Engineering an oncolytic HSV-1 for the treatment of Melanoma

Over 50,000 people die from cutaneous melanoma each year, despite it being a readily detectable skin cancer. Whilst survival has significantly improved in recent times due to the development of new treatments, severe toxicity and resistance to these therapies are common, meaning safer and more effective treatments are still needed. Most melanomas appear to carry at least one mutation that causes activation of ERK, a signaling protein that causes high levels of cell proliferation. Small molecules that inhibit MEK1/2 and mutant BRAF, signaling proteins directly upstream of ERK, are a currently approved combination therapy for melanoma. However, resistance to these inhibitors regularly occurs, with ERK frequently reactivated. DUSP6 is a protein found in human cells whose function is to specifically deactivate ERK, thus overexpression of DUSP6 stands as an interesting avenue for inhibiting ERK signaling in melanoma. To evaluate this idea, we generated several melanoma cell lines with inducible expression of DUSP6, enabling assessment of the effect DUSP6 overexpression, alone or in combination with either a BRAF or ERK inhibitor, had on BRAF mutant melanoma cells. In treatment naïve cell lines, DUSP6 overexpression and inhibitor combination led to greater suppression of ERK signaling, reduced cell proliferation, a higher proportion of apoptotic cells and/or a reduced propensity to develop resistance, than either treatment alone. Interestingly, combination treatment of DUSP6 overexpression and ERK inhibitor remained somewhat effective in cell lines that had previously developed resistance to mutant BRAF and/or MEK1/2 inhibitors. The oncolytic herpes simplex virus (HSV) T-Vec is another recently approved melanoma treatment that whilst having proven its safety, has also had limited success. The gene deletions that render T-Vec cancer specific potentially also reduce its efficacy, thus an alternate mechanism of rendering an oncolytic virus melanoma specific could increase its effectiveness. To achieve this, we attempted to generate an oncolytic HSV by replacing the promoter of UL54 with regulatory elements that would limit expression of this essential HSV gene, and thus virus replication, to melanoma cells. Usage of a tyrosinase enhancer/promoter construct was expected to limit transcription of UL54 to melanocytic cells (which includes many melanoma cells), whilst a long, structured 5’ untranslated region should restrict translation of UL54 to cancer cells (like melanoma). Evaluation of the regulatory elements indicated expression of a target gene would only occur in cells where tyrosinase was present. However, due to difficulties encountered with replacing the UL54 promoter with these elements, no virus could be produced and tested. However, the final results obtained indicated that the last strategy attempted to generate this oncolytic HSV had succeeded in producing the desired HSV recombinant genome, although further analysis is necessary to be certain

Trade-Off between Toxicity and Signal Detection Orchestrated by Frequency- and Density-Dependent Genes

Behaviors in insects are partly highly efficient Bayesian processes that fulfill exploratory tasks ending with the colonization of new ecological niches. The foraging (for) gene in Drosophila encodes a cGMP-dependent protein kinase (PKG). It has been extensively described as a frequency-dependent gene and its transcripts are differentially expressed between individuals, reflecting the population density context. Some for transcripts, when expressed in a population at high density for many generations, concomitantly trigger strong dispersive behavior associated with foraging activity. Moreover, genotype-by-environment interaction (GEI) analysis has highlighted a dormant role of for in energetic metabolism in a food deprivation context. In our current report, we show that alleles of for encoding different cGMP-dependent kinase isoforms influence the oxidation of aldehyde groups of aromatic molecules emitted by plants via Aldh-III and a phosphorylatable adaptor. The enhanced efficiency of oxidation of aldehyde odorants into carboxyl groups by the action of for lessens their action and toxicity, which should facilitate exploration and guidance in a complex odor environment. Our present data provide evidence that optimal foraging performance requires the fast metabolism of volatile compounds emitted by plants to avoid neurosensory saturation and that the frequency-dependent genes that trigger dispersion influence these processes

GWAS Meta-Analysis of Suicide Attempt: Identification of 12 Genome-Wide Significant Loci and Implication of Genetic Risks for Specific Health Factors

Objective: Suicidal behavior is heritable and is a major cause of death worldwide. Two large-scale genome-wide association studies (GWASs) recently discovered and crossvalidated genome-wide significant (GWS) loci for suicide attempt (SA). The present study leveraged the genetic cohorts from both studies to conduct the largest GWAS metaanalysis of SA to date. Multi-ancestry and admixture-specific meta-analyses were conducted within groups of significant African, East Asian, and European ancestry admixtures. Methods: This study comprised 22 cohorts, including 43,871 SA cases and 915,025 ancestry-matched controls. Analytical methods across multi-ancestry and individual ancestry admixtures included inverse variance-weighted fixed-effects meta-analyses, followed by gene, gene-set, tissue-set, and drug-target enrichment, as well as summary-data-based Mendelian randomization with brain expression quantitative trait loci data, phenome-wide genetic correlation, and genetic causal proportion analyses. Results: Multi-ancestry and European ancestry admixture GWAS meta-analyses identified 12 risk loci at p values &lt;5×10-8. These loci were mostly intergenic and implicated DRD2, SLC6A9, FURIN, NLGN1, SOX5, PDE4B, and CACNG2. The multi-ancestry SNP-based heritability estimate of SA was 5.7% on the liability scale (SE=0.003, p=5.7×10-80). Significant brain tissue gene expression and drug set enrichment were observed. There was shared genetic variation of SA with attention deficit hyperactivity disorder, smoking, and risk tolerance after conditioning SA on both major depressive disorder and posttraumatic stress disorder. Genetic causal proportion analyses implicated shared genetic risk for specific health factors. Conclusions: This multi-ancestry analysis of suicide attempt identified several loci contributing to risk and establishes significant shared genetic covariation with clinical phenotypes. These findings provide insight into genetic factors associated with suicide attempt across ancestry admixture populations, in veteran and civilian populations, and in attempt versus death.</p

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc

<div><p>The worst subtype of neuroblastoma is caused by <i>MYCN</i> oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed <i>linc00467</i> gene expression through direct binding to the <i>linc00467</i> gene promoter and reducing <i>linc00467</i> promoter activity. While N-Myc suppressed the expression of <i>RD3</i>, the protein-coding gene immediately down-stream of <i>linc00467</i> gene, through direct binding to the <i>RD3</i> gene promoter and reducing <i>RD3</i> promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of <i>linc00467</i> gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.</p></div

linc00467 enhances neuroblastoma cell survival.

<p>(<b>A</b>) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 was expressed as a percentage change in the number of viable cells after transfection with linc00467 siRNA-1, compared with control siRNA-transfected samples. (<b>B</b>) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of time and siRNAs were expressed as percentages of the number of viable cells after transfection with control siRNA for 0 hour. (<b>C</b>) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with propodium iodide, and subjected to flow cytometry analyses of the cell cycle. The percentage of cells at sub-G1 phase was calculated. (<b>D</b>) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p<0.05, and ** p<0.01.</p

Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray.

<p>Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray.</p