The second European interdisciplinary Ewing sarcoma research summit – A joint effort to deconstructing the multiple layers of a complex disease

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second “European interdisciplinary Ewing sarcoma research summit” assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.Unión Europea 261743(ENCCA)Unión Europea 259348 (ASSET

Targeting chromosomal sites with locked nucleic acid-modified triplex-forming oligonucleotides: study of efficiency dependence on DNA nuclear environment

Triplex-forming oligonucleotides (TFOs) are synthetic DNA code-reading molecules that have been demonstrated to function to some extent in chromatin within cell nuclei. Here we have investigated the impact of DNA nuclear environment on the efficiency of TFO binding. For this study we have used locked nucleic acid-containing TFOs (TFO/LNAs) and we report the development of a rapid PCR-based method to quantify triplex formation. We have first compared triplex formation on genes located at different genomic sites and containing the same oligopyrimidine•oligopurine sequence. We have shown that efficient TFO binding is possible on both types of genes, expressed and silent. Then we have further investigated when gene transcription may influence triplex formation in chromatin. We have identified situations where for a given gene, increase of transcriptional activity leads to enhanced TFO binding: this was observed for silent or weakly expressed genes that are not or are only slightly accessible to TFO. Such a transcriptional dependence was observed for integrated and endogenous loci, and chemical and biological activations of transcription. Finally, we provide evidence that TFO binding is sequence-specific as measured on mutated target sequences and that up to 50% of chromosomal targets can be covered by the TFO/LNA in living cells

Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ∼10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ∼0.1 μM for acridine-conjugated TFO/LNA (or ∼2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631

Immunogenic Eimeria tenella Glycosylphosphatidylinositol-Anchored Surface Antigens (SAGs) Induce Inflammatory Responses in Avian Macrophages

, but the ability of these proteins to stimulate immune responses in the chicken is unknown. infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity. pathogenicity associated with the endogenous second generation stages

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015

Les "Locked Nucleic Acids" pour la stratégie anti-gène (de la fixation sur l'ADN aux applications fonctionnelles)

Nous avons caractérisé des oligonucléotides capables de former une triple-hélice (OFT) chimiquement modifiés sous forme de Locked Nucleic Acids (LNA), les OFT/LNA : 1/ Propriétés anti-gènes: Les OFT/LNA sont capables de former in vitro des complexes très stables et sélectifs de la séquence d ADN cible. Cette stabilité thermodynamique permet la formation intracellulaire d un triplexe stable. 2/ Fixation dans la chromatine Nous avons montré que la fixation des OFT/LNA est spécifique de la séquence ciblée (jusqu à 50% de fixation) et que son efficacité est corrélée à l accessibilité de la chromatine, l OFT/LNA se fixant mieux sur les régions décompactées. 3/ Induction de coupures double-brin ciblées Ces ligands de l AD (OFT/LNA) ont été couplés à un agent de coupure, la phénanthroline (OP) pour obtenir des coupures double-brin de façon spécifique dans les cellules. Le taux de modification spécifique est de 50% sur cible épisomale. La coupure endogène est en cours de caractérisation.We have studied triplex-forming oligonucleotides (TFO), chemically modified as Locked Nucleic Acids (LNA), the TFO/LNA: 1/ Anti-gene properties TFO/LNA are able to form highly stable and specific complexes on the DNA target sequence in vitro. This thermodynamic stability leads to stable triplex formation in cells. 2/ Binding in the chromatine structure We have characterized TFO/LNA binding in chromatin and evaluated the cellular factors driving binding efficiency. We have shown that about 50% of the chromosomal target can be covered by the TFO specifically, and that the efficiency of TFO binding in chromatin was correlated to chromatin accessibility: TFO did bind more efficiently to unfolded chromatin regions. 3/ Induction of targeted DNA damages We have studied TFO covalently linked to a cleaving agent, the phenanthroline (OP). In cells, DNA modifications occur specifically at 50% of the episomal target plasmid. We are presently running experiments on chromosomally located target sites.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

From circRNAs to fusion circRNAs in hematological malignancies

International audienceCircular RNAs (circRNAs) represent a type of endogenous noncoding RNA generated by back- splicing events. Unlike the majority of RNAs, circRNAs are covalently closed, without a 5′ end or a 3′ poly(A) tail. A few circRNAs can be associated with polysomes, suggesting a protein-coding potential. CircRNAs are not degraded by RNA exonucleases or ribonuclease R and are enriched in exosomes. Recent developments in experimental methods coupled with evolving bioinformatic approaches have accelerated functional investigation of circRNAs, which exhibit a stable structure, a long half-life, and tumor specificity and can be extracted from body fluids and used as potential biological markers for tumors. Moreover, circRNAs may regulate the occurrence and development of cancers and contribute to drug resistance through a variety of molecular mechanisms. Despite the identification of a growing number of circRNAs, their effects in hematological cancers remain largely unknown. Recent studies indicate that circRNAs could also originate from fusion genes (fusion circRNAs, f-circRNAs) next to chromosomal translocations, which are considered the primary cause of various cancers, notably hematological malignancies. This Review will focus on circRNAs and f-circRNAs in hematological cancers