Identification and functional analysis of gene expression changes in acute myeloid leukaemia.

Abstract

Acute Myeloid Leukaemia (AML) is a malignant blood cancer characterised by uncontrolled growth of leukaemic blasts. This is associated with constitutive activation of key signalling molecules such as AKT, ERK1/2, STAT5 and NFB and with aberrant transcription factor activity, which in many cases is associated with characteristic chromosomal translocations. Aberrant receptor signaling can constitutively activate the pathways associated with the above signaling molecules. For example, autocrine interleukin-3 (IL-3), and over-expression of IL-3 receptor alpha (IL3RA/CD123) have been found in AML, as has constitutive phosphorylation of the common beta subunit (hBc) for IL-3 and granulocyte-macrophage colony-stimulating factor receptor (GMR). Also mutation of the FMS-like tyrosine kinase 3 (FLT3) receptor is common in AML (~30% of patients) and the resultant aberrant FLT3 signaling contributes to enhanced survival, growth and a block in differentiation. A focus in this thesis is the identification and dissection of the signaling pathways and downstream genes activated by a leukaemic mutant of GMR (GMR-V449E) and by the FLT3 activated mutants associated with AML. For these studies we make extensive use of the murine bi-potential myeloid cell line model FDB-1 in which these mutants induce factorindependent growth and survival and a block in differentiation. The use of this experimental approach together with bioinformatics has provided leads with regard to the role of the AKT/mTOR and ERK pathways downstream of these receptors, and important for cell proliferation, survival and differentiation. Additionally, we focused on the role of the Growth Arrest and DNA Damage 45a (Gadd45a) gene, repression of which is important for cell survival and the block in differentiation induced by the activated mutants. A second focus has been extending the bioinformatic approaches to define the gene expression and pathways associated with the abnormal growth characteristics of AML. In particular, we studied AML cases with numerical chromosomal abnormalities and translocation events. Extensive use is made of the Connectivity Map (CMAP) resource together with publicly available gene expression datasets to define agents with antileukaemic potential. We have tested drugs, selected using the inv(16) (CBFB-MYH11) and MLL AML translocation signatures, for specificity and sensitivity on AML patient samples.