Re-define Crohn’s at a molecular level to identify new biomarkers and therapies

Crohn’s disease (CD) affects 1 in 500 young adults in the UK. While 25% experience mild disease, many suffer an intractable course requiring biologic therapy and repeated surgery. Newer immunotherapies such as anti-TNF agents have improved management but these cost £10-15000 per patient per year and 20-30% of the most severely affected patients show no response. These individuals require recurrent hospitalization; consequently their care consumes over half of the £720 million/year direct healthcare cost of CD. New drugs are entering clinical trials in CD, but there is currently no way to predict which, if any, will modulate disease course and outcome. To address these issues there is an urgent need to identify strata within which distinct pathogenic mechanisms operate which represent targets for new and re-purposed therapies. Advances in large-scale molecular techniques and 3D human intestinal culture systems developed in Dr Simmons’s lab have enabled, for the first time the capture signals of genetically driven pathological pathways in patient subsets in CD, and signaling signatures that hallmark inflammatory lesions in this disease. The lab have developed a systems database that houses patient demographics, clinical phenotype, clinical investigations and genetic data for the local Oxford IBD cohort. The database is linked to large-scale systems datasets for each donor to enable computational modeling to define molecular signatures that are indicative of inflammation in specific patient subsets, or of inflammation resolution in response to immunotherapy. In this work Dr Simmons will re-stratify the Oxford IBD cohort according to: 1) Defects in pathogenic pathways, such as xenophagy, previously defined in the lab 2) Signaling signature of intestinal cell subsets defined by signal perturbation combined with phosphoproteomics and reverse phase protein arrays In addition Dr Simmons will develop, or reposition drugs designed to reverse defects in xenophagy that target key nodes in this pathway the lab have found to be defective in CD (such as activity of mitochondrial fission protein Drp1). Dr Simmons will investigate how their performance fares in reversing the inflammatory phenotype in intestinal cells in comparison with standard immunotherapies currently in the clinic, or against pipeline immunotherapies in clinical trial or supplied by our industry collaborators (Boehringer Ingelheim, Merck, UCB pharma, Lilly, Topivert etc). Stratification of Crohn’s by genetically driven pathways The strongest CD susceptibility gene remains NOD2, an intracellular sensor essential for defense against bacteria. NOD2 segregates with a specific subphenotype of CD, terminal ileal CD that is characterized by development small bowel inflammation and strictures unresponsive to anti-TNF therapy ultimately requiring surgery. Previously Dr Simmons showed NOD2 induces autophagy in human cells in a pathway essential for bacterial degradation and immune clearance. CD patients expressing polymorphisms in NOD2 or ATG16L1 show impaired autophagy, delayed bacterial destruction and defective antigen presentation. This results in reduced clearance of cytosolic microbes that could act as a trigger for inflammation in CD (1). The nominee subsequently investigated the mechanism of NOD2 mediated autophagy and signaling using quantitative phosphoproteomics to map the signaling path NOD2 activates on recognition of ligand. The lab found NOD2 activates the mitochondrial fission protein Drp1 in a pathway required for bacterial degradation. Drp1 is known to functionally interact with other CD susceptibility genes such as IRGM and LRRK2 and various kinases upstream of Drp1 could act as druggable targets in CD (2). Dr Simmons will recruit 100 CD, 100 UC and 100 healthy donors per year and use high throughput assays of autophagy including by Imagestream to measure autophagic flux or by FACS analysis of Drp1 phosphorylation on donor peripheral blood monocytes or intestinal cell subsets where NOD2 is expressed to establish the extent to which autophagy is defective in CD. This information will be matched back to donor genotype for the full complement of IBD susceptibility genes described to date and also to patient disease phenotype, results of laboratory investigation including markers of inflammation such as C-reactive protein, faecal calprotectin, serology (ASCA IgA/IgG, Atypical pANCA, Anti-OMPC, Anti-I2, Anti-CBir1, Anti-glycan Abs, serum anti-TNF drug and antibody levels (BioAnalba Ltd). and response to immunotherapy. The lab will collate clinical, serological, genetic and immunological data to determine if NOD2 mediated autophagy pathway can be used to predict CD subphenotypes and response to immunotherapy. This will determine the extent to which functional NOD2 mediated signalling defects segregate across CD as a whole and to what extent this genetically driven immunological pathway can be used to re-stratify this disease. Definition of druggable targets within the xenophagy pathway in Crohn’s Dr Simmons has set up stable cell lines expressing wild type (WT) and CD associated NOD2 that can be used for genomic and chemical library screens to define targets capable of reversing defects in autophagy found in presence of CD associated NOD2. Quantitative phosphoproteomics has revealed >1000 differentially phosphorylated proteins on NOD2 triggering in primary human cells. Dr Simmons is conducting a secondary focused siRNA library screen of those genes together with the 163 IBD susceptibility genes using Drp1 activation (ser616 phosphorylation) as a readout, to define those that operate in