The Adverse Outcome Pathway For Mucus Hypersecretion in Chronic Bronchitis Introduction Karsta Luettich1, Marja Talikka1, FrazerIntroduction J. Lowe2, Linsey E. Haswell2, Justyna Szostak1, Florian Martin1, Marianna Gaça2, Julia Hoeng1 Introduction Poster Board P225 Abstract 2823 1 Philip Morris Products S.A. (part of Philip Morris International group of companies), Neuchatel, Switzerland 2 British American Tobacco (Investments) Ltd, Southampton, UK

Introduction Adverse Outcome Pathway - Description and Evaluation Biological Network Analysis as Potential Tool for Quantitative AOP Evaluation

complex(SCOMP:"NADPH act(p(HGNC:"Proteases, composite(p(HGNC:TNF),p(HGNC:EGF)) Oxidase act(p(HGNC:ELANE)) p(HGNC:CAMP) serine")) Chronic obstructive pulmonary disease (COPD) is one of the adverse outcomes resulting from cigarette smoking composite(p(HGNC:TNF),p(HGNC:TGFA)) Complex") composite(p(HGNC:SPDEF),p(HGNC:FOXA3))p(HGNC:CLCA1) and manifests as obstruction of the small airways, chronic bronchitis, or emphysema. There is a need to establish p(HGNC:FOXA3) act(p(HGNC:P2RY2)) p(HGNC:ADAM17)p(HGNC:RPS6KA1,pmod(P)) a(CHEBI:"hyaluronic tloc(p(SFAM:"PRKC acid") a mechanistic understanding of COPD development in response to chronic exposure to inhaled toxicants for risk KE2 IL4/13 KE3 KE8 Family"),MESHCS:Cytoplasm,MESHCS:"Cell Decreased Membrane") p(SFAM:"PRKC assessment and regulatory decision-making. The Adverse Outcome Pathway (AOP) framework provides a means Transdifferentiation act(p(HGNC:FOXA3)) act(p(HGNC:ADAM17)) Apoptosis of Goblet cell Family") to outline a knowledge-driven sequence of events from exposure to adverse outcome (AO). Here, we describe CASP3↓ of Ciliated Epithelial Ciliated Epithelial metaplasia act(p(SFAM:"PRKC sec(p(HGNC:TGFA))sec(p(HGNC:AREG)) an AOP for EGFR-mediated mucus hypersecretion that leads to chronic bronchitis and COPD. The AOP is based Cells Cells p(HGNC:AGR2) Family")) on existing experimental findings with the activation of EGFR by its ligands in response to oxidative stress as the p(HGNC:TGFA) molecular initiating event (MIE). In response to EGFR activation, the key events (KEs) include decreased PI3K ↑ AKT ↑ HIF1A ↑ tloc(p(HGNC:NCF2),MESHCS:Cytosol,MESHCS:"Cell HB-EGF↑ Membrane") tloc(p(HGNC:NCF1),MESHCS:Cytosol,MESHCS:"Cell apoptosis of ciliated epithelial cells, mediated by PI3K/AKT signaling, transdifferentiation of ciliated cells into Membrane") act(p(HGNC:DUOX1)) Figure 2. Cytoscape Visualization of goblet cells by IL-4 and IL-13, and increased goblet cell proliferation. These KEs lead to goblet cell hyperplasia or MIE TGFA ↑ FOXA2 ↓ AO Goblet Cell Hyperplasia Network Model. metaplasia. Additionally, EGFR stimulation leads to the activation of SP-1 that upregulates MUC5AC, increasing AREG ↑ act(p(HGNC:EGFR)) p(HGNC:EGFR,pmod(P)) BEL statements were extracted from over mucin production by goblet cells. Together these processes ultimately result in mucus hypersecretion that, when KE1 KE5 KE6 KE9 p(HGNC:BCL2) complex(GOCC:"phosphatidylinositol act(p(HGNC:RAC1)) act(p(SFAM:"RAS NRG↑ 3-kinase act(p(HGNC:SRC)) 60 scientific publications on goblet cell p(HGNC:BAX) Family")) chronic, results in declining lung function and allows for COPD diagnosis. We describe the biological plausibility ROS Increase in Decreased complex") act(p(HGNC:NOTCH1)) ADAM↑ EGFR Transcriptional Mucus act(p(HGNC:FOXA2)) hyperplasia using BELIEF and imported Introduction RAS ↑ RAF↑ MEK1/2 ↑ ERK1/2 ↑ Mucin lung act(p(SFAM:"AKT act(p(SFAM:"RAF of the AOP and the weight of each evidence supporting the KEs and the key even relationships (KERs). Finally, we hypersecretion act(p(HGNC:BCL2)) act(p(HGNC:BAX)) act(p(HGNC:CYBB)) MMP ↑ activation activation of SP-1 Family")) Family")) a(SCHEM:NADPH) into Cytoscape for visualization of the H2O2 Production function p(HGNC:MUC5B) have evaluated the mechanisms described in the AOP with transcriptomics data from respiratory tissue of causal relationships, and