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Tremendous advances have been made in cancer immunotherapy over the last decade. Among the different steps of gene expression, translation of mRNA is emerging as an essential player in both cancer and immunity. Changes in mRNA translation are both rapid and adaptive, and translational reprogramming is known to be necessary for sustaining cancer cell proliferation. However, the role of mRNA translation in shaping an immune microenvironment permissive to tumors has not been extensively studied. Recent studies on immunotherapy approaches have indicated critical roles of mRNA translation in regulating the expression of immune checkpoint proteins, tuning the secretion of inflammation-associated factors, modulating the differentiation of immune cells in the tumor microenvironment, and promoting cancer resistance to immunotherapies. Careful consideration of the role of mRNA translation in the tumor-immune ecosystem could suggest more effective therapeutic strategies and may eventually change the current paradigm of cancer immunotherapy. In this review, we discuss recent advances in understanding the relationship between mRNA translation and tumor-associated immunity, the potential mechanisms of immunotherapy resistance in cancers linked to translational reprogramming, and therapeutic perspectives and potential challenges of modulating translational regulation in cancer immunotherapy.

Introduction Des progres significatifs ont ete realises dans le traitement du melanome avec le developpement d’immunotherapies anti-PD1. Cependant, 40 a 60 % des patients developpent des resistances au traitement. Une meilleure comprehension des mecanismes de regulation de l’axe PD1/PDL1 est donc necessaire pour optimiser les reponses therapeutiques. Le complexe eIF4F regule la traduction de l’ARNm cap-dependant et joue un role essentiel dans la biologie du cancer en controlant selectivement la synthese des proteines impliquees dans la croissance des tumeurs et la resistance aux traitements. Notre objectif etait d’identifier des ARNm dependant d’eIF4F dans un contexte de resistance au traitement par anti-PD1 et d’evaluer la reponse aux traitements anti-PD1 apres inhibition d’eIF4F dans un modele murin. Materiel et methodes Nous avons travaille initialement in vitro, sur des lignees cellulaires de melanome murin nommees BP sensibles aux anti-PD1 et BP-R1, BP-R2, resistantes. Une analyse in silico des 5′UTR des ARNm purifies a ete realisee pour estimer la minimum free energy (MFE) d’une sequence d’ARNm. Nous avons realise une analyse GSEA (Gene Set Expression Analysis), methode permettant de determiner si un sous-groupe de genes est surrepresente dans un dans un large ensemble et peut etre associe avec un phenotype, ici la resistance aux anti-PD1. Pour identifier une signature transrationnelle nous avons realise des RT-qPCR sur nos trois lignees. Pour etudier la traduction nous avons realise des profilages de Polysomes. Pour etudier l’effet de l’inhibition du complexe d’initiation de la traduction, nous avons utilise un inhibiteur d’eIF4A, le silvestrol, sur nos trois lignees. Enfin, nous avons realise une experience, de greffe tumorale chez la souris C57Bl/6 Ola/Hsd. Les souris ont ete traitees de facon quotidienne avec du silvestrol (0,5 mg/kg) ou un vehicule en intra-peritoneal. L’anti-PD1 ou l’isotype controle a ete injecte en intra-peritoneal les jours 5, 7 et 9 (100 μg/souris). Resultats Nous avons identifie un groupe d’ARN messagers dont la traduction est dependante d’eIF4F dans deux lignees cellulaires de melanome resistantes aux anti-PD1. Nous avons valide cette signature transcriptionnelle et nous l’avons correlee a l’augmentation de la traduction de ces ARNm cibles en realisant des profilages de polysome. L’augmentation de la traduction dans les deux lignees resistantes etait partiellement dependante d’eIF4F. Nous avons ensuite confirme in vivo sur des tumeurs murines que l’inhibition d’eIF4F semble augmenter la reponse aux antiPD1. Discussion L’inhibition in vitro d’eIF4F permet une diminution de la traduction de nos ARNm cibles et son inhibition in vivo semble prometteuse dans l’amelioration de la reponse aux anti-PD1 dans nos deux modeles murins.

