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Background: Inherited retinal diseases (IRD) are genetically heterogeneous disorders that cause the dysfunction or loss of photoreceptor cells and ultimately lead to blindness. To date, next-generation sequencing procedures fail to detect pathogenic sequence variants in coding regions of known IRD disease genes in about 30-40% of patients. One of the possible explanations for this missing heritability is the presence of yet unidentified transcripts of known IRD genes. Here, we aimed to define the transcript composition of IRD genes in the human retina by a meta-analysis of publicly available RNA-seq datasets using an ad-hoc designed pipeline., Results: We analysed 218 IRD genes and identified 5,054 transcripts, 3,367 of which were not previously reported. We assessed their putative expression levels and focused our attention on 435 transcripts predicted to account for at least 5% of the expression of the corresponding gene. We looked at the possible impact of the newly identified transcripts at the protein level and experimentally validated a subset of them., Conclusions: This study provides an unprecedented, detailed overview of the complexity of the human retinal transcriptome that can be instrumental in contributing to the resolution of some cases of missing heritability in IRD patients., (© 2023. The Author(s).)

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease-modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin-mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug-disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived cells and alleviate phenotype changes in mmut-deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA.

Mutated KRAS protein is a pivotal tumor driver in pancreatic cancer. However, despite comprehensive efforts, effective therapeutics that can target oncogenic KRAS are still under investigation or awaiting clinical approval. Using a specific KRAS-dependent gene signature, we implemented a computer-assisted inspection of a drug-gene network to in silico repurpose drugs that work like inhibitors of oncogenic KRAS. We identified and validated decitabine, an FDA-approved drug, as a potent inhibitor of growth in pancreatic cancer cells and patient-derived xenograft models that showed KRAS dependency. Mechanistically, decitabine efficacy was linked to KRAS-driven dependency on nucleotide metabolism and its ability to specifically impair pyrimidine biosynthesis in KRAS-dependent tumors cells. These findings also showed that gene signatures related to KRAS dependency might be prospectively used to inform on decitabine sensitivity in a selected subset of patients with KRAS-mutated pancreatic cancer. Overall, the repurposing of decitabine emerged as an intriguing option for treating pancreatic tumors that are addicted to mutant KRAS, thus offering opportunities for improving the arsenal of therapeutics for this extremely deadly disease. SIGNIFICANCE: Decitabine is a promising drug for cancer cells dependent on RAS signaling., (©2019 American Association for Cancer Research.)

Summary: Pathway-based expression profiles allow for high-level interpretation of transcriptomic data and systematic comparison of dysregulated cellular programs. We have previously demonstrated the efficacy of pathway-based approaches with two different applications: the Drug Set Enrichment Analysis and the Gene2drug analysis. Here we present a software tool that allows to easily convert gene-based profiles to pathway-based profiles and analyze them within the popular R framework. We also provide pre-computed profiles derived from the original Connectivity Map and its next generation release, i.e. the LINCS database., Availability and Implementation: the tool is implemented as the R/Bioconductor package gep2pep and can be freely downloaded from https://bioconductor.org/packages/gep2pep., Supplementary Information: Supplementary data are available at http://dsea.tigem.it/lincs., (© The Author(s) 2019. Published by Oxford University Press.)

The endocannabinoid system refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. However, the potential role of endocannabinoids in skeletal muscle disorders remains unknown. Here we report the role of the endocannabinoid CB1 receptors in Duchenne's muscular dystrophy. In murine and human models, CB1 transcripts show the highest degree of expression at disease onset, and then decline overtime. Similar changes are observed for PAX7, a key regulator of muscle stem cells. Bioinformatics and biochemical analysis reveal that PAX7 binds and upregulates the CB1 gene in dystrophic more than in healthy muscles. Rimonabant, an antagonist of CB1, promotes human satellite cell differentiation in vitro, increases the number of regenerated myofibers, and prevents locomotor impairment in dystrophic mice. In conclusion, our study uncovers a PAX7-CB1 cross talk potentially exacerbating DMD and highlights the role of CB1 receptors as target for potential therapies.

Motivation: Drug repositioning has been proposed as an effective shortcut to drug discovery. The availability of large collections of transcriptional responses to drugs enables computational approaches to drug repositioning directly based on measured molecular effects., Results: We introduce a novel computational methodology for rational drug repositioning, which exploits the transcriptional responses following treatment with small molecule. Specifically, given a therapeutic target gene, a prioritization of potential effective drugs is obtained by assessing their impact on the transcription of genes in the pathway(s) including the target. We performed in silico validation and comparison with a state-of-art technique based on similar principles. We next performed experimental validation in two different real-case drug repositioning scenarios: (i) upregulation of the glutamate-pyruvate transaminase (GPT), which has been shown to induce reduction of oxalate levels in a mouse model of primary hyperoxaluria, and (ii) activation of the transcription factor TFEB, a master regulator of lysosomal biogenesis and autophagy, whose modulation may be beneficial in neurodegenerative disorders., Availability and Implementation: A web tool for Gene2drug is freely available at http://gene2drug.tigem.it. An R package is under development and can be obtained from https://github.com/franapoli/gep2pep., Contact: dibernardo@tigem.it., Supplementary Information: Supplementary data are available at Bioinformatics online.

We performed an integrated analysis of drug chemical structures and drug-induced transcriptional responses. We demonstrated that a network representing three-dimensional structural similarities among 5452 compounds can be used to automatically group together drugs with similar scaffolds, physicochemical parameters and mode-of-action. We compared the structural network to a network representing transcriptional similarities among a subset of 1309 drugs for which transcriptional response were available in the Connectivity Map data set. Analysis of structurally similar, but transcriptionally different drugs sharing the same MOA enabled us to detect and remove weak and noisy transcriptional responses, greatly enhancing the reliability of transcription-based approaches to drug discovery and drug repositioning. Cardiac glycosides exhibited the strongest transcriptional responses with a significant induction of pathways related to epigenetic regulation, which suggests an epigenetic mechanism of action for these drugs. Drug classes with the weakest transcriptional responses tended to induce expression of cytochrome P450 enzymes, hinting at drug-induced drug resistance. Analysis of transcriptionally similar, but structurally different drugs with unrelated MOA, led us to the identification of a 'toxic' transcriptional signature indicative of lysosomal stress (lysosomotropism) and lipid accumulation (phospholipidosis) partially masking the target-specific transcriptional effects of these drugs. We found that this transcriptional signature is shared by 258 compounds and it is associated to the activation of the transcription factor TFEB, a master regulator of lysosomal biogenesis and autophagy. Finally, we built a predictive Random Forest model of these 258 compounds based on 128 physicochemical parameters, which should help in the early identification of potentially toxic drug candidates.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.

The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a "reverse" oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of "reverse" oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.

The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it)., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Mistrafficking of CFTR protein caused by F508del, the most frequent mutation in cystic fibrosis (CF), can be corrected by cell incubation at low temperature, an effect that may be mediated by altered expression of proteostasis genes., Methods: To identify small molecules mimicking low temperature, we compared gene expression profiles of cells kept at 27°C with those previously generated from more than 1300 compounds. The resulting candidates were tested with a functional assay on a bronchial epithelial cell line., Results: We found that anti-inflammatory glucocorticoids, such as mometasone, budesonide, and fluticasone, increased mutant CFTR function. However, this activity was not confirmed in primary bronchial epithelial cells. Actually, glucocorticoids enhanced Na(+) absorption, an effect that could further impair mucociliary clearance in CF airways., Conclusions: Our results suggest that rescue of F508del-CFTR by low temperature cannot be easily mimicked by small molecules and that compounds with closer transcriptional and functional effects need to be found., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Motivation: Automated screening approaches are able to rapidly identify a set of small molecules inducing a desired phenotype from large small-molecule libraries. However, the resulting set of candidate molecules is usually very diverse pharmacologically, thus little insight on the shared mechanism of action (MoA) underlying their efficacy can be gained., Results: We introduce a computational method (Drug-Set Enrichment Analysis-DSEA) based on drug-induced gene expression profiles, which is able to identify the molecular pathways that are targeted by most of the drugs in the set. By diluting drug-specific effects unrelated to the phenotype of interest, DSEA is able to highlight phenotype-specific pathways, thus helping to formulate hypotheses on the MoA shared by the drugs in the set. We validated the method by analysing five different drug-sets related to well-known pharmacological classes. We then applied DSEA to identify the MoA shared by drugs known to be partially effective in rescuing mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene function in Cystic Fibrosis., Availability and Implementation: The method is implemented as an online web tool publicly available at http://dsea.tigem.it., Contact: dibernardo@tigem.it, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press.)

