RESUMO
Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.
Assuntos
Antígenos CD/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Melanoma/imunologia , Doença Aguda , Antígenos CD/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Melanoma/genética , Melanoma/patologia , Melanoma/terapia , Mutação , Metástase Neoplásica , Receptores de Orexina , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.
Assuntos
Embaralhamento de DNA , Dependovirus/genética , Vetores Genéticos/isolamento & purificação , Genoma Viral/genética , Nanopartículas , Animais , Anticorpos/imunologia , Encéfalo/metabolismo , Capsídeo/imunologia , Cricetinae , Dependovirus/ultraestrutura , Evolução Molecular Direcionada , Biblioteca Gênica , Vetores Genéticos/genética , Humanos , Fígado/metabolismo , Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/metabolismo , Primatas , Transdução Genética , Internalização do VírusRESUMO
We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.