RESUMO
The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection.
Assuntos
Regulação da Expressão Gênica , Imunidade Inata , Proteínas de Membrana , NF-kappa B , Proteínas Oncogênicas , Proteínas de Ligação a RNA , Animais , Humanos , Sistemas CRISPR-Cas , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Imunidade Inata/genética , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Transdução de Sinais , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologiaRESUMO
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Assuntos
Antígenos de Neoplasias , Neuroblastoma , Proteínas Oncogênicas , Peptídeos , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , África/etnologia , Alelos , Sequência de Aminoácidos , Carcinogênese , Reações Cruzadas , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Neuroblastoma/genética , Neuroblastoma/imunologia , Neuroblastoma/terapia , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/imunologia , Peptídeos/antagonistas & inibidores , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/uso terapêuticoRESUMO
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos HLA/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Oncogênicas/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Antígenos de Neoplasias/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Reações Cruzadas , Apresentação Cruzada , Feminino , Antígenos HLA/metabolismo , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Humanos , Interferon gama/imunologia , Camundongos , Neoplasias/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
Increasing evidence shows that numerous cancer-testis antigens (CTAs) are uniquely overexpressed in various types of cancer and most CTAs are oncogenic. Overexpression of oncogenic CTAs promotes carcinogenesis, cancer metastasis, and drug resistance. Oncogenic CTAs are generally associated with poor prognosis in cancer patients and are an important hallmark of cancer, making them a crucial target for cancer immunotherapy. CTAs-targeted antibodies, vaccines, and chimeric antigen receptor-modified T cells (CAR-T) have recently been used in cancer treatment and achieved promising outcomes in the preclinical and early clinical trials. However, the efficacy of current CTA-targeted therapeutics is either moderate or low in cancer therapy. CTA-targeted cancer immunotherapy is facing enormous challenges. Several critical scientific problems need to be resolved: (1) the antigen presentation function of MHC-I protein is usually deficient in cancer patients, so that very low amounts of intracellular CTA epitopes are presented to tumor cell membrane surface, leading to weak immune response and subsequent immunity to CTAs; (2) various immunosuppressive cells are rich in tumor tissues leading to diminished tumor immunity; (3) the tumor tissue microenvironment markedly reduces the efficacy of cancer immunotherapy. In the current review paper, the authors propose new strategies and approaches to overcome the barriers of CTAs-targeted immunotherapy and to develop novel potent immune therapeutics against cancer. Finally, we highlight that the oncogenic CTAs have high tumor specificity and immunogenicity, and are sensible targets for cancer immunotherapy. We predict that CTAs-targeted immunotherapy will bring about breakthroughs in cancer therapy in the near future.
Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Neoplasias/terapia , Proteínas Oncogênicas/metabolismo , Linfócitos T/transplante , Testículo/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/efeitos adversos , Vacinas Anticâncer/efeitos adversos , Humanos , Imunoterapia Adotiva/efeitos adversos , Masculino , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Testículo/imunologia , Resultado do Tratamento , Microambiente TumoralRESUMO
Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.
Assuntos
Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/imunologia , Ciclina E/imunologia , Ciclinas/imunologia , Antígeno HLA-A2/imunologia , Leucemia/imunologia , Proteínas Oncogênicas/imunologia , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/farmacologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologia , Adulto JovemRESUMO
The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Ictaluridae/imunologia , Proteínas Oncogênicas/imunologia , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Quinase Syk/imunologia , Animais , Linhagem Celular , Fibroblastos , Pseudópodes/imunologia , RatosRESUMO
Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine tumor with a higher mortality rate than melanoma. Approximately 40% of MCC patients have nodal or distant metastasis at initial presentation, and one-third of patients will develop distant metastatic disease over their clinical course. Although MCC is rare, its incidence has been steadily increasing. Furthermore, the immunogenicity of MCC and its diagnostic and therapeutic application have made MCC one of the most rapidly developing topics in dermatology and oncology. Owing to the aggressive and complex nature of MCC, a multidisciplinary approach is necessary for management of this tumor, including dermatologists, surgeons, radiation oncologists, medical oncologists, pathologists, radiologists, and nuclear medicine physicians. Imaging plays a crucial role in diagnosis, planning for surgery or radiation therapy, and assessment of treatment response and surveillance. However, MCC is still not well recognized among radiologists and nuclear medicine physicians, likely owing to its rarity. The purpose of this review is to raise awareness of MCC among imaging experts by describing the epidemiology, pathophysiology, and clinical features of MCC and current clinical management with a focus on the role of imaging. The authors highlight imaging findings characteristic of MCC, as well as the clinical significance of CT, MRI, sentinel lymph node mapping, fluorine 18 fluorodeoxyglucose PET/CT, and other nuclear medicine studies such as bone scintigraphy and somatostatin receptor scintigraphy. ©RSNA, 2019.
