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Redirection of tumor-associated macrophages to eliminate tumor cells holds great promise for overcoming therapeutic resistance to rituximab and other antibody drugs. Here, we determined the expression of ectonucleotidases CD39 and CD73 in diffuse large B-cell lymphoma (DLBCL), and examined the impact of extracellular ATP (eATP) metabolism on macrophage-mediated anti-lymphoma immunity. Immunostaining of tissue microarray samples showed that CD39 (the ecto-enzyme for eATP hydrolysis) was highly expressed in tumors with the non-germinal center B-cell-like (non-GCB) subtype, and to a lesser extent tumors with the GCB subtype. By contrast, the expression of CD73 (the ecto-enzyme for adenosine generation) was undetectable in tumor cells. Pharmacological blockade of CD39 prevented eATP degradation and enhanced engulfment of antibody-coated lymphoma cells by macrophages in a P2X7 receptor-dependent manner, indicating that eATP fueled antibody-dependent cellular phagocytosis (ADCP) activity. Importantly, inhibition of CD39 augmented in vivo anti-lymphoma effects by therapeutic antibodies including rituximab and daratumumab. Furthermore, the addition of a CD39 inhibitor to anti-CD20 and anti-CD47 combination therapy significantly improved survival in a disseminated model of aggressive B-cell lymphoma, supporting the benefit of dual targeting CD39-mediated eATP hydrolysis and CD47-mediated "don't eat me" signal. Together, preventing eATP degradation may be a potential approach to unleash macrophage-mediated anti-lymphoma immunity.
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Linfoma de Células B Grandes Difuso , Macrófagos , Humanos , Rituximab/farmacología , Rituximab/uso terapéutico , Adenosina/metabolismo , Linfoma de Células B Grandes Difuso/patología , FagocitosisRESUMEN
The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.
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Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores Virales/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Inmunoterapia/métodos , Células Jurkat , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE: Resistance to anti-PD1-based immune checkpoint blockade (ICB) remains a problem for the treatment of metastatic melanoma. Tumor cells as well as host myeloid cells can express the immune checkpoint ligand CD155 to regulate immune cell function. However, the effect of tumor CD155 on the immune context of human melanoma has not been well described. This observational study characterizes tumor CD155 ligand expression by metastatic melanoma tumors and correlates results with differences in immune cell features and response to ICB. EXPERIMENTAL DESIGN: Pretreatment tumor specimens, from 155 patients with metastatic melanoma treated with ICB and from 50 patients treated with BRAF/MEK-directed targeted therapy, were assessed for CD155 expression by IHC. Intratumor T-cell features were analyzed using multiplex-immunohistofluorescence for CD8, PD1, and SOX10. Correlations were made between CD155 tumor level and bulk tumor RNA sequencing results, as well as clinical RECIST response and progression-free survival. RESULTS: High pretreatment CD155 tumor levels correlated with high parenchymal PD1+CD8+/CD8+ T-cell ratios (PD1tR) and poor response to anti-PD1 therapy. In PDL1 negative tumors, high CD155 tumor expression was associated with patients who had poor response to combination anti-PD1/CTLA4 therapy. CONCLUSIONS: Our findings are the first to suggest that tumor CD155 supports an increase in the fraction of PD1+CD8+ T cells in anti-PD1 refractory melanoma tumors and, further, that targeting the CD155 pathway might improve response to anti-PD1 therapy for patients with metastatic melanoma.
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Regulación Neoplásica de la Expresión Génica/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Melanoma/tratamiento farmacológico , Receptores Virales/genética , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Biopsia , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Melanoma/genética , Melanoma/mortalidad , Melanoma/secundario , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Supervivencia sin Progresión , Estudios Prospectivos , RNA-Seq , Criterios de Evaluación de Respuesta en Tumores Sólidos , Estudios Retrospectivos , Piel/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patologíaRESUMEN
Nicotinamide (NAM), an amide form of vitamin B3, replenishes cellular energy after ultraviolet radiation (UVR) exposure, thereby enhancing DNA repair and reducing UVR's immunosuppressive effects. NAM reduces actinic keratoses and new keratinocyte cancers in high risk individuals, but its effects on melanoma are unknown. Melanomas arising on NAM or placebo within the ONTRAC skin cancer chemoprevention trial (Oral Nicotinamide To Reduce Actinic Cancer) were examined by immunohistochemistry. The effects of NAM (50 µM, 5 mM and 20 mM) on the viability, proliferation and invasiveness of four human melanoma cell lines and on the viability and proliferation of two human melanocyte lines, with and without UV irradiation were also investigated. 50 µM NAM did not affect viability, proliferation or invasion of melanoma or melanocyte cell lines, whereas concentrations too high to be achievable in vivo reduced viability and proliferation. Nicotinamide did not enhance melanoma viability, proliferation or invasiveness in vitro, providing additional confidence in its safety for use in clinical trials in high risk patients. Peritumoral and tumour infiltrating CD4+ and CD8+ lymphocytes were significantly increased in melanomas arising on NAM compared to those arising on placebo. Given the chemopreventive activity of nicotinamide against keratinocyte cancers, its DNA repair enhancing effects in melanocytes and now its potential enhancement of tumour-infiltrating lymphocytes and lack of adverse effects on melanoma cell growth and proliferation, clinical trials of nicotinamide for melanoma chemoprevention are now indicated.
