Implication des inhibiteurs de PARP dans le cancer de l’ovaire

Le cancer épithélial de l’ovaire (EOC) est le cancer gynécologique le plus létal en Amérique du Nord. Les inhibiteurs de poly (ADP-Ribose) Polymérase (PARP) ont été utilisés pour en améliorer le traitement ; deux d’entre eux dont l’Olaparib sont approuvés par la FDA. L’Olaparib démontre une bonne efficacité (30% de réponse) en tant qu’agent unique pour les patientes atteintes du cancer de l’ovaire et encore plus pour celles présentant une mutation dans les gènes BRCA1/2 (45%). Ce résultat a été expliqué comme étant un effet de létalité synthétique entre les mutations BRCA1/2 qui sont les acteurs principaux du système de réparation de l’ADN par recombinaison homologue (RH) et l’inhibition de PARP qui est l’acteur principal du système de réparation de l’ADN par excision de base (BER). Chez les patientes ne présentant pas de mutation BRCA1/2 (25%), la réponse observée s’explique également par un effet de létalité synthétique mais cette fois-ci dû soit à la baisse d’expression des gènes BRCA1/2, soit à l’inhibition d’autres protéines spécifiques de la RH (effet BRCAness). A contrario, 55% des patientes mutées BRCA1/2 ne répondent pas au traitement. La réponse globale est seulement de 30% alors que 50% des patientes EOC séreux de haut grade (HGS) présentent une déficience en RH. Pour tenter d’expliquer cette contradiction, cette thèse cherche à définir et à améliorer la réponse à l’Olaparib dans les EOC HGS. Premièrement, il a fallu établir un modèle permettant d’étudier les différentes réponses aux inhibiteurs de PARP. Aux 12 lignées cellulaires d’EOC HGS préexistantes au laboratoire, 6 nouvelles lignées présentant des caractéristiques spécifiques à la réponse aux inhibiteurs de PARP ont été caractérisées et ajoutées [TOV2978G, TOV3041G, TOV3291G, OV866 (2), OV4453 (mutation non-sens récurrente BRCA2 d'origine germinale) et OV4485 (mutation du site d’épissage BRCA1)]. À partir de ce modèle cellulaire, un nouveau schéma prédictif de la sensibilité à l’Olaparib a été déterminé. On y expose l’idée que les patientes y répondant présentent une déficience en RH (BRCAness) mais qu’elle doit être combinée à une déficience en NER ou en MMR. Malgré les efforts pour cibler ces patientes et pour produire de nouveaux inhibiteurs de PARP plus efficaces, la réponse globale reste toujours autour de 30%, les cas de résistance acquise sont de plus en plus nombreux et le traitement drastique de maintenance reste encore le seul moyen d’obtenir une réponse clinique. Tout cela nous a amené, comme troisième objectif, à nous concentrer sur le type de destin cellulaire induit par les inhibiteurs de PARP nous permettant de déterminer une combinaison thérapeutique pour améliorer la réponse. Nous démontrons que l’Olaparib conduit à un état sénescent réversible dans les lignées cellulaires de EOC HGS malgré leurs mutations TP53. Cet état peut être ciblé par des sénolytiques (principalement un inhibiteur de Bcl2/Bcl-XL) pour créer une réponse synergique lorsqu’ils sont associés à des inhibiteurs de PARP. Les expériences in vivo confirment l’efficacité de la combinaison thérapeutique. Ces travaux ont contribué à donner une meilleure identification des patientes susceptibles de répondre aux inhibiteurs de PARP et à déterminer une nouvelle thérapie combinée particulièrement efficace.Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer in North America. Poly (ADP-Ribose) Polymerase (PARP) inhibitors have been used to improve treatment; two of which including Olaparib are approved by the FDA. Olaparib demonstrates good efficacy (30% response) as a single agent for patients with ovarian cancer and even more so for those with a mutation in the BRCA1/2 (45%) genes. This result was explained as a consequence of synthetic lethality between the mutants BRCA1/2; which are the principal actors of the DNA repair system by homologous recombination (HR), and the inhibition of PARP, which is the main actor of DNA repair by base excision repair (BER). In patients with no BRCA1/2 mutation (25%), the observed response is also explained by an effect of synthetic lethality, but this time due to either a decline in BRCA1/2 gene expression or the inhibition of other proteins specific to HR (BRCAness effect). In contrast, 55% of BRCA1/2 mutated patients did not respond to treatment. The overall response is only 30%, while 50% of high-grade serous (HGS) EOC patients have an HR deficiency. To try to explain this contradiction, this thesis seeks to define and improve the response to Olaparib in the HGS EOCs. First, a model was developed to study the different responses to PARP inhibitors. To the 12 pre-existing EOC HGS cell lines in the laboratory, 6 new lines presenting with characteristics specific to PARP inhibitors response were characterized and added [TOV2978G, TOV3041G, TOV3291G, OV866 (2), OV4453 (recurrent BRCA2 nonsense mutation of germinal origin) and OV4485 (splice-site mutation BRCA1)]. From this cell model, a new predictive pattern of susceptibility to Olaparib was determined. The idea presented is that the responding patients have an HR deficiency (BRCAness) but that it must be combined with a NER or MMR deficiency. Despite efforts to target these patients and to produce new, more effective PARP inhibitors, the overall response is still around 30%; cases of acquired resistance are increasing and drastic maintenance treatment is still the only way to obtain a clinical response. All this has led us as a third objective to focus on the type of cellular fate induced by the PARP inhibitors allowing us to determine a combination therapy for an improved response. We demonstrate that Olaparib leads to a reversible senescent state in the HGS EOC cell lines despite their TP53 mutations. This condition can be targeted by senolytics (mainly a Bcl2 / Bcl-XL inhibitor) to create a synergistic response when combined with PARP inhibitors. The in vivo experiments confirm the efficacy of the therapeutic combination. This work has contributed to the better identification of patients who are able to respond to PARP inhibitors and to determine a new and particularly effective combination therapy

