Inhibition of asparagine synthetase effectively retards polycystic kidney disease progression.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Global and precise identification of functional miRNA targets in mESCs by integrative analysis.

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

Argonaute proteins regulate a specific network of genes through KLF4 in mouse embryonic stem cells.

The Argonaute proteins (AGOs) are well known for their role in post-transcriptional gene silencing in the microRNA (miRNA) pathway. Here we show that in mouse embryonic stem cells, AGO1&2 serve additional functions that go beyond the miRNA pathway. Through the combined deletion of both Agos, we identified a specific set of genes that are uniquely regulated by AGOs but not by the other miRNA biogenesis factors. Deletion of Ago2&1 caused a global reduction of the repressive histone mark H3K27me3 due to downregulation at protein levels of Polycomb repressive complex 2 components. By integrating chromatin accessibility, prediction of transcription factor binding sites, and chromatin immunoprecipitation sequencing data, we identified the pluripotency factor KLF4 as a key modulator of AGO1&2-regulated genes. Our findings revealed a novel axis of gene regulation that is mediated by noncanonical functions of AGO proteins that affect chromatin states and gene expression using mechanisms outside the miRNA pathway.

Inhibition of FGF and TGF-ß Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor.

The fibroblast growth factor (FGF) and the transforming growth factor-ß (TGF-ß) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-ß inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-ß inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-ß signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells.

SERPINB7 Expression Predicts Poor Pancreatic Cancer Survival Upon Gemcitabine Treatment.

Stratification of patients with pancreatic ductal adenocarcinoma (PDAC) remains a key challenge in the field of clinical oncology. No predictive biomarkers have yet been found for any available treatment options. Previously, we identified SERPINB7 as a putative biomarker for PDAC and thus, herein, we aimed to validate our previous findings and assessed the predictive value of SERPINB7. Patients who underwent surgery and received gemcitabine (gem) or gemcitabine plus nab-paclitaxel (gem/nab) as adjuvant therapy, between 2011 and 2017, were included in this study (n = 57). Expression level of SERPINB7 was assessed in tumor tissue by immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH). Its association with disease-free survival (DFS) and overall survival (OS) was investigated. While IHC did not show any correlation between survival and the protein level of SERPINB7, RNA ISH revealed that expression of SERPINB7 was associated with a poor DFS (P = .01) and OS (P = .002) in the gem group but not in the gem/nab. Adjusted Cox-regression analysis confirmed the independent predictive value of SERPINB7 on OS (P = .006, HR: 3.47; 95% CI: 1.49-8.09) in the gem group. In conclusion, SERPINB7 was identified as the first predictive RNA biomarker for PDAC. This study suggests that patients who expressed SERPINB7 might receive another treatment than gem alone.

Comparative analysis of differential gene expression tools for RNA sequencing time course data.

RNA sequencing (RNA-seq) has become a standard procedure to investigate transcriptional changes between conditions and is routinely used in research and clinics. While standard differential expression (DE) analysis between two conditions has been extensively studied, and improved over the past decades, RNA-seq time course (TC) DE analysis algorithms are still in their early stages. In this study, we compare, for the first time, existing TC RNA-seq tools on an extensive simulation data set and validated the best performing tools on published data. Surprisingly, TC tools were outperformed by the classical pairwise comparison approach on short time series (<8 time points) in terms of overall performance and robustness to noise, mostly because of high number of false positives, with the exception of ImpulseDE2. Overlapping of candidate lists between tools improved this shortcoming, as the majority of false-positive, but not true-positive, candidates were unique for each method. On longer time series, pairwise approach was less efficient on the overall performance compared with splineTC and maSigPro, which did not identify any false-positive candidate.

Biochemical and genetic predictors of overall survival in patients with metastatic pancreatic cancer treated with capecitabine and nab-paclitaxel.

Pancreatic cancer is a dismal disease with a mortality rate almost similar to its incidence rate. To date, there are neither validated predictive nor prognostic biomarkers for this lethal disease. Thus, the aim of the present study was to retrospectively investigate the capability of biochemical parameters and molecular profiles to predict survival of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) who participated in a phase II clinical trial to test the safety and efficacy of the combination treatment of capecitabine plus nab-paclitaxel. Herein, we investigated the association of 18 biochemical parameters obtained from routine diagnosis and the clinical outcome of the 30 patients enrolled in the clinical trial. Furthermore, we analysed formalin-fixed paraffin-embedded (FFPE) tumour tissue to identify molecular biomarkers via RNA seq and the Illumina TruSeq Amplicon Cancer panel which covers 48 hotspot genes. Our analysis identified SERPINB7 as a novel transcript and a DNA mutation signature that might predict a poor outcome of disease. Moreover, we identified the bilirubin basal level as an independent predictive factor for overall survival in our study cohort.

Dynamics in Transcriptomics: Advancements in RNA-seq Time Course and Downstream Analysis.

Analysis of gene expression has contributed to a plethora of biological and medical research studies. Microarrays have been intensively used for the profiling of gene expression during diverse developmental processes, treatments and diseases. New massively parallel sequencing methods, often named as RNA-sequencing (RNA-seq) are extensively improving our understanding of gene regulation and signaling networks. Computational methods developed originally for microarrays analysis can now be optimized and applied to genome-wide studies in order to have access to a better comprehension of the whole transcriptome. This review addresses current challenges on RNA-seq analysis and specifically focuses on new bioinformatics tools developed for time series experiments. Furthermore, possible improvements in analysis, data integration as well as future applications of differential expression analysis are discussed.

Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution.

Phenotypic differences between closely related species are thought to arise primarily from changes in gene expression due to mutations in cis-regulatory sequences (enhancers). However, it has remained unclear how frequently mutations alter enhancer activity or create functional enhancers de novo. Here we use STARR-seq, a recently developed quantitative enhancer assay, to determine genome-wide enhancer activity profiles for five Drosophila species in the constant trans-regulatory environment of Drosophila melanogaster S2 cells. We find that the functions of a large fraction of D. melanogaster enhancers are conserved for their orthologous sequences owing to selection and stabilizing turnover of transcription factor motifs. Moreover, hundreds of enhancers have been gained since the D. melanogaster-Drosophila yakuba split about 11 million years ago without apparent adaptive selection and can contribute to changes in gene expression in vivo. Our finding that enhancer activity is often deeply conserved and frequently gained provides functional insights into regulatory evolution.

Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts.

Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca(2+) homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.