Integration of the Transcriptome and Genome-Wide Landscape of BRD2 and BRD4 Binding Motifs Identifies Key Superenhancer Genes and Reveals the Mechanism of Bet Inhibitor Action in Rheumatoid Arthritis Synovial Fibroblasts.

Fibroblast-like synoviocytes (FLS), one of the main cell types of the rheumatoid arthritis (RA) synovium, possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain family that includes BRD2, BRD3, BRD4, and BRDt, has shown efficacy in models of arthritis. We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1ß-induced RA-FLS activation and proliferation. To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling and determined BRD2 and BRD4 cistromes by identifying their global chromatin binding sites. In addition, assay for transposable accessible chromatin by high throughput sequencing was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Through an integrated analysis of expression profiling, Brd2/Brd4 cistrome data, and changes in chromatin accessibility, we found that JQ1 inhibited key BRD2/BRD4 superenhancer genes, downregulated multiple crucial inflammatory pathways, and altered the genome-wide occupancy of critical transcription factors involved in inflammatory signaling. Our results suggest a pleiotropic effect of JQ1 on pathways that have shown to be individually efficacious in RA (in vitro, in vivo, and/or in humans) and provide a strong rationale for targeting BRD2/BRD4 for disease treatment and interception.

Integrative Analysis of DiseaseLand Omics Database for Disease Signatures and Treatments: A Bipolar Case Study.

Transcriptomics technologies such as next-generation sequencing and microarray platforms provide exciting opportunities for improving diagnosis and treatment of complex diseases. Transcriptomics studies often share similar hypotheses, but are carried out on different platforms, in different conditions, and with different analysis approaches. These factors, in addition to small sample sizes, can result in a lack of reproducibility. A clear understanding and unified picture of many complex diseases are still elusive, highlighting an urgent need to effectively integrate multiple transcriptomic studies for disease signatures. We have integrated more than 3,000 high-quality transcriptomic datasets in oncology, immunology, neuroscience, cardiovascular and metabolic disease, and from both public and internal sources (DiseaseLand database). We established a systematic data integration and meta-analysis approach, which can be applied in multiple disease areas to create a unified picture of the disease signature and prioritize drug targets, pathways, and compounds. In this bipolar case study, we provided an illustrative example using our approach to combine a total of 30 genome-wide gene expression studies using postmortem human brain samples. First, the studies were integrated by extracting raw FASTQ or CEL files, then undergoing the same procedures for preprocessing, normalization, and statistical inference. Second, both p-value and effect size based meta-analysis algorithms were used to identify a total of 204 differentially expressed (DE) genes (FDR < 0.05) genes in the prefrontal cortex. Among these were BDNF, VGF, WFS1, DUSP6, CRHBP, MAOA, and RELN, which have previously been implicated in bipolar disorder. Finally, pathway enrichment analysis revealed a role for GPCR, MAPK, immune, and Reelin pathways. Compound profiling analysis revealed MAPK and other inhibitors may modulate the DE genes. The ability to robustly combine and synthesize the information from multiple studies enables a more powerful understanding of this complex disease.

Comprehensive epigenetic landscape of rheumatoid arthritis fibroblast-like synoviocytes.

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.

Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability.

Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine-specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia-inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen-independent degradation of HIF-1α. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF-1α downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF-1α stability, indicating a rate-limiting role for FAD in LSD1-mediated HIF-1α regulation. Transcriptomic analyses of patient tissues show that the HIF-1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple-negative breast cancers, reflecting the significance of FAD-dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF-mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF-1α stability.

Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma.

UNLABELLED: Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. CONCLUSION: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease.

Pyruvate kinase M2 promotes the growth of gastric cancer cells via regulation of Bcl-xL expression at transcriptional level.

PKM2 is an isoenzyme of the glycolytic enzyme pyruvate kinase that promotes aerobic glycolysis. Here, we describe an important role for PKM2 in regulating the survival of gastric cancer (GC) cells. We showed that PKM2 was overexpressed in gastric tumor tissues compared to normal tissues and its expression level was associated with poor survival of gastric cancer patients. We also showed that PKM2 affected cell survival by regulating Bcl-xL at the transcriptional level. PKM2 knockdown partially affected the stability of NF-kB subunit p65, suggesting that post-translational regulation of p65 by PKM2 is one of plausible mechanisms for the increased cell growth. Therefore, PKM2 may function as an upstream molecule that regulates p65 function and thus enhances the growth of tumor cells.

