RESUMO
Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next-generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome-wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5â39 × 10-5 , Bonferroni-adjusted), impacting leukaemia cell survival as the top-ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.
Assuntos
Leucemia Mieloide Aguda/genética , Descoberta de Drogas , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Terapia Genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/terapia , Terapia de Alvo Molecular , NADPH Oxidase 2/genéticaAssuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Neoplásicas/citologia , Idoso , Células Cultivadas , Técnicas de Cocultura , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/genética , Transcriptoma , Células Tumorais Cultivadas , Microambiente Tumoral , Adulto JovemAssuntos
Antineoplásicos/efeitos adversos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Prolinfocítica de Células T/diagnóstico , Cromossomo Filadélfia/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/efeitos adversos , Antineoplásicos/administração & dosagem , Feminino , Humanos , Imunofenotipagem , Cariótipo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patologia , Pessoa de Meia-Idade , Prognóstico , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagemRESUMO
The development of assays for evaluating the sensitivity of leukaemia cells to anti-cancer agents is becoming an important aspect of personalized medicine. Conventional cell cultures lack the three-dimensional (3D) structure of the bone marrow (BM), the extracellular matrix and stromal components which are crucial for the growth and survival of leukaemia stem cells. To accurately predict the sensitivity of the leukaemia cells in an in vitro assay a culturing system containing the essential components of BM is required. In this study, we developed a porous calcium alginate foam-based scaffold to be used for 3D culture. The new 3D culture was shown to be cell compatible as it supported the proliferation of both normal haematopoietic and leukaemia cells. Our cell differential assay for myeloid markers showed that the porous foam-based 3D culture enhanced myeloid differentiation in both leukaemia and normal haematopoietic cells compared to two-dimensional culture. The foam-based scaffold reduced the sensitivity of the leukaemia cells to the tested antileukaemia agents in K562 and HL60 leukaemia cell line model and also primary myeloid leukaemia cells. This observation supports the application of calcium alginate foams as scaffold components of the 3D cultures for investigation of sensitivity to antileukaemia agents in primary myeloid cells.
Assuntos
Alginatos , Técnicas de Cultura de Células , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia , Microambiente TumoralAssuntos
Leucemia Prolinfocítica de Células T/patologia , Linfócitos T/patologia , Idoso , Antígenos CD/genética , Doenças Assintomáticas , Aberrações Cromossômicas , Feminino , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia Prolinfocítica de Células T/sangue , Leucemia Prolinfocítica de Células T/genética , MasculinoAssuntos
Medula Óssea/metabolismo , Medula Óssea/patologia , Citometria de Fluxo , Imuno-Histoquímica , Neoplasias de Plasmócitos/diagnóstico , Plasmócitos/metabolismo , Plasmócitos/patologia , Antígenos CD/metabolismo , Biomarcadores , Biópsia , Citometria de Fluxo/métodos , Humanos , Imuno-Histoquímica/métodos , Reprodutibilidade dos TestesRESUMO
El sarcoma granulocítico (SG) es una lesión poco frecuente asociada a síndromes mielodisplásicos, mieloproliferativos o leucemias, aunque puede ser el primer hallazgo en un paciente previamente sano. Presentamos un SG que comenzó como compresión medular, en un paciente sin patología hematológica previa. Las imágenes radiológicas demostraron una lesión lítica en L1 que precisó cirugía urgente. Fue preciso realizar inmunohistoquímica de la muestra para llegar al diagnóstico. El aspirado medular no mostró evidencia de patología hematológica, siendo el SG la primera manifestación. El paciente recibió posteriormente tratamiento con quimioterapia y radioterapia, falleciendo 20 meses después del diagnóstico de una sepsis Pseudomonas aeruginosa intratratamiento de una leucemia mieloblástica. En resumen, el SG primario es un tumor infrecuente de difícil diagnóstico. Es necesario tener un alto grado de sospecha y solicitar amplios estudios inmunohistoquímicos para un diagnóstico correcto. El tratamiento debe ser precoz, agresivo e individualizado, ya que tiene mal pronóstico.
Granulocytic sarcoma (GS) is an infrequent lesion associated with myelodysplastic or myeloproliferative disorders or leukemia, although it may be the first finding in an otherwise healthy patient. A case of GS is described that presented as spinal cord compression, in a patient with no underlying hematological disorder. Imaging studies disclosed a single lytic lesion in L1, which required emergency surgery. Immunohistochemical staining of the surgical biopsy sample was needed for diagnosis. Bone marrow aspirate was unremarkable. The patient received chemo-radiotherapy, dying 20 months after diagnosis of Pseudomonas aeruginosa sepsis during treatment of acute myelogenous leukemia. In short, primary GS is an infrequent and difficult to diagnose tumor. A high degree of suspicion, along with extensive immunohistochemical studies are necessary for diagnosis. Treatment should be prompt, aggressive and individualized, since the prognosis is very poor.
