Role of the ceramide metabolism in TNF-induced melanoma dedifferentiation

Le mélanome cutané est un cancer de la peau agressif de par, notamment, son fort potentiel métastatique. L'utilisation des inhibiteurs de points de contrôle immunitaire (ICI), anti-PD-1 et anti-CTLA-4, a révolutionné la prise en charge des patients atteints de mélanome avancé. Cependant, la moitié de ces patients ne répond pas aux traitements ou rechute. Notre équipe a récemment mis en évidence que le TNF, une cytokine pro-inflammatoire produite par les cellules immunitaires, participe à la mise en place de ces résistances. En effet, l'utilisation d'anticorps ciblant le TNF améliore considérablement l'efficacité des anti-PD-1 dans un modèle murin de mélanome. Dans un contexte d'immunothérapie, le TNF peut induire la mort suite à l'activation (AICD) des lymphocytes T cytotoxiques ainsi que l'expression de points de contrôle immunitaire secondaire tel que Tim-3. De plus, le TNF peut également agir directement sur les cellules de mélanome, et entrainer leur dédifférenciation. Ce mécanisme très semblable à la transition épithélio-mesenchymateuse, se marque par la diminution du facteur de transcription MITF et de l'expression des antigènes mélanocytaires. Parallèlement, le TNF est un modulateur du métabolisme du céramide. Or, notre laboratoire a également mis en évidence que des perturbations de ce métabolisme impactent la résistance aux immunothérapies. En effet, l'inhibition de la sphingosine kinase 1, responsable de la production de S1P, ou encore la surexpression de la sphingomyélinase neutre 2, favorisant la production de céramide, améliore l'efficacité des anti-PD-1 et anti-CTLA-4 dans des modèles murins de mélanome. De plus, des études récentes suggèrent un rôle du métabolisme du céramide dans la dédifférenciation du mélanome. Au vu de l'étroite relation entre le TNF et le métabolisme du céramide, ainsi que leur rôle respectif dans la mise en place de résistance aux ICI, l'objectif général de ma thèse consistait donc à étudier le rôle du métabolisme du céramide dans la dédifférenciation du mélanome induite par le TNF. Dans un premier temps, nous avons observé que la dédifférenciation du mélanome induite par le TNF est associée à une augmentation globale de la biosynthèse des sphingolipides. Plus spécifiquement, le TNF diminue l'expression et l'activité de la céramidase acide, enzyme centrale de la dégradation du céramide. De manière intéressante, l'étude rétrospective de cohortes de patients atteints de mélanome avancé traités par anti-PD-1, met en évidence une expression de la céramidase acide diminuée dans les tumeurs classées comme transitoires ou dédifférenciées. En ce sens, nous montrons que la diminution de la céramidase acide par une stratégie de siARN, induit par elle-même la dédifférenciation du mélanome vers un état transitoire. A l'inverse, la surexpression de la céramidase acide dans une lignée de mélanome ayant un phénotype transitoire augmente l'état de différenciation et atténue la dédifférenciation du mélanome induite par le TNF. La diminution de la céramidase acide participe donc au phénomène de dédifférenciation induit par le TNF. De façon plus globale, nous montrons que le TNF induit l'accumulation de divers métabolites, dont les lactosylcéramides. Alors que le céramide et la sphingosine exogène déclenchent la dédifférenciation des cellules de mélanome, l'inhibition de la synthèse du céramide et des glycosphingolipides entrave la dédifférenciation des cellules de mélanome induite par le TNF. L'analyse du contenu plasmatique en sphingolipides chez les patients atteints de mélanome avancé indique que les lactosylcéramides sont enrichis chez les non-répondeurs à l'immunothérapie. Ainsi, cette thèse montre que les changements du métabolisme du céramide sont impliqués dans la dédifférenciation des cellules de mélanome induite par le TNF et pourraient participer à la résistance aux inhibiteurs de points de contrôle immunitaire.Cutaneous melanoma is an aggressive skin cancer with a high metastatic potential. The use of immune checkpoint inhibitors (ICI), targeting PD-1 and CTLA-4, has revolutionized the management of patients with advanced melanoma. However, half of these patients do not respond to treatment or relapse. Our team has recently demonstrated that TNF, a pro-inflammatory cytokine produced by immune cells, is involved in the development of this resistance. Indeed, the use of antibodies targeting TNF significantly improves the efficacy of anti-PD-1 in a mouse model of melanoma. In the context of immunotherapy, TNF can lead to the activation-induced cell death (AICD) of cytotoxic T cells and the expression of secondary immune checkpoints such as Tim-3. In addition, TNF can also directly act on melanoma cells, leading to their dedifferentiation. This mechanism is very similar to the epithelial to mesenchymal transition and is marked by a decreased expression of the transcription factor MITF and of melanocytic antigens. In parallel, TNF is a modulator of ceramide metabolism. Our laboratory has also demonstrated that alterations of this metabolism have an impact on the resistance to immunotherapies. Indeed, inhibition of sphingosine kinase 1, responsible for S1P production, or overexpression of neutral sphingomyelinase 2, which promotes ceramide production, improves the efficacy of anti-PD-1 and anti-CTLA-4 in murine melanoma models. In addition, recent studies suggest a role for the ceramide metabolism in melanoma dedifferentiation. Given the close relationship between TNF and the ceramide metabolism, as well as their respective roles in the development of ICI resistance, the general objective of my PhD was to investigate the role of ceramide metabolism in TNF-induced melanoma dedifferentiation. First, we observed that TNF-induced melanoma dedifferentiation is associated with an overall increase in sphingolipid biosynthesis. More specifically, TNF decreases the expression and activity of acid ceramidase, a central enzyme able to catalyse ceramide degradation. Interestingly, a retrospective study on tumours from 3 cohorts of advanced melanoma patients treated with anti-PD-1, shows a reduces expression of acid ceramidase in tumours classified as transitory or dedifferentiated. In accordance, we show that the decrease of acid ceramidase by a siRNA induces, on its own, the dedifferentiation of melanoma cells towards a more transitory state. Conversely, overexpression of acid ceramidase in a melanoma cell line with a transitory phenotype increases the differentiation state and attenuates TNF-induced melanoma dedifferentiation. The decrease of acid ceramidase thus participates in the TNF-induced dedifferentiation phenomenon. More globally, we show that TNF induces the accumulation of various/a number of metabolites, including lactosylceramides. While exogenous ceramide and sphingosine trigger melanoma cell dedifferentiation, inhibition of ceramide and glycosphingolipid synthesis impedes TNF-induced melanoma cell dedifferentiation. Analysis of plasma sphingolipid content in advanced melanoma patients indicates that lactosylceramides are enriched in non-responders to immunotherapy. Thus, our work shows that changes in ceramide metabolism are involved in the TNF-induced dedifferentiation of melanoma cells and may participate in the resistance to immune checkpoint inhibitors