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Melanoma/patología , Niacinamida/farmacología , Neoplasias Cutáneas/patología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/prevención & control , Niacinamida/química , Niacinamida/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Rayos UltravioletaRESUMEN
CD155 (PVR/necl5/Tage4), a member of the nectin-like family of adhesion molecules, is highly upregulated on tumor cells across multiple cancer types and has been associated with worse patient outcomes. In addition to well described cell-intrinsic roles promoting tumor progression and metastasis, CD155 has now been implicated in immune regulation. The role of CD155 as a potent immune ligand with diverse cell-extrinsic functions is now being defined. CD155 signaling to immune cells is mediated through interactions with the co-stimulatory immune receptor CD226 (DNAM-1) and the inhibitory checkpoint receptors TIGIT and CD96, which are differentially regulated at the cell surface on T cells and NK cells. The integration of signals from CD155 cognate receptors modifies the activity of tumor-infiltrating lymphocytes in a context-dependent manner, making CD155 an attractive target for immune-oncology. Preclinical studies suggest that targeting this axis can improve immune-mediated tumor control, particularly when combined with existing anti-PD-1 checkpoint therapies. In this review, we discuss the roles of CD155 on host and tumor cells in controlling tumor progression and discuss the possibility of targeting CD155 for cancer therapy.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inmunidad/genética , Neoplasias/inmunología , Receptores Virales/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunidad/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Receptores Virales/inmunología , Linfocitos T/inmunologíaRESUMEN
We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinflammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3-inflammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Efficacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the significant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infiltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specific T-cell transfer. SIGNIFICANCE: Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-inflammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the efficacy of adoptive T-cell transfer.This article is highlighted in the In This Issue feature, p. 1631.
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Adenosina Trifosfato/metabolismo , Antineoplásicos Inmunológicos/administración & dosificación , Apirasa/antagonistas & inhibidores , Inflamasomas/metabolismo , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Neoplasias/inmunología , Receptores Purinérgicos P2X7/metabolismo , Transducción de SeñalRESUMEN
Adjuvant immunotherapies targeting CTLA4 or PD-1 recently demonstrated efficacy in the treatment of earlier stages of human cancer. We previously demonstrated using mouse spontaneous metastasis models that neoadjuvant immunotherapy and surgery was superior, compared to surgery and adjuvant immunotherapy, in eradicating the lethal metastatic disease. However, the optimal scheduling between neoadjuvant immunotherapy and surgery and how it impacts on efficacy and development of immune-related adverse events (irAEs) remains undefined. Using orthotopic 4T1.2 and E0771 mouse models of spontaneously metastatic mammary cancer, we varied the schedule and duration of neoadjuvant immunotherapies and surgery and examined how it impacted on long-term survival. In two tumor models, we demonstrated that a short duration (4-5 days) between first administration of neoadjuvant immunotherapy and resection of the primary tumor was necessary for optimal efficacy, while extending this duration (10 days) abrogated immunotherapy efficacy. However, efficacy was also lost if neoadjuvant immunotherapy was given too close to surgery (2 days). Interestingly, an additional 4 adjuvant doses of treatment following a standard 2 doses of neoadjuvant immunotherapy, did not significantly improve overall tumor-free survival regardless of the combination treatment (anti-PD-1+anti-CD137 or anti-CTLA4+anti-PD-1). Furthermore, biochemical immune-related adverse events (irAEs) increased in tumor-bearing mice that received the additional adjuvant immunotherapy. Overall, our data suggest that shorter doses of neoadjuvant immunotherapy scheduled close to the time of surgery may optimize effective anti-tumor immunity and reduce severe irAEs.
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CD96 is a novel target for cancer immunotherapy shown to regulate NK cell effector function and metastasis. Here, we demonstrated that blocking CD96 suppressed primary tumor growth in a number of experimental mouse tumor models in a CD8+ T cell-dependent manner. DNAM-1/CD226, Batf3, IL12p35, and IFNγ were also critical, and CD96-deficient CD8+ T cells promoted greater tumor control than CD96-sufficient CD8+ T cells. The antitumor activity of anti-CD96 therapy was independent of Fc-mediated effector function and was more effective in dual combination with blockade of a number of immune checkpoints, including PD-1, PD-L1, TIGIT, and CTLA-4. We consistently observed coexpression of PD-1 with CD96 on CD8+ T lymphocytes in tumor-infiltrating leukocytes both in mouse and human cancers using mRNA analysis, flow cytometry, and multiplex IHF. The combination of anti-CD96 with anti-PD-1 increased the percentage of IFNγ-expressing CD8+ T lymphocytes. Addition of anti-CD96 to anti-PD-1 and anti-TIGIT resulted in superior antitumor responses, regardless of the ability of the anti-TIGIT isotype to engage FcR. The optimal triple combination was also dependent upon CD8+ T cells and IFNγ. Overall, these data demonstrate that CD96 is an immune checkpoint on CD8+ T cells and that blocking CD96 in combination with other immune-checkpoint inhibitors is a strategy to enhance T-cell activity and suppress tumor growth.
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Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Traslado Adoptivo , Animales , Antígenos CD/genética , Línea Celular Tumoral , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/terapiaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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Purpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.
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Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMES+CD69+CD45RO+ effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno CTLA-4/antagonistas & inhibidores , Ipilimumab/administración & dosificación , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Melanoma/tratamiento farmacológico , Nivolumab/administración & dosificación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Anciano , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígeno CTLA-4/inmunología , Resistencia a Antineoplásicos , Femenino , Humanos , Memoria Inmunológica/efectos de los fármacos , Lectinas Tipo C/inmunología , Antígenos Comunes de Leucocito/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Fenotipo , Receptor de Muerte Celular Programada 1/inmunología , Estudios Retrospectivos , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/inmunología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
AIMS: Indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme, facilitates immune escape by tumours and promotes tumour progression. IDO inhibitors with and without additional anti-PD-1 therapy have been evaluated in recent and ongoing melanoma clinical trials, but IDO expression in melanoma tumours, and therefore its potential role as a predictive biomarker remains unknown. This study sought to evaluate IDO expression in immunotherapy-naive metastatic melanoma patients in order to determine patterns of expression in corresponding primary melanomas, locoregional metastases and distant metastases. METHODS AND RESULTS: Here, we evaluated IDO expression using immunohistochemistry in 99 melanoma tumour samples from 43 immunotherapy-naive patients with metastatic melanoma to determine patterns of expression in primary melanomas (n = 29), locoregional metastases (n = 36) and distant metastases (n = 34). Thirty-seven per cent of patients demonstrated tumour IDO expression in at least one specimen. Twelve of 35 patients (34%) with longitudinal specimens (i.e. two or more separate specimens from different disease stages in the same patient) displayed heterogeneous IDO staining between samples. Tumour IDO expression positively correlated with tumour-infiltrating lymphocyte (TIL) score as well as the number of IDO-expressing mononuclear cells in the primary melanoma (P < 0.0001 and P = 0.0011, respectively) and nodal metastases (P = 0.049 and P = 0.037, respectively), but not in distant metastases. Furthermore, tumour IDO expression correlated positively with PD-L1 expression by melanoma cells among all specimens (P = 0.0073). CONCLUSIONS: Therefore, while assessment of tumour IDO expression warrants evaluation in melanoma patient cohorts treated with IDO inhibitors dosed at levels proven to inhibit the target by pharmacodynamic assessment, its utility as a biomarker may be limited by intertumoral heterogeneity.
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Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Melanoma/enzimología , Neoplasias Cutáneas/enzimología , Microambiente Tumoral , Adulto , Anciano , Biomarcadores de Tumor/análisis , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/análisis , Masculino , Persona de Mediana Edad , Melanoma Cutáneo MalignoAsunto(s)
Carcinoma Basocelular/prevención & control , Carcinoma de Células Escamosas/prevención & control , Macrófagos/efectos de los fármacos , Niacinamida/farmacología , Neoplasias Cutáneas/prevención & control , Carcinogénesis/efectos de los fármacos , Carcinoma Basocelular/epidemiología , Carcinoma Basocelular/inmunología , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/inmunología , Recuento de Células , Humanos , Incidencia , Macrófagos/inmunología , Niacinamida/uso terapéutico , Piel/citología , Piel/inmunología , Piel/patología , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/inmunologíaRESUMEN
Purpose: Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response.Patients and Methods: We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti-PD-1 therapy.Results: Increased numbers of CD69+CD103+ tumor-resident CD8+ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti-PD-1 immunotherapy.Conclusions: Tumor-resident CD8+ T-cell numbers are more prognostic than total CD8+ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti-PD-1 and anti-LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. Clin Cancer Res; 24(13); 3036-45. ©2018 AACR.
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Antígenos CD/metabolismo , Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Cadenas alfa de Integrinas/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Antígenos CD/genética , Biomarcadores de Tumor , Biopsia , Linfocitos T CD8-positivos/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunofenotipificación , Cadenas alfa de Integrinas/genética , Interleucina-15/genética , Interleucina-15/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/mortalidad , Melanoma/terapia , Terapia Molecular Dirigida , PronósticoRESUMEN
Analysis of 501 melanoma exomes revealed RGS7, which encodes a GTPase-accelerating protein (GAP), to be a tumor-suppressor gene. RGS7 was mutated in 11% of melanomas and was found to harbor three recurrent mutations (p.R44C, p.E383K and p.R416Q). Structural modeling of the most common recurrent mutation of the three (p.R44C) predicted that it destabilizes the protein due to the loss of an H-bond and salt bridge network between the mutated position and the serine and aspartic acid residues at positions 58 as 61, respectively. We experimentally confirmed this prediction showing that the p.R44C mutant protein is indeed destabilized. We further show RGS7 p.R44C has weaker catalytic activity for its substrate Gαo, thus providing a dual mechanism for its loss of function. Both of these effects are expected to contribute to loss of function of RGS7 resulting in increased anchorage-independent growth, migration and invasion of melanoma cells. By mutating position 56 in the R44C mutant from valine to cysteine, thereby enabling the formation of a disulfide bridge between the two mutated positions, we slightly increased the catalytic activity and reinstated protein stability, leading to the rescue of RGS7's function as a tumor suppressor. Our findings identify RGS7 as a novel melanoma driver and point to the clinical relevance of using strategies to stabilize the protein and, thereby, restore its function.
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Melanoma/genética , Mutación , Proteínas RGS/química , Proteínas RGS/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Disulfuros/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Enlace de Hidrógeno , Melanoma/metabolismo , Modelos Moleculares , Invasividad Neoplásica , Conformación Proteica , Estabilidad Proteica , Proteínas RGS/genéticaRESUMEN
Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Sinergismo Farmacológico , Xenoinjertos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Melanoma/enzimología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Transducción de Señal/efectos de los fármacosRESUMEN
Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAFV600E melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73+ BRAFV600E-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. Cancer Res; 77(17); 4684-96. ©2017 AACR.
Asunto(s)
Adenosina/metabolismo , Neoplasias Pulmonares/prevención & control , Melanoma/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Receptor de Adenosina A2A/química , Neoplasias Cutáneas/prevención & control , 5'-Nucleotidasa/metabolismo , Animales , Quimioterapia Combinada , Proteínas Ligadas a GPI/metabolismo , Humanos , Imidazoles/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Mutación/genética , Oximas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Receptor de Adenosina A2A/fisiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/secundarioRESUMEN
Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work substantiates CD73 as a target to combine with current immunotherapies, but its dynamic regulation suggests limited value of CD73 pretreatment expression as a biomarker to stratify melanoma patients. Cancer Res; 77(17); 4697-709. ©2017 AACR.
Asunto(s)
5'-Nucleotidasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Inmunoterapia , Inflamación/complicaciones , Melanoma/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Linfocitos T/trasplante , Adenosina/metabolismo , Traslado Adoptivo , Animales , Proteínas Ligadas a GPI/metabolismo , Humanos , Inflamación/patología , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Pronóstico , Estudios Retrospectivos , Factor de Transcripción AP-1/metabolismo , Células Tumorales CultivadasRESUMEN
INTRODUCTION: The upregulation of programmed death ligand 1 (PD-L1) is found in many cancers and contributes to evasion of the host's immune defense. In malignant pleural mesothelioma (MPM), PD-L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD-L1 expression in MPM are poorly understood. To address one possible means of PD-L1 regulation we investigated the relationship between dysregulated microRNA levels and PD-L1 expression. METHODS: PD-L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse-transcriptase quantitative polymerase chain reaction. Regulation of PD-L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD-L1 was analyzed by using argonaute-2 immunoprecipitation and a luciferase reporter assay. RESULTS: In a series of 72 patients with MPM, 18 (25%) had positive PD-L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD-L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD-L1 expression [p < 0.001]), and in multivariate analyses, PD-L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2-4.1, p < 0.01). In the same patient series, PD-L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR-15b, miR-16, miR-193a-3p, miR-195, and miR-200c were significantly lower in the PD-L1-positive samples. Transfecting MPM cell lines with mimics corresponding to miR-15a and miR-16, both of which are predicted to target PD-L1, led to downregulation of PD-L1 mRNA and protein. In addition, miR-193a-3p, with an alternative G-U-containing target site, also caused PD-L1 downregulation. CONCLUSIONS: Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD-L1 expression in MPM.