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1.
Int J Mol Sci ; 21(12)2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32549410

RESUMO

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.


Assuntos
Edição de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Histona-Lisina N-Metiltransferase/genética , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Pontos de Quebra do Cromossomo , Modelos Animais de Doenças , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3
2.
Int J Cancer ; 145(8): 2201-2208, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30485425

RESUMO

Therapeutic approaches which aim to target Acute Myeloid Leukaemia through enhancement of patients' immune responses have demonstrated limited efficacy to date, despite encouraging preclinical data. Examination of AML patients treated with azacitidine (AZA) and vorinostat (VOR) in a Phase II trial, demonstrated an increase in the expression of Cancer-Testis Antigens (MAGE, RAGE, LAGE, SSX2 and TRAG3) on blasts and that these can be recognised by circulating antigen-specific T cells. Although the T cells have the potential to be activated by these unmasked antigens, the low arginine microenvironment created by AML blast Arginase II activity acts a metabolic brake leading to T cell exhaustion. T cells exhibit impaired proliferation, reduced IFN-γ release and PD-1 up-regulation in response to antigen stimulation under low arginine conditions. Inhibition of arginine metabolism enhanced the proliferation and cytotoxicity of anti-NY-ESO T cells against AZA/VOR treated AML blasts, and can boost anti-CD33 Chimeric Antigen Receptor-T cell cytotoxicity. Therefore, measurement of plasma arginine concentrations in combination with therapeutic targeting of arginase activity in AML blasts could be a key adjunct to immunotherapy.


Assuntos
Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Arginase/antagonistas & inibidores , Arginina/sangue , Leucemia Mieloide/terapia , Doença Aguda , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Arginase/sangue , Arginase/metabolismo , Arginina/metabolismo , Azacitidina/administração & dosagem , Humanos , Imunoterapia/métodos , Células K562 , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Vorinostat/administração & dosagem
3.
Nat Commun ; 9(1): 5280, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538250

RESUMO

Acute myeloid leukaemia (AML) affects children and adults of all ages. AML remains one of the major causes of death in children with cancer and for children with AML relapse is the most common cause of death. Here, by modelling AML in vivo we demonstrate that AML is discriminated by the age of the cell of origin. Young cells give rise to myeloid, lymphoid or mixed phenotype acute leukaemia, whereas adult cells give rise exclusively to AML, with a shorter latency. Unlike adult, young AML cells do not remodel the bone marrow stroma. Transcriptional analysis distinguishes young AML by the upregulation of immune pathways. Analysis of human paediatric AML samples recapitulates a paediatric immune cell interaction gene signature, highlighting two genes, RGS10 and FAM26F as prognostically significant. This work advances our understanding of paediatric AML biology, and provides murine models that offer the potential for developing paediatric specific therapeutic strategies.


Assuntos
Leucemia Mieloide Aguda/genética , Fatores Etários , Animais , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pediatria , Prognóstico , Proteínas RGS/genética , Proteínas RGS/metabolismo
4.
Cell Death Dis ; 9(5): 443, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29670085

RESUMO

Trib2 pseudokinase is involved in the etiology of a number of cancers including leukaemia, melanoma, ovarian, lung and liver cancer. Both high and low Trib2 expression levels correlate with different types of cancer. Elevated Trib2 expression has oncogenic properties in both leukaemia and lung cancer dependent on interactions with proteasome machinery proteins and degradation of transcription factors. Here, we demonstrated that Trib2 deficiency conferred a growth and survival advantage both at steady state and in stress conditions in leukaemia cells. In response to stress, wild type leukaemia cells exited the cell cycle and underwent apoptosis. In contrast, Trib2 deficient leukaemia cells continued to enter mitosis and survive. We showed that Trib2 deficient leukaemia cells had defective MAPK p38 signalling, which associated with a reduced γ-H2Ax and Chk1 stress signalling response, and continued proliferation following stress, associated with inefficient activation of cell cycle inhibitors p21, p16 and p19. Furthermore, Trib2 deficient leukaemia cells were more resistant to chemotherapy than wild type leukaemia cells, having less apoptosis and continued propagation. Trib2 re-expression or pharmacological activation of p38 in Trib2 deficient leukaemia cells sensitised the cells to chemotherapy-induced apoptosis comparable with wild type leukaemia cells. Our data provide evidence for a tumour suppressor role of Trib2 in myeloid leukaemia via activation of p38 stress signalling. This newly identified role indicates that Trib2 may counteract the propagation and chemotherapy resistance of leukaemia cells.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide/metabolismo , Sistema de Sinalização das MAP Quinases , Mitose , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estresse Fisiológico , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
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