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ABSTRACT: Therapeutic vaccination has long been a promising avenue for cancer immunotherapy but is often limited by tumor heterogeneity. The genetic and molecular diversity between patients often results in variation in the antigens present on cancer cell surfaces. As a result, recent research has focused on personalized cancer vaccines. Although promising, this strategy suffers from time-consuming production, high cost, inaccessibility, and targeting of a limited number of tumor antigens. Instead, we explore an antigen-agnostic polymeric in situ cancer vaccination platform for treating blood malignancies, in our model here with acute myeloid leukemia (AML). Rather than immunizing against specific antigens or targeting adjuvant to specific cell-surface markers, this platform leverages a characteristic metabolic and enzymatic dysregulation in cancer cells that produces an excess of free cysteine thiols on their surfaces. These thiols increase in abundance after treatment with cytotoxic agents such as cytarabine, the current standard of care in AML. The resulting free thiols can undergo efficient disulfide exchange with pyridyl disulfide (PDS) moieties on our construct and allow for in situ covalent attachment to cancer cell surfaces and debris. PDS-functionalized monomers are incorporated into a statistical copolymer with pendant mannose groups and TLR7 agonists to target covalently linked antigen and adjuvant to antigen-presenting cells in the liver and spleen after IV administration. There, the compound initiates an anticancer immune response, including T-cell activation and antibody generation, ultimately prolonging survival in cancer-bearing mice.
Assuntos
Cisteína , Leucemia Mieloide Aguda , Humanos , Camundongos , Animais , Cisteína/uso terapêutico , Modelos Animais de Doenças , Leucemia Mieloide Aguda/tratamento farmacológico , Adjuvantes Imunológicos , Antígenos de Neoplasias , Ativação Linfocitária , Dissulfetos/uso terapêuticoRESUMO
Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)-resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.
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Interleucina-12 , Melanoma , Humanos , Interleucina-12/genética , Interleucina-7/farmacologia , Exaustão das Células T , Imunoterapia , Melanoma/patologiaRESUMO
Immune checkpoint immunotherapy (ICI) can re-activate immune reactions against neoantigens, leading to remarkable remission in cancer patients. Nevertheless, only a minority of patients are responsive to ICI, and approaches for prediction of responsiveness are needed to improve the success of cancer treatments. While the tumor mutational burden (TMB) correlates positively with responsiveness and survival of patients undergoing ICI, the influence of the subcellular localizations of the neoantigens remains unclear. Here, we demonstrate in both a mouse melanoma model and human clinical datasets of 1,722 ICI-treated patients that a high proportion of membrane-localized neoantigens, particularly at the plasma membrane, correlate with responsiveness to ICI therapy and improved overall survival across multiple cancer types. We further show that combining membrane localization and TMB analyses can enhance the predictability of cancer patient response to ICI. Our results may have important implications for establishing future clinical guidelines to direct the choice of treatment toward ICI.
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Imunoterapia , Melanoma , Animais , Humanos , Camundongos , Biomarcadores Tumorais/metabolismo , Imunoterapia/métodos , Melanoma/terapiaRESUMO
Immune stimulating agents like Toll-like receptor 7 (TLR7) agonists induce potent antitumor immunity but are limited in their therapeutic window due to off-target immune activation. Here, we developed a polymeric delivery platform that binds excess unpaired cysteines on tumor cell surfaces and debris to adjuvant tumor neoantigens as an in situ vaccine. The metabolic and enzymatic dysregulation in the tumor microenvironment produces these exofacial free thiols, which can undergo efficient disulfide exchange with thiol-reactive pyridyl disulfide moieties upon intratumoral injection. These functional monomers are incorporated into a copolymer with pendant mannose groups and TLR7 agonists to target both antigen and adjuvant to antigen presenting cells. When tethered in the tumor, the polymeric glyco-adjuvant induces a robust antitumor response and prolongs survival of tumor-bearing mice, including in checkpoint-resistant B16F10 melanoma. The construct additionally reduces systemic toxicity associated with clinically relevant small molecule TLR7 agonists.
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Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.
