Identification of Common Target Genes of the BCR/ABL and FLT3-ITD Tyrosine Kinases by Microarray Analysis.

Human myeloid leukaemias are commonly associated with activating mutations in tyrosine kinases. The BCR-ABL oncogene causes CML and is generated by the Philadelphia (Ph) chromosome translocation t(9;22)(q34;q11). The FLT3 tyrosine kinase receptor is mutated in about 30 % of all cases of AML, most often through a mechanism that involves an internal tandem duplication (ITD) of a small number of amino acid residues in the juxtamembrane domain. Both BCR/ABL and FLT3 (ITD) produce a constitutively active tyrosine kinase that enhances proliferation and viability of myeloid cells, and induce a similar myeloproliferative disorder when transplanted into mice. In an effort to identify any common downstream targets of BCR/ABL and FLT3(ITD) we generated paired Ba/F3 cell lines in which either Bcr/Abl or FLT3-ITD could be induced under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline rapidly induced expression of BCR/ABL or FLT3-ITD proteins, respectively proteins. The gene expression profile of these conditionally expressing BCR/ABL and FLT3-ITD BaF3cells was examined using the U430A gene chip (Affymetrix), which contains more than 34000 well substantiated genes. With a 2.5 fold cut-off, Bcr/Abl altered the expression of 336 genes and FLT3-ITD altered expression of 231 genes at 24 hours after induction. Fifty one genes were regulated by both Bcr/Abl and FLT3-ITD, including Cyclin G2, cyclin D2, CXCR4, osteopontin and FKBP12. Following induction of BCR/ABL and FLT3-ITD, cyclin D2, osteopontin and FKBP12 are strongly upregulated, whereas cyclin G2 and CXCR4 are downregulated. We validated the expression pattern of these genes by real-time PCR analysis. To further validate that these genes are directly regulated by the kinase activity of BCR/ABL and FLT3-ITD, cell lines were treated with specific small molecule kinase inhibitors, resulting in both cases in a significant decrease in cyclin D2, osteopontin, and FKBP12 and an increase in cyclin G2 and CXCR4 expression. The functional role of several of these genes is under investigation. For example, we found that the PI3K/AKT pathway is important for regulating osteopontin gene expression, and that osteopontin is involved in mediating the increase in cell growth caused by both BCR/ABL and FLT3-ITD. The identification of downstream targets, such as cyclins, osteopontin, and others, that are shared in common by several oncogenic tyrosine kinase oncogenes suggest that they could have value as therapeutic targets.