RESUMEN
To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.
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Melanoma , Humanos , Redes Reguladoras de Genes , Inmunoterapia , Melanocitos , Melanoma/tratamiento farmacológico , Melanoma/genética , Factor de Transcripción 4/genética , Microambiente TumoralRESUMEN
Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.
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Linfocitos T CD4-Positivos , Muerte Celular , Inmunoterapia , Inflamación , Neoplasias , Microambiente Tumoral , Humanos , Células Presentadoras de Antígenos/inmunología , Antígeno CD11c/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Muerte Celular/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunidad Innata , Inflamación/inmunología , Interferones/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Microambiente Tumoral/inmunología , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Células TH1/citología , Células TH1/inmunologíaRESUMEN
Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.
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Proliferación Celular , Melanoma , Metástasis de la Neoplasia , Animales , Comunicación Celular , Diferenciación Celular , Linaje de la Célula , Rastreo Celular , Reprogramación Celular , Células Endoteliales , Melanoma/genética , Melanoma/patología , Mesodermo/patología , Ratones , Metástasis de la Neoplasia/patología , Cresta Neural/embriología , Fenotipo , Análisis de la Célula Individual , Transcriptoma , Microambiente TumoralRESUMEN
BACKGROUND: VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically. METHODS: The expression of ten TMEMs (RTP3, SLC35G2, TMEM30B, TMEM45A, TMEM45B, TMEM61, TMEM72, TMEM116, TMEM207 and TMEM213) was measured by qPCR. T-test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools. RESULTS: We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER. CONCLUSIONS: The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas de la Membrana/genética , Receptores de Estrógenos/metabolismo , Adulto , Anciano , Carcinoma de Células Renales/patología , Análisis por Conglomerados , Biología Computacional , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Renales/patología , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Familia de Multigenes , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Oportunidad Relativa , Transporte de Proteínas , Carga TumoralRESUMEN
Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.
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Sistemas CRISPR-Cas , Neoplasias , Humanos , Linfocitos T CD8-positivos , Neoplasias/genéticaRESUMEN
Nuclear receptors (NRs) are implicated in the regulation of tumors and immune cells. We identify a tumor-intrinsic function of the orphan NR, NR2F6, regulating antitumor immunity. NR2F6 was selected from 48 candidate NRs based on an expression pattern in melanoma patient specimens (i.e., IFN-γ signature) associated with positive responses to immunotherapy and favorable patient outcomes. Correspondingly, genetic ablation of NR2F6 in a mouse melanoma model conferred a more effective response to PD-1 therapy. NR2F6 loss in B16F10 and YUMM1.7 melanoma cells attenuated tumor development in immune-competent but not -incompetent mice via the increased abundance of effector and progenitor-exhausted CD8+ T cells. Inhibition of NACC1 and FKBP10, identified as NR2F6 effectors, phenocopied NR2F6 loss. Inoculation of NR2F6 KO mice with NR2F6 KD melanoma cells further decreased tumor growth compared with NR2F6 WT mice. Tumor-intrinsic NR2F6 function complements its tumor-extrinsic role and justifies the development of effective anticancer therapies.
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Linfocitos T CD8-positivos , Melanoma , Animales , Ratones , Inmunoterapia , Melanoma/genética , Proteínas Represoras/metabolismoRESUMEN
Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.
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Formaldehído , Neoplasias , Humanos , Transcriptoma , Adhesión en Parafina , Neoplasias/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
By restoring tryptophan, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors aim to reactivate anti-tumor T cells. However, a phase III trial assessing their clinical benefit failed, prompting us to revisit the role of IDO1 in tumor cells under T cell attack. We show here that IDO1 inhibition leads to an adverse protection of melanoma cells to T cell-derived interferon-gamma (IFNγ). RNA sequencing and ribosome profiling shows that IFNγ shuts down general protein translation, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response driving activating transcription factor-4 (ATF4)high/microphtalmia-associated transcription factor (MITF)low transcriptomic signatures, also in patient melanomas. Single-cell sequencing analysis reveals that MITF downregulation upon immune checkpoint blockade treatment predicts improved patient outcome. Conversely, MITF restoration in cultured melanoma cells causes T cell resistance. These results highlight the critical role of tryptophan and MITF in the melanoma response to T cell-derived IFNγ and uncover an unexpected negative consequence of IDO1 inhibition.
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Melanoma , Triptófano , Humanos , Melanoma/patología , Interferón gamma/metabolismo , Linfocitos T/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genéticaRESUMEN
Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.
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Melanoma , Humanos , Ratones , Animales , Melanoma/patología , Células Endoteliales/metabolismo , Linfocitos T CD8-positivos , FN-kappa B/metabolismo , Autofagia , Inmunoterapia , Microambiente TumoralRESUMEN
BACKGROUND: Immune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy. METHODS: We used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs. RESULTS: Here, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry). CONCLUSION: As HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1 , Glucocorticoides , Inmunoterapia , Melanoma , Animales , Ratones , Linfocitos T CD8-positivos , Glucocorticoides/uso terapéutico , Interferón gamma/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microambiente Tumoral , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Reposicionamiento de MedicamentosRESUMEN
The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.
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Antígeno B7-H1 , Melanoma , Ratones , Animales , Antígeno B7-H1/genética , Linfocitos T , Antígenos CD58/química , Antígenos CD58/metabolismo , Melanoma/genética , Melanoma/metabolismo , Activación de LinfocitosRESUMEN
Immunotherapies, in particular immune checkpoint blockade (ICB), have improved the clinical outcome of cancer patients, although many fail to mount a durable response. Several resistance mechanisms have been identified, but our understanding of the requirements for a robust ICB response is incomplete. We have engineered an MHC I/antigen: TCR-matched panel of human NSCLC cancer and T cells to identify tumor cell-intrinsic T cell resistance mechanisms. The top differentially expressed gene in resistant tumor cells was SERPINB9. This serine protease inhibitor of the effector T cell-derived molecule granzyme B prevents caspase-mediated tumor apoptosis. Concordantly, we show that genetic ablation of SERPINB9 reverts T cell resistance of NSCLC cell lines, whereas its overexpression reduces T cell sensitivity. SERPINB9 expression in NSCLC strongly correlates with a mesenchymal phenotype. We also find that SERPINB9 is commonly amplified in cancer, particularly melanoma in which it is indicative of poor prognosis. Single-cell RNA sequencing of ICB-treated melanomas revealed that SERPINB9 expression is elevated not only in cells from post- versus pre-treatment cancers, but also in ICB-refractory cancers. In NSCLC we commonly observed rare SERPINB9-positive cancer cells, possibly accounting for reservoirs of ICB-resistant cells. While underscoring SERPINB9 as a potential target to combat immunotherapy resistance, these results suggest its potential to serve as a prognostic and predictive biomarker.
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Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Neoplasias , Serpinas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Inhibidores de Serina Proteinasa/genética , Serpinas/genética , Resistencia a Antineoplásicos/genética , Neoplasias Cutáneas , Neoplasias/genéticaRESUMEN
Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Melanoma/tratamiento farmacológico , Melanoma/genética , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Imidazoles/farmacología , Melanoma/patología , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Cresta Neural/patología , Oximas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Piridonas/farmacología , Pirimidinonas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.
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Ciclo Celular , Melanoma/patología , Proteínas de Resistencia a Mixovirus/metabolismo , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Melanoma/genética , Ratones Desnudos , Proteínas de Resistencia a Mixovirus/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
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.
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Melanoma/inmunología , Proteínas Nucleares/inmunología , Linfocitos T Reguladores/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Quimiocina CCL17/genética , Quimiocina CCL17/inmunología , Quimiocina CCL22/genética , Quimiocina CCL22/inmunología , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/inmunología , Humanos , Melanoma/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.
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Carcinogénesis/genética , Mutación con Ganancia de Función/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Oncogenes/genética , Línea Celular Tumoral , Humanos , MutaciónRESUMEN
1α,25-Dihydroxyvitamin D3 signals via the vitamin D receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As patients with melanoma commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D-VDR signaling and replicated the findings in The Cancer Genome Atlas metastases. VDR expression was independently protective for melanoma-related death in both primary and metastatic disease. High tumor VDR expression was associated with upregulation of pathways mediating antitumor immunity and corresponding with higher imputed immune cell scores and histologically detected tumor-infiltrating lymphocytes. High VDR-expressing tumors had downregulation of proliferative pathways, notably Wnt/ß-catenin signaling. Deleterious low VDR levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (<25 nmol/L â¼ 10 ng/mL) shortened survival in primary melanoma in a VDR-dependent manner. In vitro functional validation studies showed that elevated vitamin D-VDR signaling inhibited Wnt/ß-catenin signaling genes. Murine melanoma cells overexpressing VDR produced fewer pulmonary metastases than controls in tail-vein metastasis assays. In summary, vitamin D-VDR signaling contributes to controlling pro-proliferative/immunosuppressive Wnt/ß-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of a causal relationship between vitamin D-VDR signaling and melanoma survival, which should be explored as a therapeutic target in primary resistance to checkpoint blockade. SIGNIFICANCE: VDR expression could potentially be used as a biomarker to stratify patients with melanoma that may respond better to immunotherapy.
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Calcitriol/deficiencia , Melanoma/inmunología , Receptores de Calcitriol/metabolismo , Neoplasias Cutáneas/inmunología , Deficiencia de Vitamina D/inmunología , Animales , Calcitriol/sangre , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/sangre , Melanoma/mortalidad , Melanoma/patología , Ratones , Piel/metabolismo , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Factores de Tiempo , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/patología , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Systematic tumour profiling is essential for biomarker research and clinically for assessing response to therapy. Solving the challenge of delivering informative copy number (CN) profiles from formalin-fixed paraffin embedded (FFPE) material, the only likely readily available biospecimen for most cancers, involves successful processing of small quantities of degraded DNA. To investigate the potential for analysis of such lesions, whole-genome CNVseq was applied to 300 FFPE primary tumour samples, obtained from a large-scale epidemiological study of melanoma. The quality and the discriminatory power of CNVseq was assessed. Libraries were successfully generated for 93% of blocks, with input DNA quantity being the only predictor of success (success rate dropped to 65% if <20 ng available); 3% of libraries were dropped because of low sequence alignment rates. Technical replicates showed high reproducibility. Comparison with targeted CN assessment showed consistency with the Next Generation Sequencing (NGS) analysis. We were able to detect and distinguish CN changes with a resolution of ≤10 kb. To demonstrate performance, we report the spectrum of genomic CN alterations (CNAs) detected at 9p21, the major site of CN change in melanoma. This successful analysis of CN in FFPE material using NGS provides proof of principle for intensive examination of population-based samples.
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Variaciones en el Número de Copia de ADN , Neoplasias/genética , Adhesión en Parafina , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias/patología , Reproducibilidad de los Resultados , Fijación del TejidoRESUMEN
PURPOSE: Previously identified transcriptomic signatures have been based on primary and metastatic melanomas with relatively few American Joint Committee on Cancer (AJCC) stage I tumors, given difficulties in sampling small tumors. The advent of adjuvant therapies has highlighted the need for better prognostic and predictive biomarkers, especially for AJCC stage I and stage II disease. EXPERIMENTAL DESIGN: A total of 687 primary melanoma transcriptomes were generated from the Leeds Melanoma Cohort (LMC). The prognostic value of existing signatures across all the AJCC stages was tested. Unsupervised clustering was performed, and the prognostic value of the resultant signature was compared with that of sentinel node biopsy (SNB) and tested as a biomarker in three published immunotherapy datasets. RESULTS: Previous Lund and The Cancer Genome Atlas signatures predicted outcome in the LMC dataset (P = 10-8 to 10-4) but showed a significant interaction with AJCC stage (P = 0.04) and did not predict outcome in stage I tumors (P = 0.3-0.7). Consensus-based classification of the LMC dataset identified six classes that predicted outcome, notably in stage I disease. LMC class was a similar indicator of prognosis when compared with SNB, and it added prognostic value to the genes reported by Gerami and colleagues. One particular LMC class consistently predicted poor outcome in patients receiving immunotherapy in two of three tested datasets. Biological characterization of this class revealed high JUN and AXL expression and evidence of epithelial-to-mesenchymal transition. CONCLUSIONS: A transcriptomic signature of primary melanoma was identified with prognostic value, including in stage I melanoma and in patients undergoing immunotherapy.