Our long-term goals are to increase the cure rate and decrease chemotherapy-related toxicity for patients with leukemia. In particular, we are interested in patients whose leukemias express an activated FLT3 receptor, either as a result of mutation or ligand co-expression. The 20-40 percent of AML patients with mutations of the FLT3 receptor have particularly poor prognoses, with cures in the 10-20 percent range for pediatric and adult populations. This contrasts with cure rates of 40-50 percent for AML patients without mutant FLT3. This makes the development of novel strategies for these patients imperative if we are to effect improvements in their outcome. The most common form of mutations in the FLT3 receptor consists of small (18-112 bp) internal tandem duplications (lTDs) which are unique for each patient but all map to the juxtamembrane region, and point mutations affecting aspartic acid 835 in the kinase domain. Both of these mutations constitutively activate the tyrosine kinase domain of the FLT3 receptor which is required for signaling and transformation. By screening >10,000 small molecules from a library of tyrosine kinase inhibitors in a cell-based assay, we have identified several compounds that are able to completely inhibit FLT3 signaling with IC5Os in the single digit nM range. We propose to study the mechanisms by which FLT3 is constitutively activated and study how the FLT3 inhibitors interfere with these mechanisms. Responses of primary leukemic cells and cell lines to treatment with FLT3 inhibitors will be studied and the possible synergistic killing of cells when combined with chemotherapies will be explored. Signal transduction will be studied in cell lines and, in a more limited fashion, in primary cells in order to establish correlations of changes in signaling with the response of the cells to the inhibitors. Additional aliquots of cells from patient samples that respond in vitro will then be used in the NOD/SCID model of human leukemia to see if the inhibitors will also function in vivo. Cells which become resistant to FLT3 inhibitors in vitro or in vivo will be studied to determine the mechanisms that result in resistance. We hope that this work will help lead to clinical trials of these or similar compounds as novel therapeutics against leukemia.
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