Faculty Bibliography

Increased intestinal permeability and translocation of gut bacteria trigger various polyaetiological diseases associated with chronic inflammation and underlie a variety of poorly treatable pathologies. Previous studies have established a primary role of the microbiota composition and intestinal permeability in such pathologies. Using a rat model, we examined the effects of exposure to a bacteriophage cocktail on intestinal permeability and relative abundance of taxonomic units in the gut bacterial community. There was an increase in markers of impaired gut permeability, such as the lactulose/mannitol ratio, plasma endotoxin concentrations, and serum levels of inflammation-related cytokines, following the bacteriophage challenge. We observed significant differences in the alpha diversity of faecal bacterial species and found that richness and diversity index values increased following the bacteriophage challenge. There was a reduction in the abundance of Blautia, Catenibacterium, Lactobacillus, and Faecalibacterium species and an increase in Butyrivibrio, Oscillospira and Ruminococcus after bacteriophage administration. These findings provide novel insights into the role of bacteriophages as potentially pathogenic for mammals and their possible implication in the development of diseases associated with increased intestinal permeability.

Background: There are no predictive biomarkers of ipilimumab (IPI) toxicity. Of metastatic melanoma (MM) patients (pts) receiving IPI (3mg/kg), 35% require systemic therapies to treat immune-related adverse events (irAEs) and 20% must terminate treatment (Horvat et al., JCO 2015). Here we tested the hypothesis that a pre-existing autoantibody (autoAb) profile is predictive of IPI irAEs. Methods: We measured autoAb levels in pre- and post-treatment sera from mm pts who received IPI (3mg/kg) monotherapy on a proteome microarray containing ~20,000 unique full-length human proteins (HuProt array, CDI Laboratories). Clinical data were prospectively collected with protocol-driven follow-up. IrAEs were categorized by CTCAE guidelines as none (grade 0), mild (grade 1-2), or severe (grade 3-4). AutoAb levels were standardized using median quantile normalization and considered positive hits if > 2-SD above the peak array signal and differed by >=2 fold with p < 0.05 between toxicity groups (Non-parametric Analysis/Wilcox test). Results: Seventy-eight sera from 37 mm pts were analyzed. Antibodies against CTLA-4 were significantly elevated post IPI treatment (p < 0.0001), validating the assay. The pre-treatment levels of 190 IgG autoAbs were significantly diferent in pts who experienced irAEs (n = 28) compared to those with no irAEs (n = 9). Comparison of severe irAE (n = 9) and no irAE (n = 9) groups revealed 129 IgG autoAbs that significantly differed in pre-treatment sera. Localization and pathway analysis (UniProt, KEGG, Reactome) showed 81/190 (43%) of the autoAbs targeted nuclear and mitochondrial antigens and were enriched in metabolic pathways (p = 0.015). AutoAbs associated with irAEs did not correlate with treatment response. Conclusions: AutoAbs to antigens enriched in metabolic pathways prior to treatment may predict IPI-induced toxicity in MM. The subcellular localization of targeted antigens could explain the autoimmune toxicities associated with IPI. Studies in larger cohorts and in pts receiving other checkpoint inhibitors and/or combination therapies are essential to determine the validity of the data. If validated, our results would support the discovery of the first toxicity predictor in cancer immunotherapy

