Rôles régulateurs des GTPases Rho dans l'expression de ligands du système immunitaire anti-mélanome

Ma thèse porte sur l'étude des rôles régulateurs des GTPases Rho sur l'expression de ligands du système immunitaire, sur des cellules de mélanomes murins et humains et leurs conséquences sur le développement tumoral. La recherche de marqueurs liés aux capacités métastatiques des mélanomes est nécessaire, ainsi que celle de molécules pharmacologiques qui amplifieraient les réponses immunes et pourraient améliorer les thérapies du mélanome. En effet, l'incidence du mélanome est en forte augmentation et les traitements actuels ont une efficacité modérée sur les mélanomes métastatiques. Mon étude des rôles régulateurs des GTPases Rho et de leurs inhibiteurs dans le développement des mélanomes s'est décomposée en trois volets : - La mise en évidence de la surexpression membranaire des molécules de CMH de classe I et de costimulation CD80 et CD86 dans les mélanomes murin B16F10 (H-2Kb/TRP-2) et humain LB1319-MEL (HLA-A0201/MelanA-MART1). Nous avons montré que le traitement de ces mélanomes avec des inhibiteurs de GTPases Rho (statines, inhibiteur de la géranylgéranyltransférase et C3 exoenzyme) les rendaient plus immunogéniques et capables d'induire d'une part des LT CD8+ cytotoxiques anti-tumoraux à partir de PBMC naïfs et d'autre part de vacciner des souris naïves. Ce travail a été publié dans 2nd European Congress of Immunology, ECI Berlin en septembre 2009 et dans PLoS One en février 2010. - L'équipe a précédemment montré l'intérêt de l'expression ou de la sécrétion de la molécule de costimulation CD70 par des carcinomes mammaires murins dans la mise en place d'une réponse immune anti-tumorale protectrice. Nous avons recherché l'expression de CD70 sur des mélanomes et étudié son rôle dans ces tumeurs. Nous montrons pour la 1ère fois l'expression de CD70 sur des mélanomes humains (environ 30%) et sa régulation par la GTPase RhoA. Et surtout, nous décrivons un nouveau rôle non-immunologique de CD70 dans l'invasion et les capacités métastatiques des mélanomes, via son contrôle de la voie de signalisation BRAF/MEK/ERK/RhoE et la régulation des fibres de stress d'actine et des points focaux d'adhésion. Ces travaux sont soumis à PNAS. Par ailleurs, nous avons montré que BRAF régule aussi l'expression de CD70, dans une boucle de rétrocontrôle. - Nous avons enfin étudié la régulation par les GTPases Rho du ligand activateur MICA exprimé sur des mélanomes humains et reconnu par les cellules NK. Le traitement des cellules LB1319-MEL par des statines entraîne une surexpression membranaire de MICA, sans toxicité cellulaire. Cette surexpression de MICA permet d'augmenter la sensibilité à la lyse des cellules de mélanome par les cellules NK et induit un ralentissement de leur croissance tumorale sous-cutanée dans des souris NMRI nu/nu et une réduction des métastases pulmonaires. Comme nous avons montré que les GTPases Ras, RhoB, RhoC et Rac1 sont des régulateurs positifs de l'expression MICA, nous recherchons quelles autres voies métaboliques inhibées par les statines seraient capables de réguler négativement l'expression de MICA et donc d'induire sa surexpression sous traitement par les statines. Nous testerons l'implication de la production des espèces réactives oxygénées et de la voie de PPARgamma. L'ensemble de mes travaux montre que les GTPases Rho pourraient être des cibles intéressantes pour favoriser la mise en place de réponses immunes protectrices innée et adaptative dans de nouveaux protocoles d'immunothérapie du mélanome.My Ph.D subject was to study the regulatory roles of Rho GTPases in ligands expression of the immune system, in murine and human melanoma cells and their consequences in the tumor development. The search for markers linked to metastatic capacities of melanoma cells is necessary, as well as the search of pharmacological agents that would amplify the immune response and would improve melanoma therapies. Indeed, melanoma incidence is rising sharply and current treatments have moderate efficiency in metastatic melanomas. My study of regulatory roles of Rho GTPases and their inhibitors in melanoma development consists of three parts: - The demonstration of membrane overexpression of MHC class I and CD80 and CD86 costimulatory molecules in murine B16F10 (H-2Kb/TRP-2) and human LB1319-MEL (HLA-A0201/MelanA-MART1) melanoma cells. We showed that melanomas treatment with Rho GTPases inhibitors (statins, geranylgeranyltransferase inhibitor, C3 exoenzyme) made the cells more immunogenic, and able to induce on the one hand anti-tumor cytotoxic CD8 T cells from naïve PBMC and on the other hand to vaccinate naïve mice. This work was published in 2nd European Congress of Immunology, ECI Berlin in September 2009 and in PLoS One, in February 2010. - The team has previously shown the interest for the CD70 costimulatory molecule expression or secretion by murine mammary carcinoma in the setting of a protective anti-tumor immune response. We searched for CD70 expression in human melanomas and we studied its role in these tumors. We brought the first description of CD70 expression in human melanomas (around 30%) and its regulation by RhoA GTPase. Above all, we described a new non-immunologic function of CD70 in melanoma invasion and metastatic capacities, through its control of the BRAF/MEK/ERK/RhoE signaling pathway and actin stress fibers and focal adhesion regulations. This work is submitted to PNAS. Furthermore, we showed that BRAF signaling pathway also regulates CD70 expression, in a feedback loop. Finally, we studied Rho GTPases regulations of MICA expression in human melanomas. MICA is an activating ligand that is recognized by NK cells. LB1319-MEL cells treatment with statins leads to MICA membrane overexpression, without cytotoxic effect. MICA membrane overexpression enhanced the melanoma cells sensitivity to NK cells lysis and induced a subcutaneous tumor growth slowing-down in NMRI nu/nu mice and a decrease in lung metastases. As we showed that Ras, RhoB, RhoC and Rac1 GTPases are positive regulators of MICA expression, we are investigating others metabolic pathways inhibited by statins that could regulate negatively MICA expression and consequently induce MICA overexpression under statin treatment. We are currently testing the involvement of the reactive oxygen species production and of the PPARgamma pathway. Altogether my works show that Rho GTPases could be interesting targets in order to favor the setting of innate and adaptive protective immune responses in new melanoma immunotherapy protocols

