Genetics Epigenetics in IPF Katerina M Antoniou As

Genetics & Epigenetics in IPF Katerina M. Antoniou As. Professor in Thoracic Medicine Head of the Molecular & Cellular Pneumonology Lab ERS ILD Group Chair Medical School, University of Crete

UNIVERSITY OF CRETE SCHOOL OF MEDICINE What is a molecular “signature”? • It is a set of specific changes in a molecular profile, which are characteristic for a particular pathological condition. • Molecular expression profiling of human diseases have identified “signatures” associated with diagnosis, staging, progression, prognosis and response to treatment.

Common Genetic Variants in IPF associated with IPF risk or progression • Variants in several genes related to inflammation and immune response, including : • • • transforming growth factor beta-1 (TGFB 1) interleukin-1 receptor alpha (IL 1 RN) interleukin 8 (IL 8) toll-like receptor 3 (TLR 3) HLA DRB 1*150 Cell cycle progression related genes CDKN 1 A and TP 53 ERJ 2015

Surfactant protein C mutations • heterozygous missense SFTPC mutation (L 188 Q): mutated protein precursor that accumulates in the endoplasmic reticulum and causes endoplasmic reticulum stress in pts. • This protein accumulation activates the unfolded protein response, a cascade of events that, although designed to protect the cell, could lead to alveolar epithelial cell apoptosis in cases of longterm or severe activation. Thomas AQ, Lane K, Phillips J 3 rd, et al. AJRCCM 2002

In 2011: MUC 5 B gene • A common polymorphism in the promoter of the MUC 5 B gene has been associated with both sporadic and familial IPF. • Associated with a 20 -fold increased risk of IPF in subjects that were homozygous for the polymorphism and a 7 -fold increased risk in heterozygous subjects. • At least one copy of the promoter polymorphism was present in 34 -38% of IPF subjects compared with 9% of healthy controls. s. Seibold et al. NEJM 2011 s. Zhang Y, et al. NEJM 2011

The polymorphism was shown to lead to markedly increased MUC 5 B expression in the lung Control IPF MUC 5 B distribution in the cytoplasm of the secretory columnar cells of the bronchi and larger proximal bronchioles in a specimen of lung tissue. Dense accumulation of MUC 5 B: In areas of microscopical honeycombing and involved patchy staining of the metaplastic epithelia lining the honeycomb cysts (Panel B). Accumulation was also observed in the mucous plugs within the cysts (Panel C).

Hypothesis • It is not known whether this polymorphism is associated with ILD in the general population. • The relationship between ILA and the genotype at the rs 35705950 locus for a modification of effect according to age and smoking was evaluated. Hunninghake GM, et al. NEJM, June 2013

52% 41% 7% Participants with ILA were older, increased smoking history % more respiratory symptoms

Main results • ILA were found in 7% of the sample. • 50% of the participants who had ILA had reduced lung volumes • In participants with ILA: • the odds of having each copy of the minor rs 35705950 allele were increased by a factor of 2. 8, with a frequency of 10. 5% for the minor allele in the population. • About one quarter of the participants with ILA had CT abnormalities that were diagnostic of pulmonary fibrosis. • in these participants the odds of having each copy of the minor rs 35705950 allele were increased by a factor of 6. 3.

• The INSPIRE cohort was used to model the association of the MUC 5 B genotype • with survival, accounting for the effect MMP 7 blood concentration • and other demographic and clinical covariates. Peljto Anna, et al.

MUC 5 B Promoter SNP is Associated with Improved Survival Probability of Survival [INSPIRE; N=438; 73 deaths] P<0. 001 Hazard Ratio (95% CI) MUC 5 B GT 0. 46 (. 30 -. 70) MUC 5 B TT 0. 21 (. 09 -. 49) (age, gender, FVC, DLCO, and MMP 7) Time to Death (days) Peljto. JAMA 2013; 309: 2232

Results • The observed association of MUC 5 B with survival was independent of age, sex, FVC, DLCO, MMP-7, and treatment status. • MUC 5 B promoter polymorphism explains a portion of the variation in survival among IPF participants beyond that explained by MMP-7 levels.

The MUC 5 B risk variant is observed in ~19% of unaffected individuals, and approximately one-third of individuals with IIP do not have any identifiable genetic risk factors for this disease, suggesting that other genetic variants contribute to disease risk alone or in combination with the MUC 5 B variant.

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Results • • Confirmed association with: TERT at 5 p 15 MUC 5 B at 11 p 15 3 q 26 region near TERC • Seven newly associated loci : • FAM 13 A (4 q 22), DSP (6 p 24), OBFC 1 (10 q 24), ATP 11 A (13 q 34), DPP 9 (19 p 13) • chromosomal regions 7 q 22 and 15 q 14 -15.

Second Genome Association Study • 5 loci achieved genome-wide significance, including four SNPs on chromosome 11 p 15 and one on 17 q 21. • Among the 11 p 15 SNPs were MUC 5 B rs 35705950 and three SNPs within the Toll-interacting protein (TOLLIP) locus. • Linkage disequilibrium was reported to be low with rs 35705950, suggesting TOLLIP may represent an independent risk locus. • Similar to MUC 5 B rs 35705950, IPF cases with the TOLLIP risk allele (the major allele) had decreased mortality compared to minor allele carriers. Noth I, Zhang Y, Ma SF, et al. Lancet Respir Med 2013

Dysregulated lung mucins initiate or exacerbate lung fibrosis through: • Altered mucosal defense; • Interference with alveolar repair • Direct cell toxicity stimulating a fibroproliferative response initiated by unfolded intracellular MUC 5 B. • Agents that reduce MUC 5 B transcriptional activity in vitro should be tested for activity in vivo!

