Thomson Reuters Biomarker Solutions: Hepatitis C Treatment Biomarkers and special considerations in patients with Asthma

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1 : Hepatitis C Treatment Biomarkers and special considerations in patients with Asthma Abstract This case study aims to demonstrate the process of biomarker identification and validation utilizing Thomson Reuters scientific content and solutions. Through application of cross-linked manually curated biomarker information in association with biological interaction and pathway content and tools, novel potential biomarkers can be identified quickly and validated during the same process to increase productivity and enable more informed decision making. Introduction to solutions During this short case study the following Thomson Reuters solutions & content will be utilized: MetaCore with Asthma speciality Module MetaCore With Asthma Specialty Module is an integrated knowledge database and software suite for pathway analysis of experimental data and gene lists. The scope of data types includes microarray and sequence-based gene expression, SNPs and CGH arrays, proteomics, metabolomics, Co-IP pull-out, and other custom interactions. MetaCore is based on a proprietary manually curated database of human protein-protein, protein-dna, and protein compound interactions, metabolic and signaling pathways for human, mouse, and rat, supported by proprietary ontologies and controlled vocabulary. The analytical package includes easy to use, intuitive tools for search, data visualization, mapping and exchange, biological networks, and interactome. The Asthma Specialty Module is the result of the collaboration with major pharmaceutical companies and Thomson Reuters and is designed to be used by a broad range of researchers including wet lab biologists, bioinformaticians, toxicologists, biomarker researchers, and clinicians. The module integrates pathology pathways maps for disease mechanisms, highly detailed gene-disease associations (putative biomarkers), and small molecule and biologics therapies, along with an extensive OMICS data repository. Thomson Reuters Integrity with the Biomarkers Module Integrity is a unique drug discovery database integrating biological, chemical, and pharmacological data to empower your research and support better informed decision-making. It is built, populated, and updated by scientists to bring you the latest information, expert analysis and current opinion on potential and precedented targets, and translational research, including biomarkers. The Biomarkers Module of Thomson Reuters Integrity SM provides a wealth of reliable, high-quality, continually updated information supporting biomarker research at every stage of drug R&D. It is the first biomarker database to provide standardized terminology and to classify biomarkers reliably into lifecycle phases and disciplines to help you find the right biomarker with the right utility at the right time. Introduction Hepatitis C is a viral infection of the liver that affects million people globally with over 350,000 people dying of the disease every year too according the World Health Organization (WHO). Current standard of care treatment involves a daily combination of ribavirin and pegylated interferon-alpha delivered subcutaneously on a weekly schedule [Manns, MP et al, (2001) Lancet]. Current studies aim to deliver stronger viral clearance through the stimulation of the immune system but often fail due to serious adverse effects (SAEs) such as flu-like symptoms, hypotension, and lymphopenia as a result of associated pro-inflammatory response induction. In 2011 researchers at Pfizer attempted to stimulate the immune system through Toll-like receptor 7 (TLR7) agonism with a compound (PF ) designed to stimulate only the anti-viral aspect of the immune system and not the proinflammatory response [Fidock et al, (2011) Clinical Pharmacology and therapeutics]. Unfortunately, in this case the compound also induced SAEs and was withdrawn as a result. This case study aims to further understand the mechanisms by which the compound could have induced the inflammatory biomarkers and what role these biomarkers play in the anti-viral response. Furthermore, through comparison with ribavirin treatment and pathway analysis, we aim to identify potential novel biomarkers that strongly identify an anti-viral response induction. Said biomarker measurement could then be balanced with current markers for inflammatory responses allowing screening of compounds to identify further compounds that

2 trigger innate viral clearance with minimal induction of the pro-inflammatory responses. An additional aim of this study is to show how the application of Thomson Reuters biomarker and pathway content can allow such analysis to be performed and validated in silico based on high quality 100 percent manually curated information resulting in less time and resources wasted exploring unpromising avenues. Scenario: You have heard from a colleague about the Pfizer study and are looking to move into a similar field. Keen to learn from failures of the past you are looking for guidance on where your first investigatory experiments should be. Currently WHO estimates that 235 million people suffer from asthma. Given the potential for co-occurrence with HCV, and the potentially dangerous effects of inducing inflammatory responses in asthmatic patients, you are also interested in understanding any special considerations required for the use of any biomarkers identified for HCV treatment in patients who also have Asthma. This study will sequentially answer the following questions in an attempt to highlight normal research thought processes and results but is not intended as a step by step training manual: 1. What were the biomarkers measured in the study? 2. What are the potential mechanisms (both canonical and novel) by which these biomarkers could be induced by this compound? 3. Using data from the standard of care treatment can we identify an improved biomarker to highlight successful induction of antiviral actions of a compound? 4. Are there any special considerations for the use of these biomarkers in patients with Asthma and Hepatitis C? 1. What were the biomarkers measured in the study? The first step in better understanding the biomarkers used in the study is to identify them. As shown in Figure 1A is a quick search in Integrity for the compound name identifies a drug record and 4 references related to it as well as 14 biomarker records. By examining the biomarkers themselves we can see that four of them have been previously shown not to be useful as biomarkers (Figure 1 B). By selecting only those biomarkers previously shown to be useful and discounting those not shown to be of use we can ensure that we consider only those potentially useful biomarkers in our research. We can then start to gain a more in-depth understanding of the pathway context of each biomarker by exploiting the direct links to MetaCore, either individually or (by exporting the appropriate list of identifiers) as a list. By studying the larger list rather than each biomarker individually we re able to gain biological insights into what the biomarkers actually represent. A B Figure 1: Exploring biomarkers in Integrity. A) A quick search for PF shows 14 biomarkers that can be further explored by clicking on the link. B) Both positive and negative references for the biomarkers uses can easily be seen and selected appropriately.

3 2. What are the potential mechanisms (both canonical and novel) by which these biomarkers could be induced by this compound? Little information is known about PF however from the original study it was shown that it is a TLR7 agonist. How therefore can TLR7 agonism induce these biomarkers? Utilizing the shortest paths algorithm of MetaCore on the list of biomarkers, we can simply ask the question how to go from TLR7 to these biomarkers limiting the number of additional steps the system can use to get there (in this case we use 3). Figure 2 shows that all except one (IFNAR1) of the biomarkers can be induced by TLR7 agonism through the actions of MyD88 and IRF1, IRF5 and IRF7. This hypothesis relies on the correct function of MyD88 and therefore suggests a key part of the system that may affect efficacy. Indeed, linking through to the known biomarker uses of MyD88 in Integrity shows that it is already a known biomarker at the DNA level for spontaneous clearance of the disease, further highlighting its central role. The network shown in Figure 2 therefore represents the shortest route by which TLR7 agonism can induce the various inflammatory biomarkers. An understanding of the more classical (otherwise known as canonical) routes by which this signaling could occur can also easily be achieved by exploring the pathway map content. The prebuilt TLR signaling pathways map summarizes the main routes of signaling understood to result in activation of the immune system under normal conditions (Figure 3). We can see from this map that several of our biomarkers sit at the end point of this pathway causing inflammation (Figure 3A, thermometers numbered 2). We can also see that MyD88 is a key stage in TLR7 signaling classically as well as through our potential shortest routes. Further study of the pathway shows that the main anti-viral action is performed through interferon beta expression and from here we can link out to see more specific details of that anti-viral process. However, it is clear from examining this pathway that the same transcription factor (NF-KB) that triggers the anti-viral functions of TLR7 also leads to the production of the inflammatory cytokines that may be associated with the SAEs. This highlights why separating the two mechanisms is presenting the industry so many issues. Exploring the pathway of Antiviral actions of interferons linked to the TLR signaling pathway (Figure 4) gives us a better understanding of steps involved that an anti hepatitis C compound would need to stimulate. By studying where the biomarkers utilized in the study occur in the pathway we can observe that the initial trigger point of the pathway is measured for increased activity through IFN-alpha and its receptor. Furthermore one of the endpoints of the viral clearance pathway was also measured through OAS1 and OAS2. If long term immune clearance of the virus is to occur however the larger immune system will need to be involved through antigen presentation and the activation of MxA will also be required to block further replication of the virus. By studying the pathway closely we can observe that these actions could occur independent of the actions of OAS1 and 2 and therefore measurement of OAS1 and 2 may not indicate a full activation of the viral clearance mechanisms. A biomarker that captured the activation of the other side of the pathway may be informative in the study of future compounds. Figure 2: A MetaCore network showing shortest paths between TLR7 and the biomarkers exported from Integrity. Further understanding the biomarkers uses of individual entities by clicking on them and linking through via hyperlinks on the resulting entity page.