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between Programmed Death Ligand 1 (PD-L1) and Programmed Death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex regulatory mechanisms underlying its regulation are not completely understood. Here, we show that the eIF4F eukaryotic translation initiation complex, which binds the 5’ cap of mRNAs, regulates the surface expression of interferon-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the STAT1 transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacologic inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anti-ancer drugs, may also act as cancer immunotherapies. Citation Format: Michael Cerezo, Ramdane Guemiri, Isabelle Girault, Caroline Robert, Stephan Vagner. Translational control of tumor immune escape via the eIF4F-STAT1-PDL1 axis in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B40.

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-γ-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies. journal article research support, non-u.s. gov't 2018 12 2018 10 29 imported

Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth, its survival, and its motility. Emerging evidence suggests that autophagy limits several signaling pathways by degrading kinases, downstream components, and transcription factors; however, this often occurs under stressful conditions. Our recent studies revealed that constitutive autophagy temporally and spatially controls the RHOA pathway. Specifically, inhibition of autophagosome degradation induces the accumulation of the GTP-bound form of RHOA. The active RHOA is sequestered via SQSTM1/p62 within autolysosomes, and accordingly fails to localize to the spindle midbody or to the cell surface, as we demonstrate herein. As a result, all RHOA-downstream responses are deregulated, thus driving cytokinesis failure, aneuploidy and motility, three processes that directly have an impact upon cancer progression. We therefore propose that autophagy acts as a degradative brake for RHOA signaling and thereby controls cell proliferation, migration, and genome stability.

Metformin was reported to inhibit the proliferation of many cancer cells, including melanoma cells. In this report, we investigated the effect of metformin on melanoma invasion and metastasis development. Using different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration and independently of antiproliferation action. This inhibition is correlated with modulation of expression of proteins involved in epithelial–mesenchymal transition such as Slug, Snail, SPARC, fibronectin, and N-cadherin and with inhibition of MMP-2 and MMP-9 activation. Furthermore, our data indicate that this process is dependent on activation of AMPK and tumor suppressor protein p53. Finally, we showed that metformin inhibits melanoma metastasis development in mice using extravasation and metastasis models. The presented data reinforce the fact that metformin might be a good candidate for clinical trial in melanoma treatment. Mol Cancer Ther; 12(8); 1605–15. ©2013 AACR.

International audience

International audience; Cancer is the second cause of deaths worldwide and is forecasted to affect more that 22 million people in 2020. Despite dramatic improvement in its care over the last two decades, the treatment of resistant forms of cancer is still an unmet challenge. Thus, innovative and efficient treatments are still needed. In this context, we report herein the synthesis and the evaluation of a new class of bioactive molecules belonging to the N-(4-(3-aminophenyl(thiazol-2-yl)acetamide family. Structure–activity relationships could be driven and resulted in the discovery of lead compound 6b. The latter display high in vitro potency against both sensitive and resistant cancer cell lines on three models: melanoma, pancreatic cancer, and chronic myeloid leukemia (CML). 6b leads to cell death by concomitant induction of apoptosis and autophagy, shows good pharmacokinetic properties, and demonstrates a significant reduction of tumor growth in vivo on A375 xenograft model in mice.

International audience; Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.

Introduction Des progres majeurs ont recemment ete realises dans le traitement du melanome grâce aux immunotherapies ciblant l’axe PD1/PDL1. Dans le melanome, l’expression de PDL1 est essentiellement induite par l’interferon gamma secrete par les lymphocytes T, via la voie JAK/STAT activant la transcription de PDL1. Par ailleurs l’expression de PDL1 est correlee a l’activite du complexe d’initiation de la traduction eIF4F. L’objectif ici etait d’identifier de nouveaux regulateurs de PDL1, dont la traduction est controlee par le complexe eIF4F. Materiel et methodes Vingt-trois genes induits par l’interferon gamma ont une traduction dependante du complexe eIF4F dans les cellules de melanome. Nous avons etudie separement l’effet de ces 23 genes par des techniques d’interferences ARN specifiques (siRNA et shRNA) dans 3 lignees cellulaires de melanome (A375, SKMel10 et WM793) sur l’expression de PDL1 a la surface des cellules apres exposition a l’interferon gamma. Resultats Nous avons identifie TBX3 comme nouveau regulateur de PDL1. En effet, l’utilisation de siRNA et shRNA ciblant TBX3 entraine une diminution significative de l’expression de PDL1 a la surface des cellules de melanome. L’analyse du niveau d’ARNm codant pour PDL1, de l’activite du promoteur de PDL1, et des acteurs de la voie JAK/STAT en RT-qPCR et Western-Blot montrent que TBX3 n’agit pas sur la transcription de PDL1 ou sur la voie JAK/STAT/IRF1. Discussion TBX3, qui est un facteur de transcription implique dans l’embryogenese surexprime au cours de certains cancers, est implique dans l’induction de PDL1 en reponse a l’interferon gamma dans le melanome. TBX3 n’agit pas sur la transcription de PDL1 ou sur la voie JAK/STAT. Des travaux futurs vont explorer si cette regulation s’effectue en augmentant la traduction ou en diminuant la degradation de PDL1. Conclusion TBX3 est un nouveau regulateur de PDL1, dont le mecanisme d’action doit etre precise pour determiner son role dans la resistance immunitaire adaptative dans le melanome.

Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.

International audience; Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.

Introduction Les therapies ciblees et immunotherapies ont bouleverse la prise en charge des patients atteints de melanome avance. Neanmoins, les reponses sont encore inconstantes et transitoires du fait de resistances primaires ou secondaires. La necessite de decouvrir de nouveaux traitements reste pleinement d’actualite. Materiel et methodes A partir de donnees preliminaires fondamentales suggerant un effet in vitro de la metformine sur des lignees cellulaires de melanome, nous avons voulu evaluer de facon prospective et multicentrique l’efficacite et la tolerance de la metformine per os (1g 3 × /jour) chez des patients atteints de melanome avance en echec d’au moins un traitement type chimiotherapie, inhibiteur de BRAF (BRAFi) ou ipilimumab. L’evaluation tumorale etait faite selon les criteres Recist 1.1 et l’analyse de survie selon la methode de Kaplan-Meier. Resultats Dix-sept patients etaient inclus, avec un âge median de 74 ans (49–88). Les caracteristiques de la population sont detaillees dans le Tableau 1. Le taux de reponse (critere principal, reponse complete [RC] + reponse partielle [RP]) a 6 mois etait de 0 %. L’evaluation a M6 n’etait realisee que chez 1 patient. Pour les autres, il etait observe avant le 4e mois, 11 progressions, 3 deces dus a une progression et 2 interruptions de traitement dues a des effets indesirables (EI). Parmi les criteres secondaires, le meilleur taux de reponse globale etait de 5,9 % (95 CI, 0,3–30,8, methode de Wilson), la survie globale de 72,5 % (95 CI, 32,0–91,5), le taux de survie sans progression a M6 de 6,25 % (95 CI, 0,41–24,7) et la mediane de 1,94 mois. Parmi les EI rencontres (61 EI), les plus frequents etaient la diarrhee (16,4 %), les nausees (13,1 %) et la fatigue (9,8 %). Les EI de grade 3–4 concernaient 11,8 % des patients. Aucun deces lie au traitement n’etait observe. La moitie des EI ont ete consideres comme possiblement lies au traitement (Fig. 1). Discussion Il s’agit de la premiere etude prospective evaluant l’effet de la metformine chez des patients atteints de melanome metastatique. Malheureusement, les resultats sont negatifs et montrent l’absence d’efficacite de la metformine dans cette indication, du moins a cette dose et per os. D’autre part, le profil de tolerance peut aussi constituer un frein a son utilisation. Neanmoins, le rationnel scientifique a son utilisation avec de bons resultats in vitro incite a ne pas abandonner cette piste. Un derive avec une meilleure biodisponibilite et/ou son utilisation par voie intraveineuse permettraient peut-etre d’observer en clinique des resultats similaires et aussi encourageants que ceux observes in vitro. Par ailleurs, l’effet synergique avec les BRAFi demontre in vitro constitue egalement un axe de recherche particulierement interessant. Conclusion La metformine per os en monotherapie n’est pas une bonne alternative therapeutique chez les patients atteints de melanome avance. Son interet devrait etre etudie en association avec les inhibiteurs de kinases.