New concepts • Identification of the putative environmental factors should be a priority for research. • Chronic occult infections must be high on the list of suspects. • Genetic studies can provide to the understanding of complex diseases. NEJM 2013

Spagnolo P, et al. Lancet Respir 2014

The hallmarks of aging are present in IPF lung Lopez-Otin, Cell 2013 1. 2. 3. 4. 5. 6. 7. 8. 9. Altered intercellular communication Genomic instability Telomere attrition Epigenetic alterations Loss of proteostasis Deregulation of nutrient sensing Mitochondrial dysfunction Cellular senescence Stem cell exhaustion Thannickal, Biogerontology 2013; Selman, Pardo, AJRCCM 2014

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Spagnolo P, et al. Lancet Respir 2014

UNIVERSITY OF CRETE SCHOOL OF MEDICINE

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Eur Respir J 2015

UNIVERSITY OF CRETE SCHOOL OF MEDICINE What is Epigenetics? • Epigenetics: “on top of” Genetics • Modifications to nucleotides or histones that do not change the sequence but can alter gene expression • Epigenetic regulation has emerged as a key adaptive mechanism by which environmental or other stresses can induce lasting changes in the gene expression repertoire and thus in the phenotype of a cell or an organism

The Idiopathic pulmonary fibrosis transcriptome is influenced by both environmental and genetic factors Antoniou KM, et al. AJRCCM 2008 Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

Expert Opin Drug Discov 2014 1 est level 2 nd level 3 rd level

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Nucleosomes are the basic building block of Chromatin Ubiquitilation H 2 A H 3 U H 2Β H 4 Rodenhiser& Mann CMAJ 2006 174: 341

Epi-mi. RNAs: micro. RNAs and epigenetics • Investigations revealed that certain mi. RNAs (epimi. RNAs) themselves counteract Cp. G methylation. • Regulate the components of epigenetic machinery, creating a tightly controlled feedback mechanism. • Histone modification is another epigenetic mechanism that can affect mi. RNA expression as shown in breast cancer cells

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Micro. RNAs • Micro. RNAs are members of non-coding RNAs that range in size from 18 -24 nucleotides. • mi. RNAs regulate a large variety of biological functions by controlling gene expression. Apoptosis Inflammation Proliferation Development Angiogenesis Differentiation Stem cell maintenance Motility Metabolism • So far, about 2, 000 mi. RNAs have been discovered in humans. • Each mi. RNA expressed in a cell may target about 100 to 200 m. RNAs that it downregulates. • It appears that about 60% of human protein coding genes are regulated by mi. RNAs.

UNIVERSITY OF CRETE SCHOOL OF MEDICINE Expression of DNA Methyltransferases in IPF Sanders YY, et al. Am J Respir Crit Care Med, 186, 525– 535, 2012

Impaired DNA methylation studies in IPF CASZ 1 Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

Aberrant histone modifications studies in IPF sirtuin Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

Noncoding RNA regulation IPF studies Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

TGF-b mediated EMT and fibroblast activation-proliferation is reflected in the mi. RNAs affected in IPF mi. R-21 mi. R-154 cluster Targets tumor suppressors/promotes EMT Targets Inhibitors of the WNT pathway fibroblast proliferation Let-7 d Proinflammatory Activates WNT/ Targets SMADS TGF-b Targets HMG 2 allowing TWIST and SNAI EMT mi. R-31 Targets Integrin-a, Rhoa Inhibis fibroblast proliferation migration mi. R-29 Targets ECM associated types of Collagen Laminins and Integrins mi. R-155 mi. R-200 ZEB 1, ZEB 2 Promotes EMT mi. R-23 b cluster Targets SMADS Regulating TGFb Induced genes by Tsitoura E.

mir. RNA expression results • The expression of most mi. RNAs tested in BALF cells from IPF patients was down-regulated relative to controls - excluding mir-210 • mi. R-29 a and mi. R-185 were significantly down-regulated in IPF Tsitoura E. Wells A. U. , Karagiannis K. et al. submitted

mi. R-29 a expression inversely correlated with COL 1 A 1 expression Spearman correlation within IPF group r =-0. 418 P= 0. 005 **: p<0. 005 COL 1 A 1 expression is strongly associated with CPI Tsitoura E. multivariate analysis COL 1 A 1 P-value CPI 0. 005**

Selman, Pardo. AJRCCM 2014

Epigenetic modifiers of lung diseases: preclinical studies Comer SB, et al. PPT 2015

Eur Respir J 2015

Collaborators Laboratory of Cellular and Molecular pneumonology • Katerina Antoniou Eliza Tsitoura, Post doc Researcher George Margaritopoulos, Ph. D Ismini Lasithiotaki Kostas Karagiannis Athanasia Proklou Royal Brompton Hospital and Harefield NHS Trust Prof Athol Wells Elizabeth Renzoni Hiroe Sato Laboratory of clinical Virology Prof Giogros Sourvinos Eleni Bibaki Evi Vlachava Stella Sarantoulaki Melina Tseliou Stelios Michelakis Nectaria Goulidaki Eirini Varsamidi Chryssa Kokkinaki Eirini Charalambous