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

Elevated fibroblast growth factor-inducible 14 expression promotes gastric cancer growth via nuclear factor-κB and is associated with poor patient outcome.

The fibroblast growth factor-inducible 14 (Fn14) gene encodes a type I transmembrane protein that belongs to the tumor necrosis factor receptor superfamily and regulates multiple cellular processes in diverse physiological and pathological conditions, including cancer. Here, we describe an important role for Fn14 in regulating the growth of gastric cancer cells. Previous gene expression data analysis demonstrated that Fn14 was up-regulated in various tumor tissues, including gastric cancer. Using qRT-PCR, we showed that Fn14 was overexpressed in gastric tumor tissues compared to normal tissues. Furthermore, Fn14 expression levels were inversely correlated with gastric cancer patient survival. Using ectopic overexpression and shRNA-mediated knockdown of Fn14, we demonstrated that the expression level of Fn14 affected cell growth in gastric cancer. The effect of Fn14 on cell growth was mediated by the NF-κB activity and eventually by the transcriptional regulation of the anti-apoptotic Bcl-2 family gene (Bcl-xL). These results suggest that Fn14 may play an important role in gastric tumor growth by regulating NF-κB-mediated anti-apoptosis and that Fn14 may be a useful prognostic marker for gastric cancer.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer.

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

An activin receptor-like kinase 5 inhibitor reduces collagen deposition in a rat dermal incision wound healing model.

BACKGROUND: Excessive dermal scarring is characterized by an overabundant deposition of extracellular matrix caused by fibrosis. The purpose of this study was to modify a rodent model of cutaneous healing for use in the development of compounds to minimize scarring, and to test the model with a small molecule inhibitor of transforming growth factor-ß type I receptor, activin receptor-like kinase 5, because this class of inhibitors has been demonstrated to be effective in minimizing fibrosis in other organs. METHODS: The rodent model of cutaneous healing consists of uniform full-thickness incisional dermal wounds in rats. Wounds were allowed to heal by secondary intention, generally over a 14-day period. The usefulness of the model was tested by the application of an activin receptor-like kinase 5 inhibitor, CP-639180. Activin receptor-like kinase 5 inhibition antagonizes the transforming growth factor-ß pathway, and was used to determine whether there was an effect on collagen deposition in wounds. The compound was applied once per day for 7 days starting at postwounding day 0 or 7 (early or late treatment regimens). Wounds were analyzed histologically for collagen deposition and biochemically for quantification of collagen changes. RESULTS: Early and late treatment regimens with the activin receptor-like kinase 5 inhibitor significantly reduced collagen deposition without impairing wound healing. CONCLUSIONS: Application of a small molecular inhibitor of activin receptor-like kinase 5 appears to significantly reduce collagen deposition in rat dermal wounds as reported here for the first time. Activin receptor-like kinase 5 inhibition may offer a novel approach to reducing proliferative scars in humans because collagen accumulation is a core event in scarring.

Pantolactams as androgen receptor antagonists for the topical suppression of sebum production.

A series of pantolactam based compounds were identified as potent antagonists for the androgen receptor (AR). Those that possessed properties suitable for topical delivery were evaluated in the validated Hamster Ear Model. Several compounds were found to be efficacious in reducing wax esters, a major component of sebum, initiating further preclinical work on these compounds.

Liverome: a curated database of liver cancer-related gene signatures with self-contained context information.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. A number of molecular profiling studies have investigated the changes in gene and protein expression that are associated with various clinicopathological characteristics of HCC and generated a wealth of scattered information, usually in the form of gene signature tables. A database of the published HCC gene signatures would be useful to liver cancer researchers seeking to retrieve existing differential expression information on a candidate gene and to make comparisons between signatures for prioritization of common genes. A challenge in constructing such database is that a direct import of the signatures as appeared in articles would lead to a loss or ambiguity of their context information that is essential for a correct biological interpretation of a gene's expression change. This challenge arises because designation of compared sample groups is most often abbreviated, ad hoc, or even missing from published signature tables. Without manual curation, the context information becomes lost, leading to uninformative database contents. Although several databases of gene signatures are available, none of them contains informative form of signatures nor shows comprehensive coverage on liver cancer. Thus we constructed Liverome, a curated database of liver cancer-related gene signatures with self-contained context information. DESCRIPTION: Liverome's data coverage is more than three times larger than any other signature database, consisting of 143 signatures taken from 98 HCC studies, mostly microarray and proteome, and involving 6,927 genes. The signatures were post-processed into an informative and uniform representation and annotated with an itemized summary so that all context information is unambiguously self-contained within the database. The signatures were further informatively named and meaningfully organized according to ten functional categories for guided browsing. Its web interface enables a straightforward retrieval of known differential expression information on a query gene and a comparison of signatures to prioritize common genes. The utility of Liverome-collected data is shown by case studies in which useful biological insights on HCC are produced. CONCLUSION: Liverome database provides a comprehensive collection of well-curated HCC gene signatures and straightforward interfaces for gene search and signature comparison as well. Liverome is available at http://liverome.kobic.re.kr.

Rational design of a topical androgen receptor antagonist for the suppression of sebum production with properties suitable for follicular delivery.

A novel nonsteroidal androgen receptor antagonist, (R)-4-(1-benzyl-4,4-dimethyl-2-oxopyrrolidin-3-yloxy)-2-(trifluoromethyl)benzonitrile (1), for the topical control of sebum production is reported. This compound, which is potent, selective, and efficacious in the clinically validated golden Syrian hamster ear animal model, was designed to be delivered to the pilosebaceous unit, the site of action, preferentially by the follicular route.

Diphenyl ethers as androgen receptor antagonists for the topical suppression of sebum production.

A series of diphenyl ethers was prepared and evaluated for androgen receptor antagonist activity in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro activities were evaluated for topical in vivo efficacy in the Golden Syrian Hamster ear model. Several compounds showed reduction in wax esters in this validated animal model.

4-(Alkylthio)- and 4-(arylthio)-benzonitrile derivatives as androgen receptor antagonists for the topical suppression of sebum production.

The first examples of thioether-substituted benzonitriles as potential soft-drug androgen receptor antagonists are reported. A number of 4-(alkylthio)- and of 4-(arylthio)-benzonitrile analogs were evaluated in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro binding and cellular activities were evaluated for topical in vivo efficacy in the Golden Syrian hamster ear model. Analogs from both the 4-(alkylthio)- and of 4-(arylthio)-benzonitrile series showed moderate reduction of wax esters in vivo.

Synthesis and biological evaluation of amino-pyridines as androgen receptor antagonists for stimulating hair growth and reducing sebum production.

A series of amino-pyridines were synthesized and evaluated for androgen antagonist activities. Among these compounds, (R)-(+)-6-[methyl-(1-phenyl-ethyl)-amino]-4-trifluoromethyl-nicotinonitrile was the most active example of this class. This compound displayed potent androgen receptor antagonist activity as well as favorable pharmacokinetic characteristics for a potential topical agent. It also demonstrated remarkable potency for stimulating hair growth in a male C3H mouse model as well as reducing sebum production in the male Syrian hamster ear model.

Preparation of 4-aryl-2-trifluoromethylbenzonitrile derivatives as androgen receptor antagonists for topical suppression of sebum production.

A series of substituted 4-aryl-2-trifluoromethylbenzonitrile analogs were evaluated in the human androgen receptor binding and cellular functional assays. Analogs with sufficient in vitro binding and cellular potency (IC(50)<200 nM) were tested in the progesterone receptor binding assay for selectivity and in the Golden Syrian hamster ear model for in vivo efficacy. Within the series, compound 4 e was identified to be the most active analog in vivo (wax ester inhibition=86%).

(1R,2S)-4-(2-cyano-cyclohexyl-oxy)-2-trifluoromethyl-benzonitrile, a potent androgen receptor antagonist for stimulating hair growth and reducing sebum production.

Synthesis, pharmacology, and pharmacokinetic profiles of (1R, 2S)-4-(2-cyano-cyclohexyl-oxy)-2-trifluoromethyl-benzonitrile are reported. This compound demonstrated remarkable potency for stimulating hair growth in a male C3H mouse model as well as reducing sebum production in the male Syrian hamster ear model.