Siah2 control of T-regulatory cells limits anti-tumor immunity.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy

CDK4/6 Inhibitors in Advanced HR+/HER2 - Breast Cancer: A Multicenter Real-World Data Analysis

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy are considered standard-of-care for first-line therapy of patients with hormone receptor positive, HER2 negative, advanced breast cancer (HR+/HER2- ABC). Superiority of combination therapy over endocrine monotherapy has been demonstrated in a multitude of randomized controlled trials (RCTs) in phase III and IV. However, RCTs reflect clinical reality only to a limited extent, as narrow inclusion criteria lead to a selected patient collective. Here, we present real-world data (RWD) on CDK4/6i treatment in patients with HR+/HER2- ABC at four certified German university breast cancer centers.This study was supported by Novartis Pharma GmbH as part of the “ERIC” (“Excellent Researchers in Breast Cancer”) project

A role for Dynlt3 in melanosome movement, distribution, acidity and transfer

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on beta -catenin activity, revealing a function of the Wnt/beta -catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer. Aktary et al. identify novel roles for the dynein light chain Dynlt3 in melanosome transport, maturation, and transfer to keratinocytes. They also find that the Wnt/beta catenin signalling pathway controls Dynlt3 levels and thus also contributes to the regulation of melanocyte transport and skin pigmentation

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance

Mécanismes de régression spontanée du mélanome chez le porc MeLiM

Cutaneous melanoma, the most aggressive form of skin cancer, is a tumor originating from melanocytes with rapidly increasing incidence. Patients with advanced disease have a poor prognosis since melanoma is mostly resistant to current therapies. Therefore, the development of novel strategies for preventing and treating melanoma is important. To explore novel therapies we need to find appropriate targets and for that knowledge about the biology of melanoma is important. One elegant way to do so, is to study the natural but rare phenomenon of spontaneous melanoma regression. Unfortunately, complete spontaneous regression of advanced melanoma is extremely rare, and therefore hard to investigate. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a great opportunity to study spontaneous tumor regression. MeLiM pigs, suffering from hereditary melanoma, develop tumors naturally and regress them completely without external influence. Mechanisms leading to spontaneous melanoma regression are poorly understood at present. Therefore the overall objective of this PhD was to investigate mechanisms of spontaneous melanoma regression in MeLiM pigs. In an initial study we gained a global overview of differentially expressed genes between a growing and regressing tumor by conducting Subtractive Suppression Hybridization (SSH). We also performed SSH analysis of cell cultures isolated from growing and regressing melanoma to be able to distinguish between expressional changes induced by tumor microenvironment. Only a few genes were in common between these two SSH analyses. Thus we focused on the SSH results obtained from tumor tissue. We were able to identify a gene signature for growing melanoma that demonstrated common genes with human progressive melanoma (TYR, MITF, MLANA, SDCBP, SILV, TYRP and ZFP106). Genes overexpressed at the beginning of regression were mainly involved in functional classes such as differentiation, immune system, cell cycle arrest, and tumor suppression. Two genes, CD9 and RARRES1, showed a strong upregulation during early melanoma regression on the mRNA and protein level whereas CD9 is a motility-related protein and RARRES1 a putative tumor suppressor. Since spontaneous regression is a dynamic process, we performed time dependent gene expression profiling using DNA chips in a second study. We identified significant gene signatures at different stages of regression that were able to explain the corresponding phenotype. Identified gene signatures were mainly involved in the immune response, the cell cycle, and melanocyte differentiation/pigmentation processes. Interestingly, we showed an early downregulation of cell cycle related genes that could play a role in regression. Also, we identified different immune gene signatures, suggesting a major role of the host's immune system in eradicating melanoma cells. By using immunohistology and flow cytometry we characterized tumor infiltrating cells of the innate and adaptive immune system. During regression the infiltration occurred in two phases: an early phase consisting of antigen presenting cells (SWC3+), followed by a later infiltration of mainly cytotoxic T lymphocytes (CD8+). Furthermore the regression process is accompanied by the presence of big highly pigmented cells that we began to characterize. This work allowed us to point out similarities between the pig and human melanoma on the transcriptomic level along with the already observed parallels on the clinical and genetic level. These findings underline the utility for this model to study human melanoma. This work helped also to decipher the process of spontaneous regression on a cellular and molecular level. In this way we observed an early mitotic arrest of tumor cells, linked to regression, that has not been described so far, and an implication of the immune system. If the immune response is the real inducer of regression needs to be verified. Already, this work contributes to human anti-melanoma therapy as it provides potential targets that could be used for developing new strategies.Le mélanome est une tumeur originaire des mélanocytes; c'est la forme la plus agressive des cancers cutanés. Son incidence s'accroît régulièrement. De plus sa résistance aux traitements actuels (chimiothérapie, radiothérapie et immunothérapie) ne laisse qu'une faible espérance de survie aux patients présentant un stade avancé de la maladie. Aujourd'hui il est nécessaire de développer de nouveaux traitements ciblés sur la cellule tumorale. Une stratégie originale consisterait à étudier le phénomène naturel de régression spontanée des mélanomes. Malheureusement chez l'homme la régression n'est que partielle et l'extrême rareté des cas de régression totale à un stade avancé de la maladie rend impossible son étude. Cependant le modèle porcin de mélanome cutané (MeLiM) présente une opportunité unique d'étudier ces mécanismes complexes de régression spontanée. En effet, les minis porcs MeLiM developpent des mélanomes héréditaires qui régressent totalement et spontanément, indépendamment de tous facteurs externes. Actuellement, les mécanismes responsables de cette régression spontanée sont peu ou mal connus. C'est pourquoi l'objectif principal de cette thèse a été l'étude des mécanismes de la régression spontanée du mélanome dans ce modèle animal. Dans un premier temps, grace à la technique d'hybridation suppressive soustractive (HSS) nous avons comparé le transcriptome d'une tumeur en croissance et d'une tumeur en tout début de régression. Dans le but de distinguer les signaux provenant du microenvironnement tumoral de ceux propres à la cellule tumorale, nous avons également réalisé une HSS à partir des cellules isolées d'une tumeur en progression et d'une tumeur au début de la régression. Nous avons trouvé très peu de gènes en commun entre les deux HSS. Nous avons donc focalisé notre étude sur les résultats issus de la HSS à partir des tumeurs. Des gènes surexprimés chez le porc pendant la phase de proliferation tumorale sont également retrouvés dans les études d'expression conduites dans le mélanome chez l'homme (TYR, MITF, MLANA, SDCBP, SILV, TYRP and ZFP106). Au tout début du phénomène de régression, on observe une surexpression des gènes impliqués dans les fonctions de la differentiation, le système immunitaire, l'arrêt du cycle cellulaire et la suppression des tumeurs. Deux gènes CD9 et RARRES1 montrent une très forte surexpression pendant la régression tant au niveau transcriptomique que protéique. Le CD9 est lié à la motilité cellulaire et RARRES1 est un gène potentiel suppresseur de tumeur. Dans un second temps, la dynamique du processus de régression a été étudiée grâce à la technologie des puces à ADN qui a permis d'établir un profil cinétique de l'expression génique. Ainsi, pour chacun des stades de la régression une corrélation entre la signature génique et son expression phénotypique a pu être établie. La signature de la régression comprend entre autres, des gènes impliqués dans la réponse immunitaire, le cycle cellulaire et la différentiation ainsi que la pigmentation des mélanocytes. Ce travail révèle que la régression des mélanomes semblerait être lié à une sous régulation précoce du cycle cellulaire. De plus le système immunitaire semble jouer un rôle majeur dans l'éradication des cellules tumorales. Par immunohistologie et cytometrie en flux, nous avons caracterisé le phenotype des cellules infiltrant les tumeurs : les cellules de l'immunité innée et acquise. On observe deux phases pendant le processus de la régression : une phase précoce consistant principalement à une infiltration par des cellules presentatrice d'antigènes (SWC3+) puis une phase d'infiltration tardive par des lymphocytes cytotoxiques (CD8+). La régression s'accompagne également de la présence de grosses cellules hyperpigmentées que nous avons commencés à caracteriser. Ce travail a permis de montrer qu'il existe des similarités entre le transcriptome du mélanome chez le porc et chez l'homme en plus de celles déjà observées au niveau clinique et génétique ce qui permet de confirmer l'utilité de ce modèle pour l'étude du mélanome chez l'homme. Il a surtout permis de disséquer le processus de régression au niveau cellulaire et moléculaire. Ainsi on a observé un signal précoce d'arrêt en mitose des cellules tumorales, phénomene inconnue jusqu'alors, puis une implication du système immunitaire. Il reste à demontrer si ce dernier est le réel inducteur de la régression. D'ores et déjà, ce travail apporte des cibles à utiliser pour développer des traitements contre le mélanome chez l'homme