4 3. Using data from the standard of care treatment can we identify an improved biomarker to highlight successful induction of antiviral actions of a compound? Biomarkers are only useful if they are known to change in response to the condition they are being utilized to represent and can be readily measured. As PF data was unavailable we utilized data from application of the standard of care treatment, ribavirin, in a cell line model from the public data repository GEO (GSE 23031). Viral clearance with ribavirin is observed, however a sustained response is only observed in percent of the most common variant of HCV (genotype 1) [Fidock et al, (2011) Clinical Pharmacology and therapeutics]. Therefore by utilizing data from ribavirin treatment as a baseline studies, we can look to increase the anti-viral response and restrict the pro-inflammatory response to levels observed with standard of care. Interestingly, the authors of this ribavirin study, utilizing RNAi experiments, highlighted that ribavirin was acting independent of both TLR and NF- KappaB through a potentially novel innate mechanism. A better understanding of this potential mechanism could therefore aid in the design of longer term efficacy treatments. By overlaying the significant gene expression changes observed in ribavirin treated cells compared to PBS control we can see parts of the pathway that have significantly changed on the mrna level and may therefore be good biomarker targets (see thermometers numbered 1 on Figures 3 and 4). Further evaluation of the antiviral pathway with the ribavirin data overlaid (Figure 4) reveals some potential targets. The Antigen presenting components themselves (MHC-classes I and II as well as HLA-A) are significantly increased in expression as is the viral replication blocking molecule MxA. However, measuring 4 further biomarkers as well as the existing inflammatory markers adds significant cost and effort to studies as well as further complexity to data. The identification of an upstream molecule that can represent both portions of this pathway would be preferable. IRF1 (Figure 4 A) represents one such molecule. Induction of expression of IRF1 would indicate that further upstream factors such as IFN receptors and STAT1 and 2 had successfully been activated. Furthermore, the successful activation of IRF1 would lead to activation of the antigen presentation cascade through CIITA and direct HLA-A activation as well as blockage of viral replication through activation of MxA. Measuring IRF1 may therefore allow the indication of successful activation of several aspects of anti-viral pathways that would lead to longer term viral clearance by the immune system. A Figure 3: TLR signaling pathway map with known biomarkers from PF treatment (thermometers numbered 2) and fold changes gene expression data from cells treated with ribavirin (thermometers numbered 1). A) Overlap of known biomarkers with significant gene expression changes leading to potential inflammatory responses.

5 Examination of current knowledge around IRF1 as a biomarker from Integrity shows that as well as being part of several cancer biomarker panels it is indeed already in early stage investigations for Hepatitis C (Figure 5). Polymorphisms in the IRF1 gene have been shown to be useful indicators of IFN beta treatment efficacy in the past further highlighting its potential in indicating viral clearance. However, converse to our hypothesis, utilization of mrna measurement of IRF1 has been shown to be uninformative as a biomarker for ribavirin treatment in chronic Hepatitis C. This indicates that mrna measurement may not be the ideal biomarker to measure in this case. IRF1 still presents a tempting central molecule for measurement and protein abundance or transcription factor activity levels may be a potential alterative to mrna studies. From the manually curated multi-source information provided in Integrity it is possible to see that currently there have been no studies into the use of IRF1 protein abundance or activity as a biomarker for Hepatitis C indicating a potential open market for such investigations. Furthermore, Integrity shows no patents or early stage studies investigating this marker for hepatitis currently and that IRF1 protein levels have only been studied for Glioblastoma and breast cancer to date (April 2012). The experimental conditions and materials used in these protein studies could therefore present a handy head-start in the work-up of IRF1 protein abundance/activity as a hepatitis biomarker. A Figure 4: Antiviral actions of interferons pathway with biomarkers (2) and gene expression data (1) overlaid. A) IRF1 is a key hub that may be able to be measured to represent activation of several functions of the pathway at once. 4. Are there any special considerations for the use of these biomarkers in patients with Asthma and Hepatitis C? As well as detailed information on biomarkers, biological interactions and pathway biology Thomson Reuters solutions also provide a detailed knowledgebase of disease specific information. Overlaying information directly on a map in MetaCore allows visualization of cross-disease questions (Figure 6). Overlaying known molecular perturbations associated with Asthma onto this pathway allow us to see that IRF1 decreases in protein abundance and gene expression are known to be associated with Asthma. This therefore indicates that in Hepatitis C patients who also have Asthma this biomarker may be more difficult to measure due to a lower starting level of the protein. Such information provided quickly in the correct context allows the assessment of potential biomarkers in complex cross-disease scenarios accurately and efficiently. Figure 5: Exploring current biomarker uses of IRF1 shows previous hepatitis C uses.

6 Conclusions By combining manually curated biomarker, biological interaction, and pathway information in this study we have: Explored the mechanisms around TLR7 agonism by PF and the inflammatory biomarkers it induced. From the stronger understanding of what these biomarkers represent mechanistically and by overlaying gene expression data for standard of care treatment data from publically available sources we have identified a potential novel biomarker (IRF1) for further study. By examining the current knowledge around IRF1 as a biomarker we have also established that its use as an mrna marker may not be informative however little has been explored in hepatitis around IRF1 protein abundance and activity. We have found further considerations that must be taken into account for patients who also have asthma. By understanding what has been done in the past in combination with high quality biological information we have been able to identify and validate novel biomarkers for study accurately and efficiently, avoiding wasting time on avenues that have already been investigated unsuccessfully and drive our research forward. The combination of the high quality manually curated content and analysis tools from Thomson Reuters therefore allows you to make better decisions faster. Figure 6: Overlaying known expression changes associated with Asthma shows that IRF1 expression is reduced in asthmatic patients suggesting this marker may be harder to measure in such patients. THOMSON REUTERS Regional Offices North America Philadelphia Latin America Europe, Middle East and Africa Barcelona London Contact us to find out more about MetaCore or visit thomsonreuters.com/diseaseinsight Asia Pacific Singapore Tokyo For a complete office list visit: ip-science.thomsonreuters.com/ contact LS SCI - SBCS Copyright 2012 Thomson Reuters