Rôles physiologiques et potentialités thérapeutiques de la mélatonine chez l'homme

La mélatonine, hormone produite par l'épiphyse et découverte il ya bientôt 50 ans rest encore bien mystérieuse pour les chercheurs qui s'y intéressent. On sait que son rythnme de synthèse est particulier : il est dicté par l'alternance jour/nuit, de sorte que la mélatonine est le transducteur interne de la photopériode. De ce fait, la mélatonine semble être la clé de lhoméstasie car elle nous permet de vivre en harmonie avec notre environnement. Les études physiologiques révèlent que la mélatonine possède de nombreuses propriétés : anti-oxydante, hypnotique...mais sutout que cette hormone est un chronobiotique. On a donc suggéré des potentialités thérapeutiques énormes, certaines étant toutefois fantasques. Pour l'instant, aucunes d'entre elles n'ont abouti à la commercialisation de la mélationine ent tant que médicament avec une AMM, et il semble que cela ne sera d'ailleurs jamais le cas car les recherches actuelles sont orientées vers la mise au point d'analogues structuraux de la mélatonine.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

New Insights into the Role of Sphingolipid Metabolism in Melanoma

Cutaneous melanoma is a deadly skin cancer whose aggressiveness is directly linked to its metastatic potency. Despite remarkable breakthroughs in term of treatments with the emergence of targeted therapy and immunotherapy, the prognosis for metastatic patients remains uncertain mainly because of resistances. Better understanding the mechanisms responsible for melanoma progression is therefore essential to uncover new therapeutic targets. Interestingly, the sphingolipid metabolism is dysregulated in melanoma and is associated with melanoma progression and resistance to treatment. This review summarises the impact of the sphingolipid metabolism on melanoma from the initiation to metastatic dissemination with emphasis on melanoma plasticity, immune responses and resistance to treatments

Lipid metabolic Reprogramming: Role in Melanoma Progression and Therapeutic Perspectives

International audienceMetabolic reprogramming contributes to the pathogenesis and heterogeneity of melanoma. It is driven both by oncogenic events and the constraints imposed by a nutrient- and oxygen-scarce microenvironment. Among the most prominent metabolic reprogramming features is an increased rate of lipid synthesis. Lipids serve as a source of energy and form the structural foundation of all membranes, but have also emerged as mediators that not only impact classical oncogenic signaling pathways, but also contribute to melanoma progression. Various alterations in fatty acid metabolism have been reported and can contribute to melanoma cell aggressiveness. Elevated expression of the key lipogenic fatty acid synthase is associated with tumor cell invasion and poor prognosis. Fatty acid uptake from the surrounding microenvironment, fatty acid β-oxidation and storage also appear to play an essential role in tumor cell migration. The aim of this review is (i) to focus on the major alterations affecting lipid storage organelles and lipid metabolism. A particular attention has been paid to glycerophospholipids, sphingolipids, sterols and eicosanoids, (ii) to discuss how these metabolic dysregulations contribute to the phenotype plasticity of melanoma cells and/or melanoma aggressiveness, and (iii) to highlight therapeutic approaches targeting lipid metabolism that could be applicable for melanoma treatment

A fluorogenic substrate for the detection of lipid amidases in intact cells

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells

The HIV-1 nucleocapsid regulates its own condensation by phase-separated activity-enhancing sequestration of the viral protease during maturation

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.This work was supported in part by the European Project THINPAD “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance” (FP7-Grant Agreement 601969), by Foundation Clinic, by ANRS, by SIDACTION, and with federal funds from the NCI/NIH, under Contract No. HHSN261200800001E with Leidos Biomedical Research, Inc. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government (R.J.G.). S.L. acknowledges funding by the Marie-Curie IEF fellowship (FP7-Grant Agreement 237738) and is grateful to Maria Solà (IBMB-CSIC). S.K.S. and A.M. acknowledge support from amfAR Mathilde Krim Fellowship in Basic Biomedical Research number 108680 and the Spanish Ministry of Economy and Competitiveness and FEDER (Grant no. SAF2013-46077-R). S.K.S. also gratefully acknowledges support from the Volkswagen Foundation “Experiment! Funding Initiative” grant number 93874 and from the Klaus Tschira Stiftung

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti–PD-1 Therapy Efficacy

International audienceDysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1

Correction: Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

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