Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor

FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. The se receptors all contain an intrinsic tyrosine kinase domain that is critic al to signaling. Aberrant expression of the FLT3 gene has been documented i n both adult and childhood leukemias including AML, ALL and CML. In additio n, 17-27% of pediatric and adult patients with AML have small internal tand em duplication mutations in FLT3. Patients expressing the mutant form of th e receptor have been shown to have a decreased chance for cure. Our previou s study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has pro mpted us to search for inhibitors of FLT3 as a possible therapeutic approac h in these patients. AG1296 is a compound of the tyrphostin class that is k nown to selectively inhibit the tyrosine kinase activity of the PDGF and KI T receptors. Since FLT3 is a close relative of KIT, we wanted to test the p ossible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimula ted wildtype and constitutively activated FLT3. Treatment by AG1296 abolish ed IL-3-independent proliferation of Ba/F3 cells expressing the constitutiv ely activated FLT3 and thus, reversed the transformation mediated by activa ted FLT3. Inhibition of FLTB activity by AG1296 in cells transformed by act ivated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLTB in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinas es of the IL-3 pathway and other kinases further downstream involved in pro liferation intact. Several proteins phosphorylated by the activated FLTB si gnaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phospho rylated when these cells were treated with AG1296. The activity against FLT 3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase ac tivity and as a tool for studying the biology of FLTB.