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

International audienceBACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells

Identification of a novel PPARβ/δ/miR-21-3p axis in UV-induced skin inflammation.

Although excessive exposure to UV is widely recognized as a major factor leading to skin perturbations and cancer, the complex mechanisms underlying inflammatory skin disorders resulting from UV exposure remain incompletely characterized. The nuclear hormone receptor PPARβ/δ is known to control mouse cutaneous repair and UV-induced skin cancer development. Here, we describe a novel PPARβ/δ-dependent molecular cascade involving TGFβ1 and miR-21-3p, which is activated in the epidermis in response to UV exposure. We establish that the passenger miRNA miR-21-3p, that we identify as a novel UV-induced miRNA in the epidermis, plays a pro-inflammatory function in keratinocytes and that its high level of expression in human skin is associated with psoriasis and squamous cell carcinomas. Finally, we provide evidence that inhibition of miR-21-3p reduces UV-induced cutaneous inflammation in ex vivo human skin biopsies, thereby underlining the clinical relevance of miRNA-based topical therapies for cutaneous disorders

Pervasive lesion segregation shapes cancer genome evolution

Cancers arise through the acquisition of oncogenic mutations and grow through clonal expansion. Here we reveal that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterise this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions enables the accurate measurement of strand biased repair processes, quantification of oncogenic selection, and fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.This work was supported by: Cancer Research UK (20412, 22398), the European Research Council (615584, 682398), the Wellcome Trust (WT108749/Z/15/Z, WT106563/Z/14/A, WT202878/B/16/Z), the European Molecular Biology Laboratory, the MRC Human Genetics Unit core funding programme grants (MC_UU_00007/11, MC_UU_00007/16), and the ERDF/Spanish Ministry of Science, Innovation and Universities-Spanish State Research Agency/DamReMap Project (RTI2018-094095-B-I00)