Assuntos
Carcinoma de Célula de Merkel/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Anticorpos Antivirais/sangue , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/secundário , Carcinoma de Célula de Merkel/secundário , Carcinoma de Célula de Merkel/virologia , Humanos , Metástase Linfática/diagnóstico por imagem , Poliomavírus das Células de Merkel/isolamento & purificação , Estadiamento de Neoplasias , Proteínas Oncogênicas/imunologia , Infecções por Polyomavirus/diagnóstico por imagem , Infecções por Polyomavirus/virologia , Prognóstico , Compostos Radiofarmacêuticos/análise , Compostos Radiofarmacêuticos/farmacocinética , Receptores de Somatostatina/efeitos dos fármacos , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/diagnóstico por imagem , Infecções Tumorais por Vírus/virologia , Proteínas Virais/imunologiaRESUMO
Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins.
Assuntos
Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Mucoproteínas/imunologia , Mucoproteínas/metabolismo , Proteínas Oncogênicas/imunologia , Proteínas Oncogênicas/metabolismoRESUMO
BACKGROUND: In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). However, the precise mechanisms remain unclear. METHODS: Protein antibody microarray analysis and E3 ligase profiling were performed to detect the unique E3 ligase underlying E-cadherin downregulation in lung adenocarcinoma tissues. Gene knockdown was performed using viral shRNA. Immunoblotting, immunofluorescence, immunoprecipitation, and xenograft models in vivo were integratively applied to explore RNF43-induced EMT in lung adenocarcinoma cell lines. RESULTS: Protein antibody microarray analysis and E3 ligase profiling revealed that the RING finger protein 43 (RNF43) was linked to E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, the c-Src-Caspase-8 interaction markedly increased c-Src activity. Activated c-Src phosphorylated E-cadherin at the tyrosine 797 site to initiate RNF43-mediated E-cadherin ubiquitination at lysine 816 and subsequent degradation, thus allowing the nuclear translocation of ß-catenin and upregulation of Vimentin and RNF43 expression in lung adenocarcinoma cells. Decreased E-cadherin expression and increased Vimentin expression induced the EMT phenotype and promoted tumor metastasis. The Frizzled 8 (Frz8)-RNF43-induced ubiquitination of phosphorylated E-cadherin was blocked by a monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43. CONCLUSIONS: Our data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma through the ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.
Assuntos
Adenocarcinoma de Pulmão/secundário , Antígenos CD/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/patologia , Proteínas Oncogênicas/metabolismo , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Anticorpos Monoclonais , Caspase 8/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Seguimentos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Oncogênicas/imunologia , Fosforilação , Proteólise , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vimentina/metabolismo , beta Catenina/metabolismoRESUMO
Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.
Assuntos
Linfócitos B/imunologia , Proliferação de Células , Histonas/química , Histonas/imunologia , Imunidade Humoral , Lisina/imunologia , Animais , Linfócitos B/química , Linfócitos B/citologia , Ciclo Celular , Células Cultivadas , Ciclina E/genética , Ciclina E/imunologia , Desmetilação , Proteínas F-Box/genética , Proteínas F-Box/imunologia , Histonas/genética , Ferro/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/imunologia , Ativação Linfocitária , Lisina/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Regiões Promotoras Genéticas , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2 O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.
Assuntos
Células da Medula Óssea/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas de Homeodomínio/imunologia , Neutrófilos/imunologia , Proteínas Oncogênicas/imunologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/citologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Transgênicos , Neutrófilos/citologia , Proteínas Oncogênicas/genética , Espécies Reativas de Oxigênio/imunologia , Transdução de Sinais/genéticaRESUMO
Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Fagocitose/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos B/metabolismo , Humanos , Imunoglobulina M/imunologia , Proteínas Oncogênicas/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas rac1 de Ligação ao GTP/imunologiaRESUMO
Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.
Assuntos
Antígenos de Neoplasias/genética , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Fusão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/imunologia , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Sequenciamento Completo do GenomaRESUMO
The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKß. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.
Assuntos
Quinase I-kappa B/imunologia , Imunidade Inata , Proteínas Oncogênicas/imunologia , Peptídeo Sintases/imunologia , Transdução de Sinais/imunologia , Animais , Células HEK293 , Humanos , Quinase I-kappa B/genética , Camundongos , Proteína NEDD8/genética , Proteína NEDD8/imunologia , Proteínas Oncogênicas/genética , Peptídeo Sintases/genética , Fosforilação/genética , Fosforilação/imunologia , Células RAW 264.7 , Transdução de Sinais/genéticaRESUMO
Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of diverse human carcinomas and is an attractive target for the development of mAb-based therapeutics. However, attempts at targeting the shed MUC1 N-terminal subunit have been unsuccessful. We report here the generation of mAb 3D1 against the nonshed oncogenic MUC1 C-terminal (MUC1-C) subunit. We show that mAb 3D1 binds with low nM affinity to the MUC1-C extracellular domain at the restricted α3 helix. mAb 3D1 reactivity is selective for MUC1-C-expressing human cancer cell lines and primary cancer cells. Internalization of mAb 3D1 into cancer cells further supported the conjugation of mAb 3D1 to monomethyl auristatin E (MMAE). The mAb 3D1-MMAE antibody-drug conjugate (ADC) (a) kills MUC1-C-positive cells in vitro, (b) is nontoxic in MUC1-transgenic (MUC1.Tg) mice, and (c) is active against human HCC827 lung tumor xenografts. Humanized mAb (humAb) 3D1 conjugated to MMAE also exhibited antitumor activity in (a) MUC1.Tg mice harboring syngeneic MC-38/MUC1 tumors, (b) nude mice bearing human ZR-75-1 breast tumors, and (c) NCG mice engrafted with a patient-derived triple-negative breast cancer. These findings and the absence of associated toxicities support clinical development of humAb 3D1-MMAE ADCs as a therapeutic for the many cancers with MUC1-C overexpression.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoconjugados/farmacologia , Mucina-1/imunologia , Proteínas Oncogênicas/imunologia , Animais , Anticorpos Monoclonais/química , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Modelos Moleculares , Mucina-1/química , Oligopeptídeos , Proteínas Oncogênicas/química , Conformação Proteica em alfa-Hélice , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer vaccines have been developed as a new therapeutic approach, however, their clinical benefit remains limited. We previously performed a phase II study for advanced colorectal cancer (CRC) using five human leukocyte antigen (HLA-A*24:02)-restricted peptides derived from kinase of the outer chloroplast membrane 1, translocase of outer mitochondrial membrane 34 (TOMM34), ring finger protein 43 (RNF43), vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2. In the present study the relationship between overall survival (OS) and several biomarkers, including cytotoxic T lymphocyte (CTL) and immunoglobulin G (IgG) responses to these five peptides, was investigated. In 89 advanced CRC patients treated with a combination therapy consisting of these five peptides and oxaliplatin-based chemotherapy, plasma was collected before and after 3 months of vaccine administration. IgGs reactive to each of the five peptides were assessed using the multiplex bead suspension Luminex system. Antigen-specific T-cell responses were estimated by enzyme-linked immunoSpot assay. Plasma levels of TOMM34 IgG (P<0.001), RNF43 IgG (P<0.001) and VEGFR2 IgG (P<0.001) were significantly increased after vaccination and stronger VEGFR2 IgG responses correlated significantly with OS in HLA-matched patients (P=0.034). CTL responses to VEGFR1 and VEGFR2 were also significantly increased in the HLA-matched group (P=0.049 and P<0.001, respectively). However, increased CTL response did not correlate with OS. Multivariate analysis indicated that IgG responses to VEGFR2 were the most significant predictor for OS in the HLA-A*24:02-matched group (P=0.04). Our findings indicated that VEGFR2 IgG responses may be an important immunological biomarker in the early course of treatment for CRC patients treated with therapeutic epitope peptides.
Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Antígeno HLA-A24/imunologia , Imunoglobulina G/metabolismo , Idoso , Vacinas Anticâncer/imunologia , Neoplasias Colorretais/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/imunologia , Método Duplo-Cego , Epitopos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/imunologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Oncogênicas/química , Proteínas Oncogênicas/imunologia , Análise de Sobrevida , Resultado do Tratamento , Ubiquitina-Proteína Ligases , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
We previously reported a phase I clinical trial of a peptide vaccine ring finger protein 43 (RNF43) and 34-kDa translocase of the outer mitochondrial membrane (TOMM34) combined with uracil-tegafur (UFT)/LV for patients with metastatic colorectal cancer (CRC), and demonstrated the safety and immunological responsiveness of this combination therapy. In this study, we evaluated vaccination-induced immune responses to clarify the survival benefit of the combination therapy as adjuvant treatment. We enrolled 44 patients initially in an HLA-masked fashion. After the disclosure of HLA, 28 patients were in the HLA-A*2402-matched and 16 were in the unmatched group. In the HLA-matched group, 14 patients had positive CTL responses specific for the RNF43 and/or TOMM34 peptides after 2 cycles of treatment and 9 had negative responses; in the HLA-unmatched group, 10 CTL responses were positive and 2 negative. In the HLA-matched group, 3-year relapse-free survival (RFS) was significantly better in the positive CTL subgroup than in the negative-response subgroup. Patients with negative vaccination-induced CTL responses showed a significant trend towards shorter RFS than those with positive responses. Moreover, in the HLA-unmatched group, the positive CTL response subgroup showed an equally good 3-year RFS as in the HLA-matched group. In conclusion, vaccination-induced CTL response to peptide vaccination could predict survival in the adjuvant setting for stage III CRC.
