11 research outputs found
Neuroblastoma targeted therapy: employment of CRISPR gene-editing to explore relevant markers and potential targets in aggressive tumours
Neuroblastoma is a tumour originating from the sympathetic nervous system, and represents the most common extracranial solid cancer in childhood. Despite the malignancy is extremely heterogeneous, about 25% of all cases is characterized by MYCN-gene amplification, aggressive tumour and poor survival. The network of genes that are deregulated in this group of patients represents a focal point for targeted-therapy discovery. Along this research line, the first objective of the present project was to investigate the prognostic significance of a single nucleotide polymorphism (SNP) located in the promoter of ODC1, a neuroblastoma prognostic marker involved in polyamine biosynthesis. The SNP genotype was first associated with survival of a large cohort of patients with aggressive neuroblastoma. Then, CRISPR-editing revealed that the SNP genotype affects ODC1 expression and proliferation of neuroblastoma cells. At last, the SNP was found to influence cell sensibility to DFMO, an ODC1 inhibitor that is currently under trial for treatment of aggressive neuroblastoma. The second objective was to investigate the role in neuroblastoma development and progression of RUNX1T1, a poorly studied transcription repressor involved in distinct development events and cancers. Survival analysis of a cohort of neuroblastoma patients revealed that RUNX1T1 is a potential oncosuppressor. In apparent contrast, RUNX1T1 knockout by CRISPR-editing demonstrated that the gene promotes aggressiveness of neuroblastoma cells. Transcriptome analysis of the mutant cells then evidenced deregulation of a significant number of genes and pathways that are prognostic markers in neuroblastoma, therefore depicting a multifunctional regulation network that could be exploited for new therapies. The third and last objective was to test a novel therapeutic approach based on MYCN-amplification targeting via CRISPR-cleavage. In vitro experiments demonstrated that the system efficiently and specifically impairs the survival of aggressive neuroblastoma cells, thus providing a proof of principle for the development of an innovative therapy
A G316A polymorphism in the ornithine decarboxylase gene promoter modulates MYCN-driven childhood neuroblastoma
Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein
Validation of a radial diffusive sampler for measuring occupational exposure to 1,3-butadiene
1,3-Butadiene (BD) is a major industrial chemical used in the manufacture of rubbers and latexes; it is also a ubiquitous environmental pollutant whose major source is traffic. Occupational exposure to (BD) can occur both during its production and during its use as a raw material. The objective of the study was the laboratory and field validation of a new diffusive sampler for BD. The nominal sampling rate of the Radiello\uae diffusive sampler filled with Carbopack X is 30.5 cm3/min, at 0.177 mg/m3, 20 \ub0C and 50% relative humidity (RH), for an 8-h exposure time. A model can be used for calculating the sampling rate as a function of temperature, time and RH. The concentration does not affect the sampling rate above 30 \u3bcg/m3. The measurement uncertainty (k = 2), calculated both by laboratory data and by field comparison according to International Standard Organization (ISO) 13752, satisfies the EN 482:2006 requirement for measurements between 0.1 and 0.5 times the threshold limit value-time weighted average (TLV-TWA) (uncertainty < 50%). For field validation study, 38 workers exposed to BD and 20 administrative employees, as the control group, underwent environmental and biological monitoring. Personal exposure to BD was measured by diffusive samplers (Radiello\uae) in comparison with active samplers. The BD exposure levels detected for the exposed subjects were low (mean 0.059, range <0.010-1.340 mg/m3) but higher than the controls levels, all below 0.010 mg/m3. The comparison between diffusive and active (pumped) air sampling showed a good correlation, with no systematic deviation from the ideal values of the intercept and slope of the optimized regression line. The concentrations of two biomarkers were also determined on urine samples, collected at the end of the work-shift: unchanged BD, by GC-MS, and the metabolite dihydroxybutylmercapturic acid (DHBMA), by HPLC-MS/MS. The urinary excretion of the biomarkers was on average higher in the exposed group (urinary BD: mean 8.8, range <1-48.1 ng/l; DHBMA: mean 0.232, range 0.016-0.572 mg/l) than in controls (urinary BD: mean 6.4, range 2.6-14.5 ng/l; DHBMA: mean 0.205, range 0.037-0.602 mg/l), but a statistically significant difference was achieved only for unchanged BD and not for DHBMA. In conclusion, the environmental monitoring measured by diffusive samplers (Radiello\uae) appears to be a reliable method for the assessment of exposure to low levels of airborne BD and a convenient alternative to the conventional active sampling. \ua9 2014 Elsevier B.V. All rights reserved
MAX to MYCN intracellular ratio drives the aggressive phenotype and clinical outcome of high risk neuroblastoma
Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy, remarkably refractory to therapeutic treatments. One of the most powerful independent prognostic indicators for this disease is the amplification of the MYCN oncogene, which occurs at high levels in approximately 25% of neuroblastomas. Interestingly, amplification and not just expression of MYCN has a strong prognostic value, although this fact appears quite surprising as MYCN is a transcription factor that requires dimerising with its partner MAX, to exert its function. This observation greatly suggests that the role of MYCN in neuroblastoma should be examined in the context of MAX expression. In this report, we show that, in contrast to what is found in normal cells, MAX expression is significantly different among primary NBs, and that its level appears to correlate with the clinical outcome of the disease. Importantly, controlled modulation of MAX expression in neuroblastoma cells with different extents of MYCN amplification, demonstrates that MAX can instruct gene transcription programs that either reinforce or weaken the oncogenic process enacted by MYCN. In general, our work illustrates that it is the MAX to MYCN ratio that can account for tumour progression and clinical outcome in neuroblastoma and proposes that such a ratio should be considered as an important criterion to the design and development of anti-MYCN therapies
Novel Highly Potent and Selective Ï1 Receptor Antagonists Related to Spipethiane
Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novelpotent Ï1 ligands. Ï1 affinity and Ï1/Ï2 selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing Ï1 receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the Ï1 antagonist profile of this series of compounds. In particular, due to its high Ï1 affinity (pKi = 10.28) and Ï1/Ï2 selectivity ratio (29510), compound 9 might be a novel valuable tool for Ï receptor characterization and a suitable template for the rational design of potential therapeutically useful Ï1 antagonists
Novel Highly Potent and Selective Ï1 Receptor Antagonists Related to Spipethiane
Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novelpotent Ï1 ligands. Ï1 affinity and Ï1/Ï2 selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing Ï1 receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the Ï1 antagonist profile of this series of compounds. In particular, due to its high Ï1 affinity (pKi = 10.28) and Ï1/Ï2 selectivity ratio (29510), compound 9 might be a novel valuable tool for Ï receptor characterization and a suitable template for the rational design of potential therapeutically useful Ï1 antagonists
The ornithine decarboxylase G316A promoter polymorphism impacts neuroblastoma clinical outcome and may identify patients particularly susceptible to polyamine inhibition
Spliceâswitch oligonucleotideâbased combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYCâdriven hepatocellular carcinoma
Abstract Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYCâtargeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing spliceâswitch oligonucleotide (SSO) technologies with our phenotypicâanalytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine targetâtarget interactions and rank synthetic lethality targets. Our SSOâQPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYCâderegulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patientâderived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSOâQPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYCâdriven HCC
Transcriptional and epigenetic analyses of the DMD locus reveal novel cis\ue2\u80\u91acting DNA elements that govern muscle dystrophin expression
The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis\ue2\u80\u91DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies
A g316a polymorphism in the ornithine decarboxylase gene promoter modulates mycnâdriven childhood neuroblastoma
Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPRâcas9 technology was used to investigate the effects of AG clones from wildtype MYCNâamplified SKâNâBE(2)âC cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neu-roblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCNâamplified and nonâamplified patients, re-spectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to alleleâspecific regulation by the MYCN oncoprotein