RESUMO
CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Complexo Ferro-Dextran , Imunoterapia Adotiva , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.
Assuntos
Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Biomarcadores , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno , Gradação de Tumores , Metástase Neoplásica , Fenótipo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Carga Tumoral , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Reparo do DNA/efeitos da radiação , Desoxicitidina Quinase/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Desoxirribonucleosídeos/metabolismo , Instabilidade Genômica , Hematopoese/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Linfócitos T/citologia , Linfócitos T/fisiologiaRESUMO
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
Assuntos
Desoxicitidina Quinase/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/química , Arabinofuranosiluracila/metabolismo , Western Blotting , Linhagem Celular Tumoral , Desoxicitidina Quinase/genética , Feminino , Radioisótopos de Flúor/química , Células-Tronco Hematopoéticas/metabolismo , Imuno-Histoquímica , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Estimativa de Kaplan-Meier , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Endogâmicos , Camundongos Knockout , Camundongos SCID , Mutação , Timo/diagnóstico por imagem , Timo/metabolismo , Fatores de Tempo , Transplante HeterólogoRESUMO
The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronic-activated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.
Assuntos
Neoplasias Hormônio-Dependentes/etiologia , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/etiologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Sequência de Bases , Carcinógenos/metabolismo , Primers do DNA/genética , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Comunicação Parácrina , Neoplasia Prostática Intraepitelial/etiologia , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Clinical tools that measure changes in immune cell metabolism would improve the diagnosis and treatment of immune dysfunction. PET, utilizing probes for specific metabolic processes, detects regions of immune activation in vivo. In this study we investigated the immune cell specificity of PET probes for two different metabolic pathways: [18F]-2-fluorodeoxyglucose ([18F]-FDG) for glycolysis and [18F]-2-fluoro-D-(arabinofuranosyl)cytosine ([18F]-FAC) for deoxycytidine salvage. We isolated innate and adaptive immune cells from tissues of mice challenged with a retrovirus-induced sarcoma and measured their ability to accumulate FDG and FAC. We determined that the two probes had distinct patterns of accumulation: FDG accumulated to the highest levels in innate immune cells, while FAC accumulated predominantly in CD8+ T cells in a manner that correlated with cellular proliferation. This study demonstrates that innate and adaptive cell types differ in glycolytic and deoxycytidine salvage demands during an immune response and that these differential metabolic requirements can be detected with specific PET probes. Our findings have implications for the interpretation of clinical PET scans that use [18F]-FDG or [18F]-FAC to assess immune function in vivo and suggest potential applications of metabolic PET to monitor the effects of targeted immune modulation.
Assuntos
Células/imunologia , Células/metabolismo , Fenômenos do Sistema Imunitário , Redes e Vias Metabólicas , Tomografia por Emissão de Pósitrons/métodos , Animais , Proliferação de Células , Estruturas Celulares , Fluordesoxiglucose F18 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Sensibilidade e EspecificidadeRESUMO
G2A is a G-protein-coupled receptor (GPCR) involved in immune regulation. Previous studies have shown that lysophosphatidylcholine (LPC), a bioactive lipid associated with atherosclerosis and autoimmunity, acts through G2A to induce diverse biologic effects. Production of LPC during cell apoptosis serves as a chemotactic signal for macrophage recruitment. Here we demonstrate that macrophage chemotaxis to LPC is dependent on G2A function. Wild-type but not G2A-deficient mouse peritoneal macrophages migrated toward LPC. RNAi-mediated knockdown of G2A in J774A.1 macrophages abolished LPC-induced chemotaxis, whereas overexpression of G2A significantly enhanced this process. Mutation of the conserved DRY motif of G2A resulted in loss of chemotaxis to LPC, suggesting a requirement for G-protein signaling. Unlike most GPCRs, including the chemokine receptors, coupling to G(i) is not required for LPC/G2A-mediated chemotaxis, but coupling to G(q/11) and G(12/13) is necessary as judged by inhibition with dominant negative forms of these alpha subunits or with regulators of G-protein signaling (RGS) constructs. Collectively, these data establish that pertussis toxin-insensitive G2A signaling regulates macrophage chemotaxis to LPC. Defects in this signaling pathway may be related to the pathogenesis of systemic autoimmune disease.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Quimiotaxia/fisiologia , Lisofosfatidilcolinas/farmacologia , Macrófagos/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Sequência de Bases , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Primers do DNA , Regulação da Expressão Gênica , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase Cgamma2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in Btk(-)/Tec(-) mice but failed to rescue CD5(+) B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway.