Walf-Voderwüelbecke, V.; (2009) A model to investigate the oncogenic activity of MLL-fusions in Acute Myeloid Leukaemia. Doctoral thesis , UCL (University College London).

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Abstract

The MLL gene, located on 11q23, is involved in a large number of chromosomal translocations, including t(9;11)(p22;q23) and t(11;19)(p22;q23). These translocations encode the MLL-AF9 and MLL-ENL fusion transcription factors and are prevalent in infant acute leukaemia and therapy-related leukaemia. Leukaemias associated with these translocations have a particularly poor outcome. In order to conditionally express the MLL-AF9 fusion oncogene in primary haematopoietic progenitor cells, retroviral delivery of the Tet-off expression system was used. Progenitors were purified from murine bone marrow and co-infected with MSCV-TRE-fMLL-AF9 and MSCV-tTA retroviral supernatants. Using this approach, eight independent cell lines with conditional expression of MLL-AF9 and three independent cell lines with constitutive MLL-AF9 expression were generated. Treatment of the conditional cells with Doxycycline caused a decrease in MLL-AF9 mRNA and protein expression, and resulted in terminal differentiation of the cells. By analysing global changes in gene expression after treatment of cells with Doxycycline, using Mouse genome Affymetrix Gene Chips (430 2.0), we have identified a number of potential transcriptional target genes of the MLL-AF9 and MLL-ENL fusion oncogenes. In order to examine the importance of target genes for MLL-fusion mediated transformation, up-regulated target genes were knocked down in vitro. Knock-down of a small proportion of the target genes analysed caused MLL-ENL and MLL-AF9 immortalised cells to die. These data illustrate novel approaches to interfering with MLL-fusion activity in leukaemia. In order to establish the importance of MLL-fusion activity for leukaemia in vivo, and hence its dependence on the transcriptional target genes identified, MLL-ENL immortalised cell lines were chosen to be injected into primary recipients. Conditionally MLL-ENL immortalised cell lines were found to induced leukaemia in vivo. Leukaemic cells isolated from primary recipient mice were shown to have acquired additional genetic abnormalities and, when transplanted into secondary recipients, induced leukaemia with shortened latencies. However, the leukaemic cells remained dependent on MLL-ENL expression in vitro and in vivo, and its ablation resulted in regression of established leukaemias.

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