LIN28B Underlies the Pathogenesis of a Subclass of Ewing Sarcoma

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Mechanisms of action and resistance to novel inhibitors of the Hepatitis C virus RNA -dependent RNA polymerase

The hepatitis C virus (HCV) is a positive-sense RNA virus that encodes a non-structural protein, NS5B, which is an RNA-dependent RNA polymerase. NS5B is required for replication of the viral genome, making it an attractive target for antiviral drug development efforts. NS5B inhibitors are classified into two major categories: nucleoside inhibitors (NIs) and non-nucleoside inhibitors (NNIs). NIs bind to the enzyme active site and compete with the natural NTP for incorporation. In contrast, NNIs bind away from the active site and inhibit the polymerase activity allosterically, specifically during initiation of RNA synthesis by introducing conformational changes in the polymerase.The NS5B polymerase lacks proofreading ability, which results in a high rate of mutations during replication. This error prone nature of the HCV polymerase has given rise to a number of variants of the virus which are classified into different genotypes and subgenotypes. Different HCV genotypes show variations in drug susceptibility, particularly to NNIs, which limits their clinical utility. At least four distinct binding sites for NNIs have been identified on the NS5B protein and these binding sites are not necessarily conserved across the genotypes. This provides a possible explanation for the observed variations in drug susceptibility. As part of the first study described in this thesis, we tested the inhibitory activity of acyl pyrrolidine and 1,5-benzodiazepine against purified NS5B enzymes that represent the major HCV genotypes. We identified natural amino acid substitutions that contribute to resistance to the NNIs. In the second study described herein, we turned our attention to the mechanisms of action and resistance to NIs, with focus on sofosbuvir, which is part of the current standard of care in anti-HCV therapy. Previous crystallographic data of HCV NS5B in complex with an RNA primer-template has provided important information on the protein-nucleic acid interface during the elongation process. The structure points to specific interactions between residues of the nucleic acid binding channel and the 2'-hydroxyl group of the bound RNA substrate. Using templates with strategically engineered DNA-like residues, we examined the role of the 2'-hydroxyl group of the template strand in nucleotide incorporation. Our biochemical findings suggest that this 2'-hydroxyl group plays an essential role in establishing resistance to NIs, and this process may be mediated by the signature S282T mutation, which is been known to cause resistance to sofosbuvir. In the third study, we further evaluated the efficacy of novel NIs against known NI resistance-conferring mutations in NS5B. The compounds 2'F-2'-C-Me-UTP (sofosbuvir), 2'-C-Me-2'-NH2-UTP, 2'-C-Me-UTP and 2'-C-Me-(1-Thio)UTP, which were modified derivatives of sofosbuvir, were tested against mutations S282T, L159F, C316N and L320F. It was observed that S282T showed resistance to all the modified compounds. Mutations L159F, C316N and L320F showed sensitivity towards 2'-C-Me-UTP. WT NS5B, L159F, C316N and L320F showed increased IC50 values towards 2'-C-Me-2'-NH2-UTP and 2'-C-Me-(1-Thio) UTP suggesting that addition of NH2 and Thio groups do not increase the efficacy of the compounds.Overall, these studies provide a more detailed understanding of mechanisms of action and resistance to Nucleoside and Non-nucleoside inhibitors of the HCV polymerase. Our findings offer new avenues in current efforts to develop new viral polymerase inhibitors.Le virus de l'hépatite C (VHC) est un virus à ARN positif qui exprime une protéine virale non-structurale, l'ARN polymérase ARN-dépendante NS5B. Cette polymérase est requise pour la réplication du génome viral, ce qui en fait une cible attrayante pour les efforts de développement de médicaments antiviraux. Les inhibiteurs de NS5B sont classés en deux grandes catégories: les inhibiteurs nucléosidiques (INs) et les inhibiteurs non-nucléosidiques (INNs). Les INs se lient au site actif de l'enzyme et compétitionnent avec le NTP naturel pour incorporation. En revanche, les INNs se lient hors du site actif et inhibent l'activité de la polymérase par allostérie, spécifiquement lors de la phase d'initiation de la synthèse de l'ARN en provoquant des changements de conformation dans la polymérase. NS5B est dépourvue d'activité de relecture, ce qui résulte en un taux élevé de mutations lors de la réplication. Cette nature sujette aux erreurs de la polymérase du VHC a généré nombres de variantes du virus qui sont classées en différents génotypes et sous-génotypes. Les différents génotypes du VHC présentent des variations dans leur sensibilité aux médicaments, particulièrement avec les INNs, ce qui limite leur utilité clinique. Au moins quatre sites de liaison des INNs ont été identifiés sur la protéine NS5B et ces sites ne sont pas nécessairement conservés à travers les génotypes. Cela représente un possible mécanisme pour expliquer les variations observées dans la sensibilité aux médicaments. Dans le cadre de la première étude décrite dans cette thèse, nous avons testé l'activité inhibitrice de l'acyl pyrrolidine et du 1,5-benzodiazépine contre des enzymes NS5B purifiées représentant les principaux génotypes du VHC. Nous avons ainsi identifié des substitutions naturelles d'acides aminés qui contribuent à la résistance aux INNs.Dans notre seconde étude, nous nous sommes intéressés aux mécanismes d'action et de résistance aux INs, en particulier pour le sofosbuvir, qui fait partie de la norme actuelle de soins dans le traitement du VHC. Les précédentes données cristallographiques de la protéine NS5B du VHC en complexe avec une amorce-matrice d'ARN fournissent des informations importantes sur l'interface protéine-acide nucléique lors de la phase d'élongation. Cette structure indique des interactions spécifiques entre des résidus du canal de liaison à l'acide nucléique et le groupe 2'-hydroxyle du substrat ARN lié. En utilisant des matrices stratégiquement conçues avec des résidus semblables à l'ADN, nous avons examiné le rôle du groupe 2'-hydroxyle du brin matrice dans l'incorporation des nucléotides. Nos données biochimiques suggèrent que ce groupe 2'-hydroxyle joue un rôle essentiel dans l'établissement de la résistance aux INs et que ce processus pourrait être médié par la mutation signature S282T, connue pour causer de la résistance au sofosbuvir.Dans la troisième étude, nous avons examiné de manière plus approfondie l'efficacité de nouveaux INs contre des mutations dans la protéine NS5B connues comme conférant une résistance aux INs. Le 2'F-2'C-Me UTP (sofosbuvir), le 2'-C-Me-2'-NH2-UTP, le 2'-C-Me-UTP et le 2'-C-Me-(1-Thio)UTP, qui sont des dérivés modifiés du sofosbuvir, ont été testés contre les mutations S282T, L159F, C316N et L320F. La mutation S282T a montré de la résistance à l'ensemble des composés modifiés. Les mutations L159F, C316N et L320F ont montré de la sensibilité envers le 2'-C-Me-UTP. De plus, NS5B WT, L159F, C316N et L320F ont montré une augmentation des valeurs IC50 pour le 2'-C-Me-2'-NH2-UTP et le 2'-C-Me-(1-Thio) UTP, suggérant que l'addition du NH2 et du groupe Thio n'augmente pas l'efficacité de ces composés.En conclusion, ces études fournissent une compréhension plus détaillée des mécanismes d'action et de résistance aux INs et aux INNs de la polymérase du VHC. Nos résultats offrent de nouvelles avenues dans les efforts actuels pour développer de nouveaux inhibiteurs de la polymérase virale