Pharmacological inhibition of bromodomain and extra-terminal proteins induces an NRF-2-mediated antiviral state that is subverted by SARS-CoV-2 infection

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19

Extent of non-publication in cohorts of studies approved by research ethics committees or included in trial registries

BACKGROUND: The synthesis of published research in systematic reviews is essential when providing evidence to inform clinical and health policy decision-making. However, the validity of systematic reviews is threatened if journal publications represent a biased selection of all studies that have been conducted (dissemination bias). To investigate the extent of dissemination bias we conducted a systematic review that determined the proportion of studies published as peer-reviewed journal articles and investigated factors associated with full publication in cohorts of studies (i) approved by research ethics committees (RECs) or (ii) included in trial registries. METHODS AND FINDINGS: Four bibliographic databases were searched for methodological research projects (MRPs) without limitations for publication year, language or study location. The searches were supplemented by handsearching the references of included MRPs. We estimated the proportion of studies published using prediction intervals (PI) and a random effects meta-analysis. Pooled odds ratios (OR) were used to express associations between study characteristics and journal publication. Seventeen MRPs (23 publications) evaluated cohorts of studies approved by RECs; the proportion of published studies had a PI between 22% and 72% and the weighted pooled proportion when combining estimates would be 46.2% (95% CI 40.2%-52.4%, I2 = 94.4%). Twenty-two MRPs (22 publications) evaluated cohorts of studies included in trial registries; the PI of the proportion published ranged from 13% to 90% and the weighted pooled proportion would be 54.2% (95% CI 42.0%-65.9%, I2 = 98.9%). REC-approved studies with statistically significant results (compared with those without statistically significant results) were more likely to be published (pooled OR 2.8; 95% CI 2.2-3.5). Phase-III trials were also more likely to be published than phase II trials (pooled OR 2.0; 95% CI 1.6-2.5). The probability of publication within two years after study completion ranged from 7% to 30%. CONCLUSIONS: A substantial part of the studies approved by RECs or included in trial registries remains unpublished. Due to the large heterogeneity a prediction of the publication probability for a future study is very uncertain. Non-publication of research is not a random process, e.g., it is associated with the direction of study findings. Our findings suggest that the dissemination of research findings is biased

Tears evoke the intention to offer social support: A systematic investigation of the interpersonal effects of emotional crying across 41 countries

Tearful crying is a ubiquitous and likely uniquely human phenomenon. Scholars have argued that emotional tears serve an attachment function: Tears are thought to act as a social glue by evoking social support intentions. Initial experimental studies supported this proposition across several methodologies, but these were conducted almost exclusively on participants from North America and Europe, resulting in limited generalizability. This project examined the tears-social support intentions effect and possible mediating and moderating variables in a fully pre-registered study across 7007 participants (24,886 ratings) and 41 countries spanning all populated continents. Participants were presented with four pictures out of 100 possible targets with or without digitally-added tears. We confirmed the main prediction that seeing a tearful individual elicits the intention to support, d = 0.49 [0.43, 0.55]. Our data suggest that this effect could be mediated by perceiving the crying target as warmer and more helpless, feeling more connected, as well as feeling more empathic concern for the crier, but not by an increase in personal distress of the observer. The effect was moderated by the situational valence, identifying the target as part of one's group, and trait empathic concern. A neutral situation, high trait empathic concern, and low identification increased the effect. We observed high heterogeneity across countries that was, via split-half validation, best explained by country-level GDP per capita and subjective well-being with stronger effects for higher-scoring countries. These findings suggest that tears can function as social glue, providing one possible explanation why emotional crying persists into adulthood.</p

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors