Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/patologia , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas/genética , Proteína 1 Parceira de Translocação de RUNX1/fisiologia , Fatores de Transcrição/genética , Fatores Etários , Animais , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos , Camundongos Knockout , Translocação GenéticaRESUMO
Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.
Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Granulócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Proteína 1 Parceira de Translocação de RUNX1/fisiologia , Transcriptoma , Proliferação de Células/genética , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Granulócitos/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucopoese/genética , Monócitos/fisiologia , Mielopoese/genética , Proteínas de Fusão Oncogênica/genética , Oncogenes/fisiologia , Proteína 1 Parceira de Translocação de RUNX1/genética , Transcriptoma/genética , TransfecçãoAssuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Camundongos , Proteínas Tirosina Fosfatases/genética , Taxa de SobrevidaRESUMO
The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Neoplásicas/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Proteína 1 Parceira de Translocação de RUNX1/fisiologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Microambiente Tumoral/genéticaRESUMO
Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.