A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin

International audienceThe hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2′-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2′-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively

A tumor-specific endogenous repetitive element is induced by herpesviruses

Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases

Rigid 2′,4′-Difluororibo­nucleosides: Synthesis, Conformational Analysis, and Incorporation into Nascent RNA by HCV Polymerase

We report on the synthesis and conformational properties of 2′-deoxy-2′,4′-di­fluoro­uridine (2′,4′-diF-rU) and cytidine (2′,4′-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereo­electronic effects induced by the two fluorines essentially “lock” the conformation of the sugar in the North region of the pseudo­rotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2′,4′-diF-rU 5′-triphosphate (2′,4′-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2′,4′-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC₅₀ > 50 μM). 2′,4′-diF-rU/C represents rare examples of “locked” ribonucleoside mimics that lack a bicyclic ring structure

Transcriptomic Analysis of Laser Capture Microdissected Tumors Reveals Cancer- and Stromal-Specific Molecular Subtypes of Pancreatic Ductal Adenocarcinoma

International audienceAbstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival. Experimental Design: LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples. Results: Gene expression profiling identified four subtypes of cancer cells (C1–C4) and three subtypes of cancer-adjacent stroma (S1–S3). These stroma-specific subtypes were associated with DFS (P = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; P < 0.02). Conclusions: Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target

Metastatic dissemination contributes to the lethality in pancreatic ductal adenocarcinoma (PDAC). Here, the authors perform RNA-sequencing on patient derived circulating tumor cells (CTCs) and identify three major CTC subgroups, and show the therapeutic potential of targeting LIN28B/let-7 pathway to halt cancer metastasis