xenophagy. This will priortise a list of candidates to which chemical probes can be designed, in collaboration with Professors Knapp and Brennan, SGC, Oxford. These chemical probes will then be interrogated in model and primary cell systems for their ability to repair CD associated xenophagy defects withNOD2 autophagy pathway, or healthy control donors with no autophagy defects. These cells will be exposed to chemical probes for dose response and time course pre and post activation of NOD2. Cells wil be assayed for their ability to induce autophagy following exposure to chemical probes by Imagestream or FACS analysis of Drp1 activation. Those successful in acheiving this effect will be screened for toxicity and off target effects in peripheral blood or intestinal cell systems using broad spectrum readouts such as for cell death and inflammatory mediator release. Drug repositioning to repair xenophagy in Crohn’s NIH and Johns Hopkins drug libraries available at the TDI, Oxford will be utilized by Dr Simmons, to establish whether existing drugs are capable of reversing defects in NOD2 mediated autophagy observed in the presence of CD associated NOD2. Using Thp-1 stably expressing CD associated NOD2, compounds/drugs able to induce Drp1 phosphorylation by FACS analysis following PRR triggering will be documented. Those drugs successful in activating Drp1 and autophagy in the presence of defective NOD2 will be analysed further for their ability to reverse defects observed in patient’s cells. In cases where the lab find established therapies with acceptable side effect profile that successfully repair NOD2 autophagy defects we will design experimental medicine studies to establish whether these medications can be repurposed for use in CD.Stratification of inflammation in IBD by systems analysis of intestinal lesions An alternative approach to stratification will involve definition of signatures indicative of specific forms of inflammation in IBD, rather than driven by genetic signatures. Following more than 5 years experience in optimizing phosphoproteomic approaches (3) the lab will utilize results of quantitative phosphoproteomics +/- signal perturbation obtained in key reference cases to guide design of reverse phase protein arrays or FACS markers able to capture signatures of specific inflammatory lesions in intestinal mucosa. The lab have developed a number of strategies to produce relevant intestinal cells for such studies from biopsy material obtained at endoscopy, such as expansion of intestinal stromal cells or intestinal epithelial organoids. Stromal cells or epithelial organoids will be expanded from pooled biopsies obtained at endoscopy, stimulated with PRR ligands and lysed. Samples will be analysed using reverse-phase protein lysate microarrays to analyse targets of interest when array-compatible antibodies are available. In addition release of panels of cytokines and pro-inflammatory factors will be assayed. This will give information of signatures that hallmark specific subtypes of inflammatory lesion across a spectrum of IBD patients. We will define signatures of health and inflammation using this methodology that can be used to model response to standard or novel immunotherapies in vitro, prior to matching back to clinical outcome of each patient in terms of disease course and response to immunotherapy. Similar approaches to this have been successfully used to define optimal combinations of anti-cancer therapies to treat resistant breast cancer (4). Dr Simmons is collaborating with Professor Doug Lauffenburger, Head of Bioengineering Massachusett’s Institute of Technology, Cambridge, MA, USA in this work. Patient recruitment, donor sampling and generation of intestinal cell cultures In order to define druggable pathways in poorly accessible intestinal cell subsets the lab have generated methods to expand mucosal cell populations derived from gut biopsies obtained at endoscopy including 3D cultures of stromal, immune and epithelial cells (Figure 2) and intestinal epithelial organoids (5). This enables growth of mucosal cells without changing their phenotype to cell numbers required for proteomic and genomic experiments. This work is currently supported by a translational team embedded within the lab including a research nurse, clinical fellow, a research assistant and bioinformatics post-doc co-supervised by Professor Douglas Lauffenburger (MIT). Each donorhas DNA and serum stored and biopsy samples processed to expand intestinal crypts/stromal cells. A database houses patient demographics, clinical phenotype, clinical investigations and genetic data. The database is capable of storing large-scale systems datasets including WG sequence data, metagenomic, siRNA library, phosphoproteomic data for each donor.Healthy donor samples will be obtained at screening colonoscopy for colonic polyps. Donors will be off medication and have no significant co-morbidity. IBD donors will be new diagnosis and off immunotherapy at the time of sampling. Samples will be taken from terminal ileum, right colon and left colon and from inflamed and non-inflamed lesions. A blood sample will also be taken to generate DNA, RNA and serum. Gut biopsies will be processed using Gentlemacs digestion protocol optimized in the lab plus collagenase. For intestinal epithelial organoids a minimal digestion protocol will be followed by culture in recombinant stem cell growth factors ie noggin to generate intact intestinal crypt structures. For 3D organoids cell culture plates will be coated with collagen matrix, seeded with stromal and epithelial cells for expansion. Table 1 indicates number of biopsy samples and location for each donor LaySummary>