Role of tumor plasticity in immune escape and immunotherapy resistance in melanoma

Le mélanome est le cancer de la peau le plus agressif. La prise en charge des patients atteints de mélanome métastatique a été améliorée grâce à l’essor de stratégies d’immunothérapie ciblant les points de contrôle du système immunitaire (ICI). Si des réponses durables sont observées chez une partie des patients, près de 60% des patients sont encore résistants aux ICI, tels que les anti-PD1. Si l'efficacité des immunothérapies dans le mélanome métastatique repose bien entendu sur la composition du microenvironnement immunitaire, il apparaît que des mécanismes intrinsèques aux cellules cancéreuses joueraient également un rôle clé dans l'échappement et la résistance immunitaire, mais les interactions complexes entre ces différentes populations cellulaires restent à mieux caractériser. Dans ce contexte, l’expression du facteur de transcription inducteur d’EMT ZEB1, est fréquemment réactivée dans le mélanome, où elle soutient la plasticité des cellules de mélanome et la résistance aux thérapies ciblées. Nous démontrons à présent qu’une forte expression de ZEB1 dans les cellules tumorales modifie la composition et la fonction du microenvironnement immunitaire dans le mélanome. L’analyse spatiale de l’infiltrat immunitaire dans des cohortes d’échantillons de mélanomes humains a ainsi révélé une corrélation entre une forte expression de ZEB1 par les cellules tumorales et une diminution de l'infiltration par les lymphocytes T CD8+ ainsi qu’une augmentation des macrophages (TAM) pro-tumoraux de type M2. De plus, des expériences de gain ou de perte de fonction dans des modèles murins de mélanome ont confirmé que ZEB1 entrave le recrutement des lymphocytes T CD8+ et favorise la polarisation des TAM de type M2, via une régulation directe de la production de chimiokines et cytokines. L’expression de ZEB1 dans les cellules tumorales entraîne ainsi une évasion immunitaire de la tumeur et une résistance au blocage des points de contrôle immunitaire in vivo. Si l’expression de ZEB1 seule ne suffit pas à prédire la réponse aux ICI dans les cohortes de patients traités par ICI, la perte d’expression de SOX10, associée à la dédifférenciation tumorale, est en revanche associée à la résistance aux ICI. De plus, nous identifions une population de TAM de type M1 exprimant PD-L1 comme un biomarqueur prédictif de la réponse à l’immunothérapie et proposons donc d’intégrer les informations spatiales de l’infiltrat immunitaire avec un score de plasticité tumorale afin de mieux prédire la réponse aux ICI. Enfin, le ciblage de ZEB1 améliore l'efficacité de l'immunothérapie anti-PD-1 in vivo, ce qui laisse entrevoir une nouvelle cible thérapeutique dans le mélanome métastatique.Melanoma is the most aggressive type of skin cancer. The treatment of metastatic melanoma has been recently improved with the development of immunotherapy strategies targeting immune checkpoints (ICI). While long-term responses may be observed in some patients, nearly 60% of patients are still resistant to anti-PD1. The efficacy of immunotherapies in metastatic melanoma depends on the composition of the immune microenvironment, but cancer cell-intrinsic mechanisms are believed to participate to immune escape and resistance, while the complex interactions between these cell subpopulations remain to be precisely characterized. In this context, the EMT-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, favoring resistance to targeted therapies. Here we demonstrate the major role of melanoma cell intrinsic ZEB1 expression in shaping an immunosuppressive microenvironment in melanoma. Spatial analysis of the immune infiltrate in human melanoma samples revealed a correlation between high ZEB1 expression in tumor cells with decreased CD8+ T cell infiltration and increased M2-like macrophages (TAMs). Furthermore, gain-or-loss-of-function experiments in mouse models of melanoma confirmed that ZEB1 impairs CD8+ T cell recruitment and promotes M2-like TAM polarization, through regulation of chemokine/cytokines production. ZEB1 expression thus leads to tumor immune evasion and resistance to immune checkpoint blockade in vivo. While ZEB1 expression alone is not sufficient to predict ICI response in cohorts of patients treated by ICI, SOX10 down-regulation, linked with dedifferentiation, is associated with resistance to ICI. Moreover, we identified PD-L1+ M1 TAM as predictive of ICI response and thus propose to integrate spatial information with a score of tumor cell plasticity in order to better predict ICI. Finally, targeting ZEB1 improves the efficacy of anti-PD-1 immunotherapy, suggesting a new therapeutic target in metastatic melanoma

Étude du facteur de transcription ZEB1, son rôle dans l'échappement au système immunitaire et la résistance à l'immunothérapie dans le mélanome

Le mélanome est le cancer de la peau le plus agressif. L’essor de stratégies d’immunothérapie pour le traitement des patients atteints de mélanome métastatique a permis d’obtenir des réponses sur le long terme chez une partie des patients. Cependant, près de 60 % des patients sont encore résistants, de manière innée ou acquise, aux anti-PD-1. L'efficacité des immunothérapies dans le mélanome métastatique dépend d'une infiltration robuste des lymphocytes T. Par conséquent, il est crucial de définir les mécanismes intrinsèques des cellules cancéreuses induisant l'exclusion des lymphocytes T et la résistance immunitaire. Le facteur de transcription ZEB1, qui induit l'EMT, est un régulateur majeur de la plasticité des cellules de mélanome, qui entraîne une résistance aux thérapies ciblées.Nous démontrons ici le rôle majeur de l’expression de ZEB1, intrinsèque aux cellules de mélanome, dans la prévention de l'infiltration des lymphocytes T dans le mélanome. L’analyse spatiale des infiltrats immunitaires dans des échantillons de mélanomes humains a révélé une corrélation entre une forte expression de ZEB1 dans les cellules tumorales et une diminution de l'infiltration des lymphocytes T CD8+, indépendamment de l'activation de la voie de la ß-caténine. De plus, des expériences de gain ou de perte de fonction dans des modèles murins de mélanome ont montré que ZEB1 entrave le recrutement des lymphocytes T CD8+ en supprimant la production de chimiokines attirant les lymphocytes T, ce qui entraîne une évasion immunitaire de la tumeur et une résistance au blocage des points de contrôle immunitaire. De plus, le ciblage de ZEB1 améliore l'efficacité de l'immunothérapie anti-PD-1, ce qui laisse entrevoir une nouvelle cible thérapeutique dans le mélanome métastatique. À l'avenir, le développement d'analyses transcriptomiques et spatiales à l'échelle de la cellule unique sera crucial pour l’identification de biomarqueurs de la réponse aux immunothérapies

Cancer Cell Phenotype Plasticity as a Driver of Immune Escape in Melanoma

International audienceImmunotherapies blocking negative immune checkpoints are now approved for the treatment of a growing number of cancers. However, even in metastatic melanoma, where sustained responses are observed, a significant number of patients still do not respond or display resistance. Increasing evidence indicates that non-genetic cancer cell-intrinsic alterations play a key role in resistance to therapies and immune evasion. Cancer cell plasticity, mainly associated with the epithelial-to-mesenchymal transition in carcinoma, relies on transcriptional, epigenetic or translational reprogramming. In melanoma, an EMT-like dedifferentiation process is characterized by the acquisition of invasive or neural crest stem cell-like features. Herein, we discuss recent findings on the specific roles of phenotypic reprogramming of melanoma cells in driving immune evasion and resistance to immunotherapies. The mechanisms by which dedifferentiated melanoma cells escape T cell lysis, mediate T cell exclusion or remodel the immune microenvironment will be detailed. The expanded knowledge on tumor cell plasticity in melanoma should contribute to the development of novel therapeutic combination strategies to further improve outcomes in this deadly metastatic cancer

Efficacy of Targeted Radionuclide Therapy Using [131I]ICF01012 in 3D Pigmented BRAF- and NRAS-Mutant Melanoma Models and In Vivo NRAS-Mutant Melanoma

