The role of MLL/AF4 in leukaemic cell biology

Abstract

T(4;11) acute lymphoblastic leukaemia (ALL) presenting the fusion gene MLL/AF4 is a hallmark of infant ALL. The 5 year event free survival is less than 40%. It was shown that MLL/AF4 is important for cell cycle progression, proliferation, clonogenicity, engraftment in mice and repression of apoptosis. However, in order to improve treatment MLL/AF4 needs to be examined in contexts closer to the leukaemic situation in vivo. To investigate the fusion gene, MLL/AF4 positive SEM, generated from an ALL patient, were depleted for MLL/AF4 by RNAi. The consequential changes in gene expression patterns were analyzed using cDNA- and Oligo- arrays. These gene expression patterns were assigned to biological functions and pathways using the Ingenuity platform. A set of significantly differently expressed genes such as N-CADHERIN and FGFR1, both described for the adherence and regulation of haematopoietic stem cells (HSC), was identified and validated by qRT-PCR. The HSC niche context was further investigated by establishment of a leukaemic - bone marrow feeder cell-to-cell interaction assay. Increased cell death, cell cycle arrest and prolonged growth curves caused by MLL/AF4 depletion in SEM could also be shown on feeders. Additionally, culturing of MLL/AF4 positive patient material on feeders allowed for long-term culture. An imaging method using fluorescent MLL/AF4 positive cells in mouse xenograft models was employed to monitor the distribution and biology of MLL/AF4 positive cells, showing colonisation of bone marrow rich spinal, femoral and cranial bones. Finally, in order to analyze the function of identified target genes of MLL/AF4 by overexpression, a novel lentiviral expression system allowing transduction of stem cells and tightly regulating induction of expression, was initiated. In conclusion, these data indicate a central role of MLL/AF4 in leukaemogenisis while the systems established in this work are of relevance for drug development assays.