Abstract
The tyrosine kinase FLT3 has received much attention as a potential drug target in acute myeloid leukemias. FLT3 is expressed on hematopoietic precursors and its activation plays a role in cell survival and proliferation as well as prevention of differentiation. Activating mutations of FLT3 are found in 30% of acute myeloid leukemias (AMLs) and can be associated with an unfavorable prognosis. The majority of leukemic cells also express both FLT3 and FLT3-ligand enabling a self-activation loop.
Several micro array analyses have shown that MLL leukemias, harboring a rearrangement of the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23, are characterized by an exceptionally high level of FLT3 mRNA expression. Other studies have shown that FLT3 activating mutations are present in 10 to 20% of MLL leukemias. These findings suggest that the FLT3 receptor could be a valid drug target to fight these MLL leukemias against which current treatments are by enlarge inefficient.
We have developed a bone marrow retroviral transduction/transplantation model to study the mechanisms of transformation by MLL fusion genes. We measured the transcript level of FLT3 using real time PCR and found that cells transformed by MLL fusion genes expressed considerably higher levels of FLT3 in vitro and in vivo compared to controls. We then investigated whether FLT3 signaling contributed to the pathogenesis of MLL leukemias in our model. We co-transduced the constitutively active FLT3ITD mutant with MLL-ENL in C57Bl6 bone marrow and compared the properties of the resulting cells in vitro and in vivo to cells solely infected with MLL-ENL. We found that FLT3ITD did not confer any advantage in terms of growth factor dependence or rate of proliferation and that the degree of myeloid maturation of the cells in culture was unaffected. Co-transduced cells inoculated to irradiated recipient mice induced acute myeloid leukemias with similar characteristics and latency as well. We next investigated if MLL-ENL could transform cells in vitro or in vivo in the absence of FLT3 signaling. Bone marrow from flt3 knock out or flt3 +/+ mice were transduced in parallel with MLL-ENL and studied in vitro and in vivo. We found that flt3−/− bone marrow could be immortalized and displayed the same properties as MLL-ENL flt3+/+ cell lines. flt3+/+ irradiated mice inoculated with flt3−/− or flt3+/+ MLL-ENL-transduced bone marrow progenitors also developed indistinguishable acute myeloid leukemias. Finally we studied the effect of the FLT3 inhibitors PKC412 (100 nM) and AG1296 (1 mM) on cells immortalized by MLL-ENL in culture. We found that both these drugs inhibited the proliferation and induced the myeloid maturation of MLL-ENL cells in culture.
Surprisingly, we observed identical activities of these FLT3 inhibitors on flt3−/− cells immortalized by MLL-ENL. This can likely be accounted for by the fact that PKC412 and AG1296 are known to be inhibitors of multiple kinases, including some thought to be important for carcinogenesis. Altogether, our findings indicate that FLT3 may not be a relevant drug target for all MLL leukemias and that FLT3 inhibitors’ activity in this model of MLL leukemia is mediated by interference on other signaling pathways via alternative kinase inhibition, yet to be identified.
Analysis of p73 expression pattern in acute myeloid leukemias: lack of ΔN-p73 expression is a frequent feature of acute promyelocytic leukemia