Assuntos
Neoplasias Colorretais/mortalidade , Proteínas de Ligação a DNA/imunologia , Proteínas de Transporte da Membrana Mitocondrial/imunologia , Proteínas Oncogênicas/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinação , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Método Duplo-Cego , Feminino , Antígeno HLA-A24/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Estadiamento de Neoplasias , Ubiquitina-Proteína LigasesRESUMO
OBJECTIVE: The nuclear oncoprotein DEK is an autoantigen associated with juvenile idiopathic arthritis (JIA), especially the oligoarticular subtype. DEK is a secreted chemotactic factor. Abundant levels of DEK and DEK autoantibodies are found in inflamed synovium in JIA. We undertook this study to further characterize the nature of DEK autoantibodies in screening serum samples from 2 different cohorts that consisted mostly of patients with JIA. METHODS: DEK autoantibody levels were analyzed in sera from 33 JIA patients, 13 patients with other inflammatory conditions, and 11 healthy controls, as well as in 89 serum samples from JIA patients receiving anti-tumor necrosis factor (anti-TNF) therapy. Recombinant His-tagged full-length DEK protein (1-375 amino acids [aa]) and the 187-375-aa and 1-350-aa His-tagged DEK fragments made in a baculovirus system were used for enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The C-terminal 25-aa fragment of DEK was expressed in a glutathione S-transferase-tagged vector. ELISA results were calculated as area under the curve by the trapezoidal rule. RESULTS: DEK autoantibody levels were significantly higher in patients with polyarticular JIA than in those with oligoarticular JIA, and were higher in patients with polyarticular JIA who had more active disease after cessation of anti-TNF therapy. Immunoblotting against the C-terminal 25-aa fragment of DEK confirmed that this section of the DEK molecule is the most immunogenic domain. CONCLUSION: DEK autoantibody levels are higher in patients with polyarticular JIA than in those with oligoarticular JIA, and higher in patients who have disease flares after cessation of anti-TNF therapy. The C-terminal 25-aa fragment is the most immunogenic portion of DEK. These findings are significant with respect to the nature of DEK autoantibodies, their contribution to JIA pathogenesis, and their implications for JIA management.
Assuntos
Antirreumáticos/imunologia , Artrite Juvenil/sangue , Autoanticorpos/sangue , Proteínas Cromossômicas não Histona/imunologia , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adolescente , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Exacerbação dos Sintomas , Suspensão de TratamentoRESUMO
There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.
Assuntos
Antineoplásicos Imunológicos/imunologia , Mutação , Proteínas Oncogênicas/imunologia , Proteínas ras/imunologia , Animais , Antineoplásicos Imunológicos/análise , Linhagem Celular Tumoral , Fibroblastos , Humanos , Hibridomas , Camundongos , Proteínas Oncogênicas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno , Proteínas ras/genéticaRESUMO
Adaptor proteins non-catalytic region of tyrosine kinase (NCK) and Abelson interactor (ABI) are crucial for disease response. NCK1 was identified to be a candidate gene for enteric septicemia of catfish (ESC) disease resistance, and was speculated to play similar roles during ESC and enteropathogenic Escherichia coli (EPEC) pathogenicity. ABI1 was reported as a positional candidate gene for bacterial cold water disease (BCWD) resistance in rainbow trout. In this study, three NCK genes and six ABI genes were identified in the channel catfish (Ictalurus punctatus) genome and blue catfish (I. furcatus) transcriptome, and annotated by domain structures, phylogenetic and syntenic analyses. Their expression patterns were examined in the intestine and liver of catfish after challenge with Edwardsiella ictaluri. In the intestine, NCK1, ABI2a, ABI2b, ABI3a were differentially expressed after E. ictaluri infection. In the liver, NCK2a, NCK2b, ABI1b, ABI2a, ABI2b were significantly upregulated in ESC susceptible fish. In general, the NCK and ABI genes, with exception of ABI3a gene and NCK1 gene, were expressed at higher levels in susceptible fish after infection than in control fish, but were expressed at lower levels in resistant fish than in the control fish. Taken together, these results support the notion that NCK and ABI genes are involved in disease processes facilitating pathogenesis of the E. ictaluri bacteria.