to verify content act(p(SFAM:"MEK1/2 a(CHEBI:"hydrogen KE4 KE7 act(p(HGNC:CASP3)) human smokers and rodents exposed to cigarette smoke. These results can be further compared to Family")) peroxide") and directionality. Increased experimental in vitro data in the context of the AOP for translational purposes to reduce animal experimentation Goblet cell a(CHEBI:"reactivep(HGNC:SRC,pmod(P)) Proliferation of oxygen species") in toxicological assessment. hyperplasia translocation (tloc), secretion (sec) Epithelial Cells chemical p(HGNC:MMP9) complex(p(HGNC:CD44),p(HGNC:EGFR)) p(HGNC:AREG) biological process (bp) Decreased protein abundance (p) act(p(SFAM:"MAPK act(p(HGNC:MMP9)) p(HGNC:EGFR) p(HGNC:CD44) protein activity (act) Bcl-2↑ Bax↓ Apoptosis of Erk1/2 Family")) complex Goblet Cells p(HGNC:IL8) complex(p(HGNC:BCL2L11),p(HGNC:BCL2L1)) composite Methods p(HGNC:HIF1A) p(HGNC:PARP1)act(p(HGNC:SP1)) act(p(HGNC:CD44)) Figure 1. Schematic presentation of the decreased lung function AOP. MIE; molecular initiating event, KE; key event, AO; adverse outcome, ROS; reactive oxygen species, H O ; hydrogen peroxide, ADAM; a disintegrin and increases 2 2 bp(GOBP:"neutrophil act(p(SFAM:"KLK act(p(HGNC:HIF1A)) act(p(HGNC:PARP1)) directly increases activation") p(HGNC:BCL2L11) Family")) metalloproteinase, MMP; matrix metalloproteinase, HB-EGF; heparin-binding EGF-like growth factor, TGFA; transforming growth factor alpha, AREG; amphiregulin, NRG; neuregulin, EGFR; epidermal growth factor receptor, PI3K; p(HGNC:MUC2) p(HGNC:BCL2L1) decreases directly decreases bp(GOBP:"programmed phosphoinositide 3-kinase, AKT; protein kinase B, CASP3; caspase 3, RAS; p21/Ras GTPase, RAF; RAF proto-oncogene serine/threonine-protein kinase, MEK1/2; mitogen-activated protein kinase kinase 1/2, ERK1/2; extracellular p(HGNC:MUC5AC) p(HGNC:TNF) The AOP for decreased lung function that arises from oxidative stress-mediated EGFR activation in the airway cell death") p(HGNC:EGF) non-causal relationships, e.g. between protein epithelium was developed based on the guidance provided by the Organisation for Economic Cooperation and signal–regulated kinases 1/2, HIF1A; hypoxia-inducible factor 1 alpha, Bcl-2; B-cell lymphoma 2, Bax; bcl-2-like protein 4, IL4; interleukin 4, IL13; interleukin 13, HIF1A; hypoxia-inducible factor 1-alpha, FOXA2; forkhead box A2 p(HGNC:BCL2L11,pmod(P)) abundance and protein activity or complex and bp(GOBP:"regulation of lung goblet cell act(p(HGNC:PTPN1)) its component Development (OECD) using currently available mechanistic evidence from in vitro, in vivo and clinical studies. differentiation") Table 1. Summary overview of biological plausibility and empirical support of KERs, essentiality of KEs and applicability of AOP. The evidence has been summarized and evaluated using a modified weight-of-evidence approach and will be bp(GOBP:"mucus secretion") available for review and comment on the AOPwiki (www.aopwiki.org). Biological Plausibility of KERs Empirical support for KERs Essentiality of KEs

A computable network model that describes the biological signaling pathways regulating the increase in the Is there a mechanistic relationship between KEs consistent with established biological knowledge? Does the empirical evidence support that a change in KEup leads to an appropriate change in Are KEsdown and/or the AO prevented if a KEup is blocked? number of airway goblet cells by increased proliferation or transdifferentiation, clinically known as goblet cell KEdown? Temporality? Inconsistencies? A *o*k* B C hyperplasia (GCH) was constructed using a semi-automated knowledge extraction workflow (BELIEF). BELIEF MIE KE1: Oxidative stress directly leading to EGFR High: Considered critical for mucus hypersecretion and 1 Strong Strong KE1 allows for the transformation of unstructured information available in the literature and published datasets activation GCH/GCM into a structured, cause-effect, scientific