Structural determinants of lipoprotein(a) pathogenicity Marlys L. - PowerPoint PPT Presentation

Structural determinants of lipoprotein(a) pathogenicity Marlys L. Koschinsky, PhD FAHA FNLA Scientific & Executive Director Robarts Research Institute Professor, Dept. of Physiology & Pharmacology Schulich School of Medicine & Dentistry The University of Western Ontario @RobartsDirector

Disclosures Marlys L. Koschinsky holds/has held research grants from Canadian Institutes of Health Research, Heart and Stroke Foundation of Canada, Natural Sciences and Engineering Research Council of Canada, and Pfizer; is a member of advisory boards for Sanofi and Amgen; has received speaker’s honoraria/consulting fees from Amgen, Regeneron, and Eli Lilly; and holds/has held research contracts with Sanofi, Ionis, Eli Lilly, and Abcentra (Cardiovax).

Acknowledgements Team Koschinsky Alumni UWO University of Washington Dr. Amer Youssef Dr. Corey Scipione Dr. Robert Hegele Dr. Santica Marcovina Julia St. John Dr. Rocco Romagnuolo Dr. Murray Junop Matthew Borrelli Jackson McAiney Robert Szabla University of Helsinki Justin Clark Matthew Gemin Dr. Kati Öörni Bella Xing Dr. Martina Lorey Ahmed Habib IRCM University of Amsterdam Dr. Nabil Seidah Dr. Erik Stroes University of California, San Diego Team Boffa Dr. Annik Prat Renate Hoogeveen Dr. Sam Tsimikas Tasnim Reza Dr. Joe Witztum Abdullah Masoodi Kevin Zhang John Ackersviller

Lp(a) assembly – the science is not settled

Models from in vivo kinetics Model I Model IIa Lp(a) formed Some in or on contribution hepatocytes from circulating LDL Model IIb Model III Lp(a)-apoB Apo(a) recycled recycled Reyes-Soffer G, et al. J Lipid Res 2017;58:1756

Evidence for coupling of apo(a) and Lp(a)-apoB biosynthesis • Oleate stimulates apo(a) secretion from HepG2 cells • Nassir F, et al. J Biol Chem 1998;273:17793 • Lp(a) levels are reduced by an MTP inhibitor (lomitapide) and by apoB antisense oligonucleotide (mipomersen) • Samaha FF, et al. Nat Clin Pract Cardiovasc Med 2008;5:497 • Santos RD, et al. ATVB 2015;35:689 • PCSK9 inhibitor (evolocumab) monotherapy reduces apo(a) PR • Watts GF et al. Eur Heart J 2018;39:2577

PCSK9 enhances apoB secretion HepG2 - 17K J. Clark

J. Clark PCSK9 increases apo(a) secretion HepG2 - 17K

J. Clark PCSK9 effect dependent on apo(a):apoB interaction HepG2 - 17K ∆ LBS 7,8 HepG2 - 17K

J. Clark Lomitapide decreases apoB secretion HepG2 - 17K

J. Clark Lomitapide decreases apo(a) secretion HepG2 - 17K

J. Clark Lomitapide effect dependent on apo(a):apoB interaction HepG2 - 17K ∆ LBS 7,8

Effect of sortilin overexpression and knockdown on apo(a) secretion M. Gemin HepG2 - 17K

M. Gemin Sortilin effect dependent on apo(a):apoB interaction HepG2 - 17K ∆ LBS 10 HepG2 - 17K ∆ LBS 7,8

J. Clark Effect of SORT1 variants on apo(a) secretion apo(a) Dr. R. Hegele

J. Clark Effect of sortilin overexpression on apoB secretion HepG2 - 17K HepG2

Co-IP of apo(a) and apoB from lysates – REDUCING CONDITIONS Dr. A. Youssef Glycine ε -ACA ⍺ 1-4- anti-apo(a) ⍺ 1-4- anti-apo(a) Poly anti-ApoB Poly anti-ApoB HepG2 - 17K IP: Inp Inp -ve -ve [aa] Apo(a) 100 mM Lysates IB: A5 Anti-apo(a) [aa] Apo(a) 200 mM [aa] Media Apo(a) 200 mM

Dr. A. Youssef Co-IP of apo(a) and apoB from lysates – NON-REDUCING CONDITIONS HepG2 - 17K

Dr. A. Youssef Colocalization of apo(a) and apoB intracellularly Triple Colocalization DAPI Apo(a) ApoB Calnexin Merged Merged DAPI Apo(a) ApoB TGN46 Merged DAPI Apo(a) ApoB LAMP1 Apo(a) ApoB EEA1 Merged DAPI

Mechanism? modified from Fisher E, et al. J Biomed Res 2014;28:178

OxPL on apo(a) as a unifying hypothesis for the pathogenic effects of Lp(a) Boffa MB, Koschinsky ML Nat Rev Cardiol 2019, in press

OxPL on apo(a) and the NLRP3 inflammasome Dr. M. Lorey dissertation: Secretome analysis of human macrophages activated by microbial stimuli, http://urn.fi/URN:ISBN:9 78-951-51-3522-3

Inflammasome induction in THP-1 macrophages Dr. K. Öörni 8 0 0 M. Borrelli Dr. A. Youssef 6 0 0 R N A e x p r e s s i o n ( f o l d ) a l i z e d t o G A P D H Dr. M. Lorey 4 0 0 I L - 1 β I L - 8 I L - 1 8 2 0 0 n o r m m 5 . 0 2 . 5 0 . 0

Structure-function relationships in KIV 10 Thr64 (Met) Arg10 His31 (Gln) His3 His33 Trp72 (mutated in non- human primates) pdb: 3kiv ε -ACA Asp56 (mutated in non- human primates)

M. Borrelli Mutation of His33 prevents apo(a) secretion 17K 17K H33A 17K (mature) 17K (immature) 250 kDa medium lysate 6K (mature) 250 kDa 6K (immature)

M. Borrelli Thr64: enhanced oxPL modification? KIV 10 -KV di-kringle constructs M64T D56A M64T D56A DTT - + - + DTT - + - + protein stain IB: E06 IB: poly- α -oxPL clonal α -apo(a) re-probe D56A Wt M64T ε -ACA Elution progression

Modeling of M64T substitution in KIV 10 R71 H31 H33 T64 model (pdb: 3kiv) M64 R. Szabla Dr. M. Junop M64 model T64 ε -ACA • Energy minimization: Rosetta Relax • Structure visualization: PyMOL

High-level challenges Establish plausibility/mechanisms of phenomena arising from in • vivo kinetic studies of Lp(a) production and clearance (e.g. recycling of apo(a) and/or apoB; role of individual receptors) Tease apart lysine-binding and oxPL-binding functions of KIV 10 • Establish tractable animal model for Lp(a) pathogenicity • Corroborate pathogenic mechanisms in vivo •