FL-Dependent Wild-Type FLT3 Signals Reduce the Inhibitory Effects of FLT3 Inhibitors On Wild-Type and Mutant FLT3 Co-Expressing Cells.

FLT3 mutation, which is found in about 30% of adult AML patients, is involved in the signaling pathway of autonomous proliferation and differentiation block in leukemia cells. Since FLT3 mutation is associated with a poor prognosis in AML patients, mutated FLT3 serves as an important molecular target in the treatment of leukemia. However, it is notable that AML cells harboring FLT3 mutation show the various degree of the allelic ratio of mutant to wild-type (wt) FLT3. To date, several FLT3 kinase inhibitors have been developed, while their inhibitory effects were mainly evaluated in mutant FLT3 alone-expressing cells. In this study, we analyzed how FL-dependent wt-FLT3 signal affects the inhibitory effect of FLT3 inhibitors on wt- and mutant FLT3-expressing cells.

We first established wt-FLT3, mutant FLT3, extra cellular domain-lacking mutant FLT3 (cy-mutant FLT3), wt- and mutant FLT3 co-expressing and wt-and cy-mutant FLT3 co-expressing 32D cells. Furthermore, we also established membrane bound FL-expressing Cos7 cells. Like mutant FLT3, cy-mutant FLT3 was constitutively phosphorylated resulting in autonomous proliferation of cy-mutant FLT3-expressing 32D cells. Using these cells, we evaluated the effects of FL-dependent wt-FLT3 signal on growth inhibition, apoptosis induction and signal inhibition induced by FLT3 inhibitors.

The FL-stimulation reduced growth inhibitory and apoptosis induction effects by FLT3 inhibitors in wt- and mutant FLT3 co-expressing 32D cells in a dose-dependent manner, while those reducing effects were little on mutant FLT3 alone-expressing 32D cells. In consistent with the reducing effect on the growth inhibition, FL-stimulation broadly reduced the de-phosphorylation levels of FLT3, STAT5, AKT and MAPK by FLT3 inhibitors, and the strongest reduction was observed in the de-phosphorylation of MAPK. These reduced effects were more evident in the continuous FL-stimulation by the co-culture with FL-expressing Cos7 cells than the transient stimulation by the addition of soluble FL. In the transient stimulation by the soluble FL at 50 ng/ml, the growth inhibitory effects of FLT3 inhibitors were reduced to about 50% of those without the FL-stimulation. In contrast, the continuous FL-stimulation by the co-culture with FL-expressing Cos7 cells reduced the inhibitory effects to less than 40% of the co-culture with parental Cos7 cells. Of note is that the reduction level of inhibitory effects by the FLT3-selective inhibitors, such as FI-700 was greater than that by multi-kinase inhibitors, such as sunitinib. Furthermore, these FL-dependent reducing effects was also observed in wt- and cy-mutant FLT3 co-expressing cells at the same level as wt- and mutant FLT3 co-expressing cells, indicating that the FL-stimulation reduced the inhibitory effects of FLT3 inhibitors through the interaction with wt-FLT3.

We demonstrated that FL-dependent wt-FLT3 signals reduced the inhibitory effects of FLT3 inhibitors. Since FLT3 expresses on normal hematopoietic progenitors, the lower potency against FL-dependent wt-FLT3 kinase is thought to be an advantage for avoiding the bone marrow suppression in the clinical use. However, our results suggest that the potency against both wt- and mutant FLT3 kinases is required for the sufficient efficacy of leukemia cells, which express both wt- and mutant FLT3. Furthermore, we should consider the allelic ratio of mutant to wt-FLT3 and the selectivity of FLT3 inhibitors for their clinical applications.

Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Consultancy; Novartis Pharma Co. Ltd.: Research Funding. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.