Carboplatin sensitivity in epithelial ovarian cancer cell lines: The impact of model systems

ABSTRACT: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America, underscoring the need for the development of new therapeutic strategies for the management of this disease. Although many drugs are pre-clinically tested every year, only a few are selected to be evaluated in clinical trials, and only a small number of these are successfully incorporated into standard care. Inaccuracies with the initial in vitro drug testing may be responsible for some of these failures. Drug testing is often performed using 2D monolayer cultures or 3D spheroid models. Here, we investigate the impact that these different in vitro models have on the carboplatin response of four EOC cell lines, and in particular how different 3D models (polydimethylsiloxane-based microfluidic chips and ultra low attachment plates) influence drug sensitivity within the same cell line. Our results show that carboplatin responses were observed in both the 3D spheroid models tested using apoptosis/cell death markers by flow cytometry. Contrary to previously reported observations, these were not associated with a significant decrease in spheroid size. For the majority of the EOC cell lines (3 out of 4) a similar carboplatin response was observed when comparing both spheroid methods. Interestingly, two cell lines classified as resistant to carboplatin in 2D cultures became sensitive in the 3D models, and one sensitive cell line in 2D culture showed resistance in 3D spheroids. Our results highlight the challenges of choosing the appropriate pre-clinical models for drug testing

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases

Genomic selection for growth traits in Pacific oyster (Crassostrea gigas): Potential of low-density marker panels for breeding value prediction

<p>Pacific oysters are a key aquaculture species globally, and genetic improvement via selective breeding is a major target. Genomic selection has the potential to expedite genetic gain for key target traits of a breeding program, but has not yet been evaluated in oyster. The recent development of SNP arrays for Pacific oyster (Crassostrea gigas) raises the opportunity to test genomic selection strategies for polygenic traits. In this study, a population of 820 oysters (comprising 23 full-sibling families) were genotyped using a medium density SNP array (23 K informative SNPs), and the genetic architecture of growth-related traits [shell height (SH), shell length (SL), and wet weight (WW)] was evaluated. Heritability was estimated to be moderate for the three traits (0.26 ± 0.06 for SH, 0.23 ± 0.06 for SL and 0.35 ± 0.05 for WW), and results of a GWAS indicated that the underlying genetic architecture was polygenic. Genomic prediction approaches were used to estimate breeding values for growth, and compared to pedigree based approaches. The accuracy of the genomic prediction models (GBLUP) outperformed the traditional pedigree approach (PBLUP) by ∼25% for SL and WW, and ∼30% for SH. Further, reduction in SNP marker density had little impact on prediction accuracy, even when density was reduced to a few hundred SNPs. These results suggest that the use of genomic selection in oyster breeding could offer benefits for the selection of breeding candidates to improve complex economic traits at relatively modest cost.</p

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

<p>Abstract</p> <p>Background</p> <p>Sessile bivalves of the genus <it>Mytilus </it>are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of <it>M. galloprovincialis</it>, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.</p> <p>Results</p> <p>We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in <it>M. galloprovincialis</it>. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with <it>Vibrio splendidus </it>at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the <it>Vibrio</it>-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.</p> <p>Conclusions</p> <p>The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on <it>Vibrio</it>-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the <it>Mytilus </it>species to an evolving microbial world.</p

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene.

BACKGROUND: Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. METHODS: Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. RESULTS: In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7-19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. CONCLUSIONS: This is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families

Design status of ASPIICS, an externally occulted coronagraph for PROBA-3

The "sonic region" of the Sun corona remains extremely difficult to observe with spatial resolution and sensitivity sufficient to understand the fine scale phenomena that govern the quiescent solar corona, as well as phenomena that lead to coronal mass ejections (CMEs), which influence space weather. Improvement on this front requires eclipse-like conditions over long observation times. The space-borne coronagraphs flown so far provided a continuous coverage of the external parts of the corona but their over-occulting system did not permit to analyse the part of the white-light corona where the main coronal mass is concentrated. The proposed PROBA-3 Coronagraph System, also known as ASPIICS (Association of Spacecraft for Polarimetric and Imaging Investigation of the Corona of the Sun), with its novel design, will be the first space coronagraph to cover the range of radial distances between ~1.08 and 3 solar radii where the magnetic field plays a crucial role in the coronal dynamics, thus providing continuous observational conditions very close to those during a total solar eclipse. PROBA-3 is first a mission devoted to the in-orbit demonstration of precise formation flying techniques and technologies for future European missions, which will fly ASPIICS as primary payload. The instrument is distributed over two satellites flying in formation (approx. 150m apart) to form a giant coronagraph capable of producing a nearly perfect eclipse allowing observing the sun corona closer to the rim than ever before. The coronagraph instrument is developed by a large European consortium including about 20 partners from 7 countries under the auspices of the European Space Agency. This paper is reviewing the recent improvements and design updates of the ASPIICS instrument as it is stepping into the detailed design phase