representation in Biological Expression Language (BEL; Szostaket al.). KE1 KE2: EGFR activation indirectly leading to decreased Moderate: While the evidence supports essentiality of EGFR Moderate Moderate KE2 *o * k* To date, the network model contains causal relationships from over 60 scientific publications and focuses on epithelial cell apoptosis activation for decreased ciliated cell apoptosis, there is also 1 EGFR-mediated goblet cell proliferation and mucin production. KE2 KE3: Decreased epithelial cell apoptosis directly evidence supporting decreased apoptosis in airway goblet 0.75 Moderate Weak There is only correlative evidence for this KER. KE3 Publicly available gene expression datasets were used to validate the network model using the NPA algorithm leading to transdifferentiation into goblet cells cells in vitro.

(Martinet al.). Dataset 1 (GSE22430), summarizes the effects of pyocyanin (PYO), a redox-active toxin, on gene KE1 KE4: EGFR activation directly leading to increased 0.75 *o k* Moderate Moderate KE4 High: Blocking EGFR signaling suppresses GCH/GCM. *o*k* 1 expression in H292 lung epithelial cells. Dataset 2 (GSE5264) reflects gene expression changes in airway basal epithelial cell proliferation 0.5 epithelial cells undergoing mucociliary differentiation at early, intermediate and late time points, respectively. Moderate: The evidence suggests that other transcription NPA Score 0.5 Dataset 3 (GSE37693) was derived from bronchial epithelial cells treated with IL-13. KE1 KE5: EGFR activation directly leading to Sp-1 activation Moderate Weak There is little direct evidence for this KER. KE5 0.75 factors could contribute to increased MUC5AC expression. *o * k* NPA Score Additional datasets were then used to test the model, including gene expression data from nasal  *o*k*

KE1 KE6: EGFR activation indirectly leads to increased 0.5 epithelia of Apoe-deficient mice exposed to cigarette smoke (dataset 4; Phillips et al.), large Moderate Strong KE6 High 0.25

mucin production 0.25 airway epithelial cells of smokers and never-smokers (dataset 7; GSE16008) and COPD patients *o k* NPA Score *o * k* k* Inferred: The term ‘hyperplasia’ refers to an increase in a tissue 0.25 and controls (dataset 5; DOI:10.1038/sdata.2014.9), and from small airway epithelial cells from *k* k* k* KE4 KE7: Increased epithelial cell proliferation directly or organ that is linked to an increase in cell number or cell size. k* 0 0 smokers and never-smokers (dataset 8; GSE19667) and COPD patients and controls (dataset 6; Moderate Weak KE7 Moderate 0 leading to GCH Therefore, increased proliferation can be considered a root GSE10006/GSE11906/GSE11952/GSE13933/GSE19407/GSE19667/GSE20257/GSE5058/GSE8545). cause of GCH. Dataset 1 2 3 1 2 3 6 8 months Dataset 5 6 7 8 -/- References: Inferred: Following injury, airway epithelial repair is 1: PYO Treated vs Untreated (GSE22430) Dataset 4: Nasal epithelium, Apoe mouse, exposed 5: Large airways, COPD vs control (DOI: 10.1038/sdata.2014.9) OECD. Users' handbook supplement to the Guidance document for developing and assessing adverse outcome pathways. KE3 KE8: Transdifferentiation into goblet cells directly accomplished by (transient) remodeling processes. In the 2: Epithelial cell differentiation, early (left bar) , to mainstream smoke from 3R4F reference cigarettes 6: Small airways, COPD vs control (GSE10006/GSE11906/ Moderate Weak KE8 Moderate intermediate (middle bar) and late time points GSE11952/GSE13933/GSE19407/ GSE19667/GSE20257/ https://aopkb.org/common/AOP_Handbook.pdf, 2014. leading to GCM absence of cell proliferation, this remodeling is thought to be (right bar; GSE5264) GSE5058/GSE8545) Martin F, Sewer A, Talikka M, Xiang Y, Hoeng J, Peitsch MC. Quantification of biological network perturbations for mechanistic insight facilitated by transdifferentiation. 3: IL13 Treated vs Untreated (GSE37693) 7: Large airways CS vs NS (GSE16008) and diagnostics using two-layer causal models. BMC Bioinformatics. 