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a class of factors that are important for regulating development and cancer. Computational prediction of lncRNAs from ultra-deep RNA sequencing has been successful in identifying candidate lncRNAs. However, the complexity of handling and integrating different types of genomics data poses significant challenges to experimental laboratories that lack extensive genomics expertise. RESULT: To address this issue, we have developed lncRNA-screen, a comprehensive pipeline for computationally screening putative lncRNA transcripts over large multimodal datasets. The main objective of this work is to facilitate the computational discovery of lncRNA candidates to be further examined by functional experiments. lncRNA-screen provides a fully automated easy-to-run pipeline which performs data download, RNA-seq alignment, assembly, quality assessment, transcript filtration, novel lncRNA identification, coding potential estimation, expression level quantification, histone mark enrichment profile integration, differential expression analysis, annotation with other type of segmented data (CNVs, SNPs, Hi-C, etc.) and visualization. Importantly, lncRNA-screen generates an interactive report summarizing all interesting lncRNA features including genome browser snapshots and lncRNA-mRNA interactions based on Hi-C data. CONCLUSION: lncRNA-screen provides a comprehensive solution for lncRNA discovery and an intuitive interactive report for identifying promising lncRNA candidates. lncRNA-screen is available as open-source software on GitHub.

BACKGROUND: Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. RESULTS: We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival. CONCLUSIONS: The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

BACKGROUND: Chromatin conformation capture techniques have evolved rapidly over the last few years and have provided new insights into genome organization at an unprecedented resolution. Analysis of Hi-C data is complex and computationally intensive involving multiple tasks and requiring robust quality assessment. This has led to the development of several tools and methods for processing Hi-C data. However, most of the existing tools do not cover all aspects of the analysis and only offer few quality assessment options. Additionally, availability of a multitude of tools makes scientists wonder how these tools and associated parameters can be optimally used, and how potential discrepancies can be interpreted and resolved. Most importantly, investigators need to be ensured that slight changes in parameters and/or methods do not affect the conclusions of their studies. RESULTS: To address these issues (compare, explore and reproduce), we introduce HiC-bench, a configurable computational platform for comprehensive and reproducible analysis of Hi-C sequencing data. HiC-bench performs all common Hi-C analysis tasks, such as alignment, filtering, contact matrix generation and normalization, identification of topological domains, scoring and annotation of specific interactions using both published tools and our own. We have also embedded various tasks that perform quality assessment and visualization. HiC-bench is implemented as a data flow platform with an emphasis on analysis reproducibility. Additionally, the user can readily perform parameter exploration and comparison of different tools in a combinatorial manner that takes into account all desired parameter settings in each pipeline task. This unique feature facilitates the design and execution of complex benchmark studies that may involve combinations of multiple tool/parameter choices in each step of the analysis. To demonstrate the usefulness of our platform, we performed a comprehensive benchmark of existing and new TAD callers exploring different matrix correction methods, parameter settings and sequencing depths. Users can extend our pipeline by adding more tools as they become available. CONCLUSIONS: HiC-bench consists an easy-to-use and extensible platform for comprehensive analysis of Hi-C datasets. We expect that it will facilitate current analyses and help scientists formulate and test new hypotheses in the field of three-dimensional genome organization.

Cell-specific gene expression programs rely on the orchestrated function of transcription factors, co-activators, the transcriptional machinery and non-coding RNA elements at enhancer and promoter elements. Mediator is a large co-activator complex that bridges promoters with transcription factors bound to cell-specific enhancers. Here we describe Med12, a member of the Mediator complex kinase module, as an indispensable regulator of hematopoietic stem cell (HSC) maintenance and differentiation. Conditional deletion of Med12 rapidly exhausts adult bone marrow HSCs leading to lethality. Conversely, HSC-specific deletion of other members of the Mediator complex kinase module, Med13, CyclinC and CDK8 are dispensable for HSC function. We also show that Med12 is an enhancer element that co-localizes with essential hematopoietic transcription factors and enhancer-specific histone marks. In this context, we demonstrate that Med12 loss in HSCs results in rapid dysregulation of stemness signatures, as well as failure of enhancer activation by depletion of H3K27Ac. Finally, we find that Med12 promotes stabilization of p300 at enhancers, thereby contributing to the preservation of HSC homeostasis. Collectively, our data describes a novel, kinase-independent function for Med12 in the regulation of HCS enhancer elements and the maintenance of normal stem cell function. As a result, we believe that alterations in the Med12-dependent regulation of HSC enhancers may contribute to malignant transformation and disease