RESUMEN
Our focus is the identification, characterisation and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions.
Asunto(s)
Elementos Transponibles de ADN , Vectores Genéticos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Transfección/métodos , Transposasas/metabolismo , Animales , Línea Celular , Proliferación Celular , Supervivencia Celular , Clonación Molecular , Elementos Transponibles de ADN/genética , Fibroblastos/metabolismo , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Factores de Tiempo , Transcripción Genética , Translocación Genética , Transposasas/genéticaRESUMEN
Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.
Asunto(s)
Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Recombinación Genética , Translocación Genética , Enfermedad Aguda , Adulto , Biopsia , Médula Ósea/química , Médula Ósea/patología , Niño , Rotura Cromosómica , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/ultraestructura , Biología Computacional , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Duplicación de Gen , N-Metiltransferasa de Histona-Lisina , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The human mixed lineage leukemia (MLL) gene is frequently involved in genetic rearrangements with more than 55 different translocation partner genes, all associated with acute leukemia. Reciprocal chromosomal translocations generate two MLL fusion alleles, where 5'- and 3'-portions of MLL are fused to gene segments of given fusion partners. In case of t(4;11) patients, about 80% of all patients exhibit both reciprocal fusion alleles, MLL.AF4 and AF4.MLL, respectively. By contrast, 20% of all t(4;11) patients seem to encode only the MLL.AF4 fusion allele. Here, we analyzed these 'MLL.AF4(+)/AF4.MLL(-)' patients at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found in this group of t(4;11) patients. Reciprocal MLL fusions with novel translocation partner genes, for example NF-KB1 and RABGAP1L, were identified and actively transcribed in leukemic cells. In other patients, the reciprocal 3'-MLL gene segment was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, patients were identified that encode only solitary 3'-MLL gene segments on the reciprocal allele. Based on these data, we propose that all t(4;11) patients exhibit reciprocal MLL alleles, but due to the individual recombination events, provide different pathological disease mechanisms.