Cystic fibrosis (CF) is caused by mutations in CF transmembrane conductance regulator (CFTR). The most frequent mutation (F508del-CFTR) results in altered proteostasis, that is, in the misfolding and intracellular degradation of the protein. The F508del-CFTR proteostasis machinery and its homeostatic regulation are well studied, while the question whether 'classical' signalling pathways and phosphorylation cascades might control proteostasis remains barely explored. Here, we have unravelled signalling cascades acting selectively on the F508del-CFTR folding-trafficking defects by analysing the mechanisms of action of F508del-CFTR proteostasis regulator drugs through an approach based on transcriptional profiling followed by deconvolution of their gene signatures. Targeting multiple components of these signalling pathways resulted in potent and specific correction of F508del-CFTR proteostasis and in synergy with pharmacochaperones. These results provide new insights into the physiology of cellular proteostasis and a rational basis for developing effective pharmacological correctors of the F508del-CFTR defect.

Summary: Elucidation of molecular targets of a compound [mode of action (MoA)] and its off-targets is a crucial step in drug development. We developed an online collaborative resource (MANTRA 2.0) that supports this process by exploiting similarities between drug-induced transcriptional profiles. Drugs are organized in a network of nodes (drugs) and edges (similarities) highlighting 'communities' of drugs sharing a similar MoA. A user can upload gene expression profiles before and after drug treatment in one or multiple cell types. An automated processing pipeline transforms the gene expression profiles into a unique drug 'node' embedded in the drug-network. Visual inspection of the neighbouring drugs and communities helps in revealing its MoA and to suggest new applications of known drugs (drug repurposing). MANTRA 2.0 allows storing and sharing user-generated network nodes, thus making MANTRA 2.0 a collaborative ever-growing resource., Availability and Implementation: The web tool is freely available for academic use at http://mantra.tigem.it., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Mendelian disorders are mostly caused by single mutations in the DNA sequence of a gene, leading to a phenotype with pathologic consequences. Whole Exome Sequencing of patients can be a cost-effective alternative to standard genetic screenings to find causative mutations of genetic diseases, especially when the number of cases is limited. Analyzing exome sequencing data requires specific expertise, high computational resources and a reference variant database to identify pathogenic variants., Results: We developed a database of variations collected from patients with Mendelian disorders, which is automatically populated thanks to an associated exome-sequencing pipeline. The pipeline is able to automatically identify, annotate and store insertions, deletions and mutations in the database. The resource is freely available online http://exome.tigem.it. The exome sequencing pipeline automates the analysis workflow (quality control and read trimming, mapping on reference genome, post-alignment processing, variation calling and annotation) using state-of-the-art software tools. The exome-sequencing pipeline has been designed to run on a computing cluster in order to analyse several samples simultaneously. The detected variants are annotated by the pipeline not only with the standard variant annotations (e.g. allele frequency in the general population, the predicted effect on gene product activity, etc.) but, more importantly, with allele frequencies across samples progressively collected in the database itself, stratified by Mendelian disorder., Conclusions: We aim at providing a resource for the genetic disease community to automatically analyse whole exome-sequencing samples with a standard and uniform analysis pipeline, thus collecting variant allele frequencies by disorder. This resource may become a valuable tool to help dissecting the genotype underlying the disease phenotype through an improved selection of putative patient-specific causative or phenotype-associated variations.