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Proteínas Quinases Dependentes de AMP Cíclico , Proteína de Ligação a Fosfatidiletanolamina , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Humanos , Masculino , Células PC-3 , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Neoplasias da Próstata/metabolismo , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
Immune-checkpoint inhibitors have shown modest efficacy against immunologically 'cold' tumours. Interleukin-12 (IL-12)-a cytokine that promotes the recruitment of immune cells into tumours as well as immune cell activation, also in cold tumours-can cause severe immune-related adverse events in patients. Here, by exploiting the preferential overexpression of proteases in tumours, we show that fusing a domain of the IL-12 receptor to IL-12 via a linker cleavable by tumour-associated proteases largely restricts the pro-inflammatory effects of IL-12 to tumour sites. In mouse models of subcutaneous adenocarcinoma and orthotopic melanoma, masked IL-12 delivered intravenously did not cause systemic IL-12 signalling and eliminated systemic immune-related adverse events, led to potent therapeutic effects via the remodelling of the immune-suppressive microenvironment, and rendered cold tumours responsive to immune-checkpoint inhibition. We also show that masked IL-12 is activated in tumour lysates from patients. Protease-sensitive masking of potent yet toxic cytokines may facilitate their clinical translation.
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Interleucina-12 , Melanoma , Animais , Citocinas , Imunoterapia , Interleucina-12/farmacologia , Camundongos , Peptídeo Hidrolases , Microambiente TumoralRESUMO
The ten-eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AT-hook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2-TET1-HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2-TET1-HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Proteína HMGA2/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxigenases de Função Mista , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNAi(Met) in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNAi(Met) significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.
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Neoplasias da Mama/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Aminoacil-RNA de Transferência/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA de Transferência/genética , TranscriptomaRESUMO
Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.
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Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/análogos & derivados , Neovascularização Patológica/metabolismo , Neoplasias da Próstata/fisiopatologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Western Blotting , Linhagem Celular Tumoral , AMP Cíclico/farmacologia , Ensaio de Imunoadsorção Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/fisiopatologia , Neoplasias da Próstata/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
O presente trabalho consiste em uma pesquisa bibliométrica e bibliográfica, que investigou os resultados do impacto da exposição à violência interparental em crianças. Realizou-se uma busca eletrônica em duas diferentes bases de dados, BVS e Psyc Info, com associação dos descritores "conflito familiar" e "criança" e "domestic violence" com "child", respectivamente. Foram selecionados apenas artigos entre 2005 e 2010, observando-se autor, periódico, metodologia e resultados principais. Dos 211 artigos coletados, foram selecionados 15, destacando país e periódico com mais números de publicações (EUA, 12; Canadian Psychology, Journal of Youth Adolescence e Journal of Family Psychology, dois, respectivamente). Os resultados apontaram que o impacto desse tipo de violência assistida por crianças é evidente no curso de seu desenvolvimento, ocasionando sintomas depressivos e queda no desempenho escolar. Destaca-se a necessidade de novas pesquisas relativas ao tema, cuja produção é ainda incipiente.
This work consists of a bibliometric and bibliographic research which investigated the results of the impact of children witnessing interparental violence. An electronic search was conducted in two different databases, BVS and Psyc Info, with the descriptors "conflito familiar" (family conflict) with "criança" (child) and "domestic violence" with "child", respectively. Only articles written from 2005 and 2010 were selected, taking into account the author, the journal, the methodology, and the main results. From the 211 articles collected, 15 were selected, highlighting country and journal with more publications (USA, 12; Canadian Psychology, Journal of Youth Adolescence and Journal of Family Psychology, two, respectively). The results suggested that the impact of this type of violence observed by children is evident in the course of their development, causing depressive symptoms and decrease of school performance. The need for new researches related to the issue is highlighted, for its production is still incipient.
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Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.
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Fatores de Troca do Nucleotídeo Guanina/metabolismo , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Trombospondinas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Western Blotting , Células Endoteliais/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Camundongos Nus , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
As bactérias Salmonella e Escherichia coli têm sido responsáveis por surtos de doenças transmitidas por alimentos em diversos países. As hortaliças, em especial as alfaces (Lactuca sativa), estão entre os alimentos mais implicados. Durante os meses de agosto a outubro de 2002, foram coletadas e analisadas 42 amostras de alface comercializadas em quatro feiras livres e em um mercado do município de São Luís, MA. As amostras foram submetidas a análises microbiológicas, seguindo as metodologias descritas no APHA (1984) e API-20E, bioMérieux. Os resultados constataram a ausência de Salmonella e a presença de Escherichia coli em 29 amostras. Considerando-se que este tipo de hortaliça é consumido in natura, a presença desta bactéria pode favorecer a ocorrência de toxinoses alimentares.
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Escherichia coli , Contaminação de Alimentos , Incidência , Lactuca , SalmonellaRESUMO
ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.