RESUMO
Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.
Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais , Proteínas de Membrana , Mesotelioma Maligno , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Proteínas de Membrana/agonistas , Receptores de Antígenos QuiméricosRESUMO
While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate upregulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+ T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interleukin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Glioma/terapia , Imunoterapia , Antígeno MART-1/metabolismo , Melanoma/terapia , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Técnicas de Cocultura , DNA Topoisomerases/metabolismo , Daunorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/imunologia , Glioma/patologia , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Antígeno MART-1/genética , Antígeno MART-1/imunologia , Melanoma/imunologia , Melanoma/patologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transgenes/genéticaRESUMO
Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy.
Assuntos
Antígenos de Neoplasias/metabolismo , Sistema de Sinalização das MAP Quinases , Melanócitos/metabolismo , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Transcrição Gênica , Butadienos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Antígeno MART-1 , Fator de Transcrição Associado à Microftalmia/fisiologia , Nitrilas/farmacologia , Compostos Orgânicos/farmacologia , Polimorfismo de Fragmento de Restrição , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linfócitos T Citotóxicos/fisiologia , TransfecçãoRESUMO
In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Melanoma Experimental/patologia , Antígenos Específicos de Melanoma/genética , Linfócitos T/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoterapia , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Mutação , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Melanoma/imunologia , Linfócitos T/imunologia , Especificidade de Anticorpos , Antígenos de Neoplasias/biossíntese , Linhagem Celular Tumoral , Clonagem Molecular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genes Reporter/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoterapia , Células Jurkat , Lentivirus/genética , Antígeno MART-1/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines. We have now identified IFN-beta as an additional stimulus to TAA expression, including Melan-A/MART-1, gp100, and MAGE-A1. IFN-beta (but neither IFN-alpha nor IFN-gamma) augmented both protein and mRNA expression of melanocytic TAA in 15 melanoma lines (irrespective of initial Ag-expression levels). Treatment of low Ag melanoma lines with IFN-beta increased expression of melanocyte-lineage Ags, inducing susceptibility to lysis by specific CTLs. Treatment with IFN-beta also enhances expression of class I HLA molecules, thereby inducing both nominal TAA and the presenting HLA molecule. Data from fluorescent cellular reporter systems demonstrated that IFN-beta triggers promoter activation, resulting in augmentation of Ag expression. In addition to enhancing TAA expression in melanomas, IFN-beta also stimulated expression of the melanocytic Ag gp100 in cells of other neural crest-derived tumor lines (gliomas) and certain unrelated tumors. Because IFN-beta is already approved for human clinical use in other contexts, it may prove useful as a cotreatment for augmenting tumor Ag expression during immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/imunologia , Interferon beta/farmacologia , Melanoma/imunologia , Evasão Tumoral/imunologia , Antígenos de Neoplasias/biossíntese , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/imunologia , Glioma/metabolismo , Glioma/terapia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia , Interferon beta/imunologia , Interferon beta/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Melanoma/metabolismo , Melanoma/terapia , RNA Mensageiro/biossíntese , RNA Mensageiro/imunologia , RNA Neoplásico/biossíntese , RNA Neoplásico/imunologia , Evasão Tumoral/efeitos dos fármacosRESUMO
Defining the hardwiring of transcription factors to their cognate genomic binding sites is essential for our understanding of biological processes. We used scanning chromatin immunoprecipitation to identify in vivo binding regions (E boxes) for c-Myc in three target genes as a model system. Along with other c-Myc target genes that have been validated by chromatin immunoprecipitation, we used the publicly available genomic sequences to determine whether experimentally derived in vivo binding sites might be predictable from nonexonic sequence conservation across species. Our studies revealed two classes of target genomic binding sites. Although the majority of target genes studied [class I: B23 (NPM1), CAD, CDK4, cyclin D2, ID2, LDH-A, MNT, PTMa, ODC, NM23B, nucleolin, prohibitin, SHMT1, and SHMT2] demonstrate significant sequence conservation of the E boxes and flanking regions, several genes (cyclin B1, JPO1, and PRDX3) belong to a second class (class II) that does not display sequence conservation at and around the site of c-Myc binding. On the basis of our model, we propose a strategy for predicting transcription factor binding sites using phylogenetic sequence comparisons, which will select potential class I target genes among the many emerging candidates from DNA-microarray studies for experimental validation by chromatin immunoprecipitation.