Summary Metformin is the most widely used antidiabetic drug that belongs to the biguanide class. It is very well tolerated and has the major clinical advantage of not inducing hypoglycemia. Metformin decreases hepatic glucose production via a mechanism requiring liver kinase B1, which controls the metabolic checkpoint, AMP-activated protein kinase-mammalian target of rapamycin and neoglucogenic genes. The effects of metformin on this pathway results in reduced protein synthesis and cell proliferation. These observations have given the impetus for many investigations on the role of metformin in the regulation of tumor cell proliferation, cell-cycle regulation, apoptosis, and autophagy. Encouraging results from these studies have shown that metformin could potentially be used as an efficient anticancer drug in various neoplasms such as prostate, breast, lung, pancreas cancers, and melanoma. These findings are strengthened by retrospective epidemiological studies that have found a decrease in cancer risk in diabetic patients treated with metformin. In this review, we have focused our discussion on recent molecular mechanisms of metformin that have been described in various solid tumors in general and in melanoma in particular.

International audience; Several reports have demonstrated the inhibitory effect of metformin, a widely used drug in the treatment of type 2 diabetes, on the proliferation of many cancers including melanoma. Recently, it has been shown that metformin is able to modulate the cAMP level in the liver. As cAMP has a crucial role in melanin synthesis and skin pigmentation, we investigated the effect of metformin on melanogenesis both in vitro and in vivo. We showed that metformin led to reduced melanin content in melanoma cells and in normal human melanocytes by decreasing cAMP accumulation and cAMP-responsive element-binding protein phosphorylation. This inhibitory effect is correlated with decreased expression of master genes of melanogenesis, microphthalmia-associated transcription factor, tyrosinase, dopachrome tautomerase, and tyrosinase-related protein 1. Furthermore, we demonstrated that the antimelanogenic effect of metformin is independent of the AMPK pathway. Interestingly, topical application of metformin induced tail whitening in mice. Finally, we confirmed the antimelanogenic effect of metformin on reconstituted human epidermis and on human skin biopsies. These data emphasize the depigmenting effect of metformin and suggest a clinical strategy for using metformin in the topical treatment of hyperpigmentation disorders.

Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance. Cancer Res; 73(14); 4311–22. ©2013 AACR.

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.

Degradation of signaling proteins is one of the most powerful tumor suppressive mechanisms by which a cell can control its own growth, its survival, and its motility. Emerging evidence suggests that autophagy limits several signaling pathways by degrading kinases, downstream components, and transcription factors; however, this often occurs under stressful conditions. We show here that the constitutive autophagy is required for fine-tuning the RHOA pathway. Specifically, inhibition of autophagosome degradation induces the accumulation of the GTP-bound form of RHOA. The active RHOA is sequestered via SQSTM1/p62 within autolysosomes, and accordingly fails to localize to the lamella of crawling cells, one region where RHOA inactivation is essential in cell migration. As a result, all autophagy compromises deregulate cell motility. We therefore propose the term “signalphagy” to indicate a dedicated type of macroautophagy that degrades and thereby maintains the appropriate level of active signaling proteins to achieve tumor suppression. In this model, the remarkable dynamics of autophagy together with its integration of extracellular cues might dictate the time and place where RHOA is active to ensure directed cell motility. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A224. Citation Format: Amine Belaid, Papa Diogop N'diaye, Michaël Cerezo, Patrick Brest, Daniel J. Klionsky, Georges F Carle, Paul Hofman, Baharia Mograbi. Autophagy and SQSTM1 on the RHOA(d) again. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A224.

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacological and genetic evidence indicate that autophagy plays pleiotropic functions in cellular homeostasis, development, survival and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by M-CSF. We have previously shown, using pharmacological inhibitors, siRNA approaches and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper CSF-1-driven differentiation of human and murine monocytes. We have also unraveled a role for autophagy in macrophage acquisition of phagocytic functions. Here, we determine that the CaMKKb-AMPKa1-ULK1 pathway is required for CSF-1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2Y6 to the induction of autophagy, and we decipher the signalling network that links the CSF-1 receptor to P2Y6-mediated autophagy and monocyte differentiation. Importantly, we show that the physiological P2Y6 ligand UDP and the specific P2Y6 agonist MRS2693 restore normal monocyte differentiation through re-induction of autophagy in primary myeloid cells from chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for P2Y6-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML. Disclosures No relevant conflicts of interest to declare.