RESUMO
Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin γ RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation.
Assuntos
Aminopiridinas/farmacologia , Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Leucemia Mieloide Aguda/metabolismo , Triazinas/farmacologia , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
Faithful modeling of mixed-lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid, or mixed-lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenvironmental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML.
Assuntos
Linhagem da Célula , Transformação Celular Neoplásica/patologia , Células-Tronco Fetais/patologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Multipotentes/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Aneuploidia , Animais , Antígenos CD34/análise , Apoptose , Técnicas de Cultura de Células , Linhagem Celular Transformada , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cromossomos Humanos Par 11 , Meio Ambiente , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Células-Tronco Fetais/imunologia , Células-Tronco Fetais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Multipotentes/imunologia , Células-Tronco Multipotentes/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais , Especificidade da Espécie , Transplante de Células-Tronco , Fatores de Tempo , Transdução Genética , Translocação Genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismoAssuntos
Ilhas de CpG/genética , Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Adaptadoras de Transdução de Sinal , Quimiocinas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , Fenótipo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genéticaRESUMO
The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.