Sustained activation of the Aryl hydrocarbon Receptor (AhR) transcription factor promotes resistance to BRAF inhibitors in melanoma

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.status: accepte

Mechanisms of spontaneous melanomia regression in melim pigs

Le mélanome est la forme la plus agressive des cancers cutanés et les formes invasives sont résistantes aux thérapies classiques. L objectif principal de cette thèse a été l étude des mécanismes de la régression spontanée dans un modèle porcin de mélanome cutanée (MeLiM). Par une approche de biologie intégrative utilisant des techniques d étude du transcriptome (HSS, puce ADN) et de caractérisation phénotypique des cellules du microenvironnement tumoral nous avons disséqué le processus de régression au niveau cellulaire et moléculaire. Ce travail a également permis de montrer qu il existe des similarités entre le transcriptome du mélanome chez le porc et chez l homme en plus de celles déjà observées au niveau clinique et génétique. D ores et déjà, ce travail apporte des cibles à utiliser pour développer des traitements contre le mélanome chez l homme.Melanoma is the most aggressive form of skin cancer. The invasive form of the disease exhibits resistance to classical therapy. The overall objective of this PhD was to investigate mechanisms of spontaneous tumor regression using a porcine melanoma model (MeLiM). We applied an approach of integrative biology composed of transcriptomic techniques (SSH and DNA chips) and the phenotypic characterization of cells in the tumor microenvironment. By these means, we deciphered the process of regression on a cellular and molecular level. This work allowed us to point out similarities between porcine and human melanoma on the transcriptomic level along with the already observed parallels on the clinical and genetic level. Already, this work contributes to human anti-melanoma therapy as it provides potential targets that could be used for developing new strategies.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF