Analysis of the Constitutively Active Wild-Type FLT3 Kinase Mediating Signaling.

[Background] FLT3 is a type 3 receptor tyrosine kinase and is widely expressed in both AML and ALL cells. FLT3 ligand stimulates the proliferation and inhibits the apoptosis of leukemia cells. Thus FL/FLT3 signaling is important for survival of leukemia cells. We previously reported that some AML cells express high level of wild-type (wt) FLT3 transcripts, whose products are constitutively activated (Ozeki K et al, Blood, 2004). In addition, potent FLT3 inhibitors were cytotoxic to those AML cells, indicating that FLT3 inhibitors are also applicable for those patients. However, biological significance of constitutively active wt-FLT3 mediating signaling remains unclear.

[Purpose] The purpose of this study is to clarify the constitutively active wt-FLT3 mediating signaling and its biological significance.

[Material and method] In this study, we first established 32D cell line expressing both wt-FLT3 and FL as a model of constitutively active wt-FLT3. We evaluated its growth potential by MTT assay, methylcellulose colony assay, and transplantation to NOG mice in comparison with wt- and mutant FLT3-expressing cells. Signal transduction pathway and sensitivities to several inhibitors were also examined.

[Results] FL/FLT3 co-expressing 32D (FL/FLT3) cells showed IL3-independent proliferation and constitutive phospholylation of wt-FLT3. In the methylcellulose colony assay, both FLT3/ITD and FL/FLT3 32D cells formed colonies without cytokine. FL/FLT3 cells also revealed leukemia when inoculated into NOG mice as with mutant FLT3, but after a longer latency. Engraftment rate of FL/FLT3 cells in bone marrow and spleen at day 10 was less than those of mutant FLT3 expressing cells.

We, then, examined the downstream signaling pathways of FLT3 activation in those cells. Constitutive activations of AKT, MAPK and STAT5 were observed both in FL/FLT3 expressing- and mutant FLT3 expressing-32D cells, while the STAT3 activation was observed only in the former. FLT3 inhibitor (AG1296) was cytotoxic to both cell lines, but not were MEK (PD98059) and PI3K (LY294002) inhibitors. HSP90 inhibitor (geldanamycin) was only toxic to mutant FLT3 expressing-32D cells.

[Discussion] FL/FLT3 co-expressing 32D cell is transformed in vitro and leukemogenic in vivo, and thus can be used as a model of leukemia with constitutive activity of wt-FLT3 kinase. Constitutively active wt-FLT3 mediating signaling may be involved in the pathophysiology of leukemia development as well as mutant FLT3, though the leukemogenic potency is relatively lower than that of mutant FLT3. Activation of STAT3-related signaling might be a distinguish mechanism between constitutively active wt- and mutant FLT3 kinases, though further analysis is required. To date, several reports showed the synergistic effect of FLT3 inhibitors and HSP90 inhibitors on AML cells harboring mutant FLT3, while the present study demonstrated that constitutively active wt-FLT3 is not a client of HSP90, and HSP90 inhibitors are not suitable for the combination therapy with FLT3 inhibitors. In conclusion, FL/FLT3 co-expressing 32D cell line is a useful tool not only to analyze the biology of constitutively active wt-FLT3 kinase but also to evaluate the efficacy of FLT3 inhibitors.