Horton, S.J.; (2005) Establishment of an in vitro model to identify the molecular mechanism of immortalisation by MLL-ENL. Doctoral thesis , University of London.

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Abstract

The t (l I 19)(p22 q23) translocation, which gives rise to the MLL-ENL fusion protein is commonly found in infant acute leukaemias of both the myeloid and lymphoid lineage. In order to study the molecular mechanism of haematopoietic progenitor cell (HPC) immortalisation by MLL-ENL, a conditional system of MLL- ENL expression was established in primary murine HPCs. This was achieved by delivering the Tet-Off inducible expression system to primary cells using two retroviral expression constructs. Several conditional immortalised myeloid cell lines were generated in vitro which were dependent on continued MLL-ENL expression for their survival and proliferation. The immortalised cells either terminally differentiated or died when MLL-ENL expression was turned off with doxycycline. Since several Hox genes are targets of MLL, the expression profile of all 39 murine Hox genes was analysed in the MLL-ENL immortalised cell lines by real-time quantitative PCR. Loss of MLL-ENL expression resulted in a decrease in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte-colony-stimulating factor, we found that reduced Hox expression was specific to loss of MLL-ENL expression and was not a consequence of differentiation. Affymetrix microarray analysis revealed that MLL-ENL may maintain or activate the expression of the transcription factor Sdccag33 and the serine / threonine kinase Pim-2, which confers protection from apoptosis. The analysis also demonstrated that MLL-ENL may repress the expression of apoptosis promoting genes such as Ddb2 and Aatk. In summary, MLL-ENL is required to initiate and maintain the immortalisation of myeloid progenitors and may contribute to immortalisation by aberrantly maintaining the expression of multiple Hoxa genes. The pathways regulated by multiple Hoxa genes and the MLL-ENL target genes identified by Affymetrix analysis represent new possibilities for therapies which may combat these aggressive leukaemias.

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