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1.
Nat Commun ; 14(1): 2068, 2023 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-37045815

RESUMO

The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Interleucina-12/genética , Molécula de Adesão da Célula Epitelial , Imunoterapia Adotiva , Neoplasias/genética , Neoplasias/terapia , Antígenos de Neoplasias/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente Tumoral
2.
Nature ; 526(7572): 273-276, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26416749

RESUMO

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.


Assuntos
Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Animais , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Proteínas Nucleares/antagonistas & inibidores , Compostos Policíclicos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
3.
Exp Hematol ; 40(4): 307-17.e3, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22155722

RESUMO

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-Cre-inducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling Lin(-)Sca-1(+)c-kit(+) (LSK) cells is increased in Fak(-/-) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from Fak(-/-) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs (Lin(-)Sca-1(+)c-kit(+)CD34(-)Flk-2(-) cells) sorted from Fak(-/-) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of Fak(fl/fl)Mx1-Cre(+) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady-state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.


Assuntos
Quinase 1 de Adesão Focal/deficiência , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco/fisiologia , Animais , Animais Congênicos , Antígenos de Diferenciação/análise , Transplante de Medula Óssea , Comunicação Celular , Ciclo Celular , Linhagem da Célula , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/fisiologia , Genes Reporter , Hematopoese/genética , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/enzimologia , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação , Células Estromais/fisiologia
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