International audiencePurpose: To assess the efficiency of targeted radionuclide therapy (TRT), alone or in combination with MEK inhibitors (MEKi), in melanomas harboring constitutive MAPK/ERK activation responsible for tumor radioresistance.Methods: For TRT, we used a melanin radiotracer ([131I]ICF01012) currently in phase 1 clinical trial (NCT03784625). TRT alone or combined with MEKi was evaluated in three-dimensional melanoma spheroid models of human BRAFV600E SK-MEL-3, murine NRASQ61K 1007, and WT B16F10 melanomas. TRT in vivo biodistribution, dosimetry, efficiency, and molecular mechanisms were studied using the C57BL/6J-NRASQ61K 1007 syngeneic model.Results: TRT cooperated with MEKi to increase apoptosis in both BRAF- and NRAS-mutant spheroids. NRASQ61K spheroids were highly radiosensitive towards [131I]ICF01012-TRT. In mice bearing NRASQ61K 1007 melanoma, [131I]ICF01012 induced a significant extended survival (92 vs. 44 days, p < 0.0001), associated with a 93-Gy tumor deposit, and reduced lymph-node metastases. Comparative transcriptomic analyses confirmed a decrease in mitosis, proliferation, and metastasis signatures in TRT-treated vs. control tumors and suggest that TRT acts through an increase in oxidation and inflammation and P53 activation.Conclusion: Our data suggest that [131I]ICF01012-TRT and MEKi combination could be of benefit for advanced pigmented BRAF-mutant melanoma care and that [131I]ICF01012 alone could constitute a new potential NRAS-mutant melanoma treatment

Immune checkpoint inhibitors reverse tolerogenic mechanisms induced by melanoma targeted radionuclide therapy

International audienceIn line with the ongoing phase I trial (NCT03784625) dedicated to melanoma targeted radionuclide therapy (TRT), we explore the interplay between immune system and the melanin ligand [131I]ICF01012 alone or combined with immunotherapy (immune checkpoint inhibitors, ICI) in preclinical models. Here we demonstrate that [131I]ICF01012 induces immunogenic cell death, characterized by a significant increase in cell surface-exposed annexin A1 and calreticulin. Additionally, [131I]ICF01012 increases survival in immunocompetent mice, compared to immunocompromised (29 vs. 24 days, p = 0.0374). Flow cytometry and RT-qPCR analyses highlight that [131I]ICF01012 induces adaptive and innate immune cell recruitment in the tumor microenvironment. [131I]ICF01012 combination with ICIs (anti-CTLA-4, anti-PD-1, anti-PD-L1) has shown that tolerance is a main immune escape mechanism, whereas exhaustion is not present after TRT. Furthermore, [131I]ICF01012 and ICI combination has systematically resulted in a prolonged survival (p < 0.0001) compared to TRT alone. Specifically, [131I]ICF01012 + anti-CTLA-4 combination significantly increases survival compared to anti-CTLA-4 alone (41 vs. 26 days; p = 0.0011), without toxicity. This work represents the first global characterization of TRT-induced modifications of the antitumor immune response, demonstrating that tolerance is a main immune escape mechanism and that combining TRT and ICI is promising

ZEB1 controls a lineage-specific transcriptional program essential for melanoma cell state transitions

International audienceCell plasticity sustains intra-tumor heterogeneity and treatment resistance in melanoma. Deciphering the transcriptional mechanisms governing reversible phenotypic transitions between proliferative/differentiated and invasive/stem-like states is required. Expression of the ZEB1 transcription factor is frequently activated in melanoma, where it fosters adaptive resistance to targeted therapies. Here, we performed a genome-wide characterization of ZEB1 transcriptional targets, by combining ChIP-sequencing and RNA-sequencing, upon phenotype switching in melanoma models. We identified and validated ZEB1 binding peaks in the promoter of key lineage-specific genes crucial for melanoma cell identity. Mechanistically, ZEB1 negatively regulates SOX10-MITF dependent proliferative/melanocytic programs and positively regulates AP-1 driven invasive and stem-like programs. Comparative analyses with breast carcinoma cells revealed lineage-specific ZEB1 binding, leading to the design of a more reliable melanoma-specific ZEB1 regulon. We then developed single-cell spatial multiplexed analyses to characterize melanoma cell states intra-tumoral heterogeneity in human melanoma samples. Combined with scRNA-Seq analyses, our findings confirmed increased ZEB1 expression in Neural-Crest-like cells and mesenchymal cells, underscoring its significance in vivo in both populations. Overall, our results define ZEB1 as a major transcriptional regulator of cell states transitions and provide a better understanding of lineage-specific transcriptional programs sustaining intra-tumor heterogeneity in melanom

ZEB1 transcription factor promotes immune escape in melanoma

International audienceBackground The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy. Methods We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8 + , CD4 + T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated. Results Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8 + T cell infiltration, independently of β-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8 + T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, Zeb1 knock-out, by promoting CD8 + T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression. Conclusions We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma. Trial registration number NCT02828202