A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma

NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1–585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3′ acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1–585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity

T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

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Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells.

LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

Targeted SMN exon skipping: A useful control to assess in vitro and in vivo splice-switching studies

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species

RNA therapeutics in the treatment of retinal disease - delivering the potential

Purpose : Treatment options for inherited retinal diseases (IRD), the leading cause of vision loss in persons aged 15 - 45 years have been limited, however, novel gene and molecular therapeutics are now demonstrating significant potential in the treatment of IRDs. RNA therapeutics hold unique promise in these diseases; although achieving safe and efficient delivery of molecular drugs to the retina and the retinal pigmented epithelium in particular, remains a significant obstacle to clinical application. Antisense oligomers (AO) are a well-established class of RNA therapeutic whose potential is yet to be fully realised due to this delivery challenge. We report an AO conjugate that traffics through the vitreous after intravitreal administration, reaching the deepest layers of the retina and localising to the nuclei to modulate gene expression. This class of therapeutic holds substantial promise in the treatment of IRDs. Methods : We exploit peptide libraries derived from 82 microorganism genomes and 118 synthetic viral genes to identify cell penetrating peptides (CPP) to deliver AO cargos to cells in vitro and to tissues and organs in vivo. The CPPs were screened initially against mammalian cells using a cytosolic extraction method, followed by next generation sequencing and selection using a combination of algorithms known to produce a favourable toxicology and efficacy profile in the eye. Results : The CPPs were conjugated to an antisense morpholino oligomer designed to mediate exon selection in a reporter mRNA. Standout performance in the latter assay, when administered via intravitreal injection, and a clean toxicology profile identified a lead peptide for our retinal disease program. CPP conjugation to our candidate AO therapeutic and evaluation in IRD patient-derived retinal pigmented epithelium rescued target gene expression and improved cell function. Conclusions : Notable CPPs in pre-clinical and clinical development include chemical stabilisation or poly-arginine that can limit efficacy or increase toxicity. Our discovery peptides are derived from nature, lack chemical modifications, and yield optimal amino acid sequences with enhanced efficacy and toxicity performance. The lead CPP, HPG_0031, traffics the AO through the vitreous, into the retinal pigment epithelium with no evidence of retinal damage, resulting in enhanced exon skipping and 6-fold lower cytotoxicity than the competitor CPP

Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production

A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy