Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Proteínas de Ligação a DNA/genética , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Translocação Genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Proteínas de Ligação a DNA/fisiologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Humanos , Leucemia/patologia , Terapia de Alvo Molecular/métodos , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas Nucleares/fisiologia , Sítios de Splice de RNA/genética , Sítios de Splice de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Elongação da TranscriçãoRESUMO
The most frequent MLL-gene rearrangement found in leukemia is a reciprocal translocation with AF4 on chromosome 4 resulting in the formation of the MLL-AF4 and the AF4-MLL fusion genes. The oncogenic role of MLL-AF4 is documented but the significance of the reciprocal product - AF4-MLL in leukemia is less clear. In the human leukemia cell lines - RS4;11 and SEMK2-M1, both of which express MLL-AF4 and AF4-MLL, we knocked down the expression of AF4-MLL using siRNA. Loss of AF4-MLL had no effect on the growth of either RS4;11 or SEMK2-M1 cells. Furthermore, in SEMK2-M1 cells there were no changes in cell cycle or apoptosis with loss of AF4-MLL. In contrast, knockdown of MLL-AF4 significantly inhibited growth of both RS4;11 and SEMK2-M1. Additionally, in SEMK2-M1 cells, loss of MLL-AF4 led to G2/M cell cycle arrest and increased apoptosis. Overall, these results demonstrate that in t(4;11) leukemia, the MLL-AF4 fusion protein is critical for leukemia cell proliferation and survival while the AF4-MLL fusion product is dispensable.
Assuntos
Apoptose , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Proteínas de Ligação a DNA/genética , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética/genética , Western Blotting , Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Histona-Lisina N-Metiltransferase , Humanos , Leucemia/patologia , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas de Fusão Oncogênica/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Elongação da Transcrição , Células Tumorais CultivadasRESUMO
Leukemias with MLL rearrangements are characterized by high expression of the homeobox gene MEIS1. In these studies, we knocked down Meis1 expression by shRNA lentivirus transduction in murine Mll-AF9 leukemia cells. Meis1 knockdown resulted in decreased proliferation and survival of murine Mll-AF9 leukemia cells. We also observed reduced clonogenic capacity and increased monocytic differentiation. The establishment of leukemia in transplantation recipients was significantly delayed by Meis1 knockdown. Gene expression profiling of cells transduced with Meis1 shRNA showed reduced expression of genes associated with cell cycle entry and progression. shRNA-mediated knockdown of MEIS1 in human MLL-fusion gene leukemia cell lines resulted in reduced cell growth. These results show that MEIS1 expression is important for MLL-rearranged leukemias and suggest that MEIS1 promotes cell-cycle entry. Targeting MEIS1 may have therapeutic potential for treating leukemias expressing this transcription factor.
Assuntos
Proteínas de Homeodomínio/genética , Leucemia/genética , Leucemia/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Introdução de Genes , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Lentivirus/genética , Camundongos , Camundongos Mutantes , Proteína Meis1 , Transplante de NeoplasiasRESUMO
The pathways by which oncogenes, such as MLL-AF9, initiate transformation and leukemia in humans and mice are incompletely defined. In a study of target cells and oncogene dosage, we found that Mll-AF9, when under endogenous regulatory control, efficiently transformed LSK (Lin(-)Sca1(+)c-kit(+)) stem cells, while committed granulocyte-monocyte progenitors (GMPs) were transformation resistant and did not cause leukemia. Mll-AF9 was expressed at higher levels in hematopoietic stem (HSC) than GMP cells. Mll-AF9 gene dosage effects were directly shown in experiments where GMPs were efficiently transformed by the high dosage of Mll-AF9 resulting from retroviral transduction. Mll-AF9 upregulated expression of 192 genes in both LSK and progenitor cells, but to higher levels in LSKs than in committed myeloid progenitors.
Assuntos
Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Animais , Divisão Celular , Dosagem de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Retroviridae/genética , Células-Tronco/citologiaRESUMO
The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-), PAX5(+), TdT(+), IgH rearranged)/myeloid (CD11b/Mac1(+), c-fms(+), lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model.
Assuntos
Neoplasias Hematológicas/etiologia , Linfócitos/patologia , Células Mieloides/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Animais , Linhagem da Célula , Neoplasias Hematológicas/patologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Transgênicos , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Fatores de TempoRESUMO
PURPOSE: We conducted studies to evaluate the hypothesis that FLT3 is a client of heat shock protein (Hsp) 90 and inhibitors of Hsp90 may be useful for therapy of leukemia. EXPERIMENTAL DESIGN: The effects of the Hsp90-inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on cell growth, expression of signal transduction kinases, apoptosis, FLT3 phosphorylation and interaction with Hsp90 was determined in FLT3(+) human leukemias. RESULTS: We found that FLT3 is included in a multiprotein complex that includes Hsp90 and p23. 17-AAG inhibited FLT3 phosphorylation and interaction with Hsp90. FLT3(+) leukemias were significantly more sensitive to the Hsp90 inhibitors 17-AAG and Herbimycin A in cell growth assays than FLT3-negative leukemias. Cells transfected with FLT3 became sensitive to 17-AAG. Cell cycle inhibition and apoptosis were induced by 17-AAG. Cells with constitutive expression of FLT3, as a result of internal tandem duplication, were the most sensitive; cells with wild-type FLT3 were intermediate in sensitivity, and FLT3-negative cells were the least sensitive. 17-AAG resulted in reduced cellular mass of FLT3, RAF, and AKT. The mass of another Hsp, Hsp70, was increased. The expression level of MLL-AF4 fusion protein was not reduced by 17-AAG in human leukemia cells. CONCLUSIONS: FLT3(+) leukemias are sensitive to 17-AAG and Herbimycin A. 17-AAG inhibits leukemia cells with either FLT3-internal tandem duplication or wild-type FLT3, in part through destabilization of client kinases including FLT3, RAF, and AKT. 17-AAG is potentially useful for therapy of FLT3-expressing leukemias, including the mixed lineage leukemia fusion gene leukemias.