Zebrafish model of MLL leukemogenesis; implication for fish favorable model of MLL-related leukemia development

During these years of experimental work, my research has been addressed to the investigation of the mixed lineage leukemia (MLL) gene functions in acute leukemias, and in particular, to explore the viability of the zebrafish (Danio rerio) as a model to use for understand the role of human MLL in normal and malignant hematopoiesis. The MLL gene at human chromosome band 11q23 is an important oncogene that is frequently disrupted by chromosomal translocations with more than 50 partner genes in a variety of high-risk acute leukemias of either myeloid or lymphoid derivation, hence the name MLL. These rearrangements are present at significantly high incidence in infants and in the majority of patients with therapy-related leukemia induced by inhibitors of topoisomerase II. MLL is a large multi-domain protein that has a global role in the regulation of transcription. In particular MLL is required for normal hematopoiesis and implicated in the maintenance of Hox genes expression. In this PhD thesis the research aim to study three different aspects of MLL oncogene in acute leukemia. 1. The observation that MLL rearrangements and disease itself may initiate within an undifferentiated hematopoietic stem cell. 2. The assessment of MLL genomic breakpoints distribution within the breakpoint cluster region (bcr) in particular in secondary leukemias samples. 3. The versatile biology and genetic flexibility of zebrafish organism as a vertebrate model for studying human hematopoiesis. 1. The investigation of the cell origin of MLL translocations, was studied with a case of a patient diagnosed with pre-pre-B ALL/t (4;11) leukemia, which during the treatment and after matched bone marrow transplantation (BMT), underwent two consecutive switches from lymphoid to myeloid lineage and vice versa. The high expression of HOXA9 and FLT3 genes remaining genotypically stable in leukemia throughout phenotypic switches, suggests that this leukemia may have originated as a common B/myeloid progenitors. This part of the work has been performed by morphology and flow cytometry analyses combined with the microarray analysis, in order to evaluate gene expression during different phases of disease. 2. The work about the localization of MLL genomic breakpoint junction indicates that translocations in treatment related leukemias occur mainly near precise or precise interchromosomal DNA recombination at the sequence level, and confirms a translocation breakpoint hotspot at 3' region in the bcr intron 8. This part of work has been performed by panhandle PCR analysis, a technique that allows the amplification of the MLL genomic breakpoint junction from a stem-loop template using primers all from MLL. The panhandle PCR also identified a breakpoint junction of the uncovered ARMC3 from band 10p12 and MLL intron 9 in a case of treatment-related myeloid leukemia. ARMC3 protein contains Arm repeats similar to catenin proteins (eg. b-catenin), plakophilins and the tumor suppressor APC. ARMC3 is the first gene of this type disrupted by MLL translocation. 3. The overall high-grade conservation of the molecular pathways governing hematopoiesis between mammals and zebrafish, as well as the identification of several well conserved zebrafish transcription factors mammalian orthologs, permitted the identification and characterization of a 12657 bp cDNA sequence which represents a candidate zebrafish orthologue of the human MLL gene. The major advantages of this system include robust experimental techniques in both genetics and embryology, which have been utilized to model many aspects of human development and diseases. This part of the work has been performed by bioinformatics and classical molecular analyses. After sequencing, the zebrafish mll nucleotide sequence, exon-intron structure, amino acid sequence, and conserved domains were analyzed via the zebrafish databases. The temporal pattern of mll mRNA expression was examined using quantitative RT-PCR analysis and whole-mount in situ hybridization analysis. These findings indicate that there is a single mll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcripts to the embryo, indicates that mll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the starting point to use zebrafish for studying MLL in hematopoiesis and leukemia.