2014 Jul 11;15:238. Moderate: It is currently unclear whether chronic mucus 8: Small airways, CS vs NS (GSE19667) KE5 KE6: Sp-1 activation directly leading to increased Szostak J, Ansari S, Madan S, Fluck J, Talikka M, Iskandar A, De Leon H, Hofmann-Apitius M, Peitsch MC, Hoeng J. Construction of Moderate Moderate KE9 hypersecretion alone is sufficient to affect a decrease in biological networks from unstructured information based on a semi-automated curation workflow. Database (Oxford). 2015 Jun mucin production Figure 3. NPA analysis confirmed that the model contains appropriate biological information, is specific for EGFR signaling and sensitive to an increase in 17;2015:bav057. lung function goblet cell numbers. A) Network Validation: Pyocyanin treatment of H292 cells results in significant and maximum network perturbation, while IL-13 treatment Inferred: Increased mucin production is a requirement in states Phillips B, Veljkovic E, Boué S, Schlage WK, Vuillaume G, Martin F, Titz B, Leroy P, Buettner A, Elamin A, Oviedo A, Cabanski M, KE6 KE9: Increased mucin production directly leading to Applicability of bronchial epithelial cells does not, confirming that the network is specific for EGFR-mediated signaling that results in GCH. The model is also sensitive toward De León H, Guedj E, Schneider T, Talikka M, Ivanov NV, Vanscheeuwijck P, Peitsch MC, Hoeng J. An 8-Month Systems Toxicology Moderate Weak of mucus hypersecretion to restore depleted mucin stores (Rose mucus hypersecretion the physiological increase in goblet cell numbers occurring during mucociliary differentiation of bronchial epithelial cells. B) Network Testing: Exposure of Inhalation/Cessation Study in Apoe-/- Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified et al.). The majority of KEs are preserved across small rodents and humans. Apoe-deficient mice to mainstream cigarette smoke results in remodeling of the nasal epithelium which is reflected by significant perturbation of the GCH model, Risk Tobacco Product, THS 2.2, Compared With Conventional Cigarettes. Toxicol Sci. 2016 Feb;149(2):411-32. However, the link between mucus hypersecretion and airflow Inferred: According to Rose et al., “Secretory cell hyperplasia is a indicating the utility of the model in systems toxicological assessment. C) Network Testing: GCH is a common feature in the airways of asymptomatic smokers and KE7 KE9 and KE8 KE9: GCH/GCM directly leading to obstruction is much less supported by studies in laboratory animals Moderate Weak prerequisite for sustained mucus hypersecretion/mucin COPD patients. Small airway epithelial cell transcriptomic profiles can be used to score the extent of GCH network perturbations in those populations. mucus hypersecretion where the human disease phenotype cannot be modelled in its Abbreviations: PYO, pyocyanin; IL13, interleukin 13; CS, current smoker; NS, never-smoker. overproduction”. Society of Toxicology 56th Annual Meeting entirety. KL, MT, JS, FM and JH are full-time employees of Philip Morris International (PMI). March 12 - 16, 2017 In general, the exposures resulting in oxidative stress to subsequently induce EGFR activation apply to adults who are more likely to be FJL, LEH and MG are full-time employees of British American Tobacco (Investments) Ltd. KE9 AO: Chronic mucus hypersecretion directly leads to The work presented in this poster was fully sponsored by PMI and BAT. Baltimore, MD, USA Moderate Moderate exposed to these stressors. decreased lung function No part of these materials may be reproduced in any form or by any means without the prior The available in vivo and clinical evidence in support of the proposed written consent of the sponsors and no responsibility or liability is accepted for any third party AOP suggest that there is no remarkable gender difference. www.pmiscience.com reliance on any data stated. www.bat-science.com