18602-18612.

J Lipid Res

Steinberg D, et al.

6.

. 2009;50:S172-S177.

Proc Natl Acad Sci

Zhang DW, et al.

. 2009;106:9546-

J Biol Chem.

Genetic Variants of PCSK9 Demonstrate Its Importance in Regulating LDL Levels PCSK9 Gain of Function = Less LDLRs 1 PCSK9 Loss of Function = More LDLRs 1 Recycling of LDLR Lysosomal degradation of LDLR Gain-of-function PCSK9 Loss-of-function PCSK9

Mutations in the human

PCSK9

gene that lead to a loss of PCSK9 function are found in 1% to 3% of the representative populations 2,3

1.

Steinberg D, et al.

PNAS.

2009;106:9546-9547.

2.

Cohen JC, et al.

N Engl J Med

. 2006;354:1264-1272.

3.

Benn M, et al.

J Am Coll Cardiol

. 2010;55:2833-2842.

7

Gain-of-Function Mutations in PCSK9 Cause Familial Hypercholesterolemia* † PCSK9 Variant Population Clinical/Biochemical Characteristics

D374Y 1 S127R 1 British, Norwegian families, 1 Utah family French, South African, Norwegian families Tendon xanthomas, severe hypercholesterolemia Tendon xanthomas • R218S 2 Associated with: – – High serum LDL-C 1 French families Tendon xanthomas, arcus corneae In vitro testing in many identified mutations shows decreased levels of LDLRs 3 *Autosomal Dominant Hypercholesterolemia

1.

Abifadel M, et al.

Hum Gen

. 2009;30:520-529.

2.

Lopez D.

Biochem Biophys Acta

. 2008;1781:184-191.

3.

Cameron J, et al.

Hum Mol Genet

. 2006;15:1551-1558.

8

† For a full list of ADH mutations, please refer to Abifadel reference.

Loss-of-Function Mutations in PCSK9 Are Associated With Decreased LDL-C

• •

PCSK9 Variant Population LDL-C

R46L Y142X or C679X ARIC ARIC 1 1 , DHS , DHS 2 2 ↓ 15% 1 ↓ 28%–40% 1 • R46L CGPS 3 ↓ 11% Heterozygous LOF mutations found in 1% to 3% of representative populations 1,3 3 Associated with – Lower serum LDL-C 1 PCSK9 null individual identified (compound heterozygote for two inactivating mutations) – No detectable circulating PCSK9 with strikingly low LDL-C (14 mg/dL) 4 LOF = loss of function ARIC = Atherosclerosis Risk in Communities (N ~ 4,000); DHS = Dallas Heart Study (N = 3,553); CGPS = Copenhagen General Population Study (N = 26,013)

1.

Cohen JC, et al.

N Engl J Med

. 2006;354:1264-1272.

2.

Cohen J, et al

. Nat Genet

. 2005;37:161-165

3.

Benn M, et al.

J Am Coll Cardiol

. 2010;55:2833-2842.

4.

Zhao Z, et al

. Am Journal of Hum Gen

. 2006;79:514-534.

9

LDLR and PCSK9 Expression Are Both Upregulated When Intracellular Cholesterol Levels Are Low

SREBP = sterol regulatory element-binding protein

1.

Goldstein JL, et al.

Arterioscler Thromb Vasc Biol

. 2009;29:431-438.

2.

Dubuc G, et al

. Arterioscler Thromb Vasc Biol

. 2004;24:1454-1459.

10

Expression of PCSK9 Depends on Intracellular Cholesterol Levels - Cholesterol Depletion* - Statins ↑ SREBP-2 ↑ PCSK9 - Dietary Cholesterol - Cellular Cholesterol ↓ SREBP-2

*Intracelullar Cholesterol Depletion

1.

Abifadel M, et al. In: Toth PP.

The Year in Lipid Disorders.

Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23. 11

↓ PCSK9

Summary

• LDLR and PCSK9 Expression Are Both Regulated by Intracellular Cholesterol Levels 1,2 • Genetic Variants of PCSK9 Support its Role in Regulating LDL Levels 2 – – Gain-of-function mutations result in increased LDL-C 3,4 Loss-of-function mutations are associated with decreased LDL-C 5

1.

Dubuc G, et al

. Arterioscler Thromb Vasc Biol

. 2004;24:1454-1459.

2.

Abifadel M, et al. In: Toth PP.

The Year in Lipid Disorders.

Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23.

3.

Abifadel M, et al.

Hum Mutat.

2009;30:520-529.

4.

Med

. 2006;354:1264-1272. Horton JD, et al.

J Lipid Res

12

5.

Cohen JC, et al.

N Engl J