Targeting FLT3 Phosphorylation and Signaling in Acute Myeloid Leukemia.

Children with acute myeloid leukemia (AML) have 50% overall survival despite aggressive chemotherapy and bone marrow transplantation. Similarly, only one third of adults diagnosed with AML will be cured. AML blast cells from approximately 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains internal tandem duplications in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and members of the VEGF and PDGF receptor families. We previously demonstrated that ABT-869 in vitro induces apoptosis of AML cell lines harboring the FLT3-ITD and primary AML cells, and in vivo in tumors from MV-411 xenograft models. Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and propidium iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. Toxic effects were not observed on normal hematopoietic progenitor cells in methylcellulose-based colony assays at concentrations that were effective in AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-411 with oral preparations of ABT-869. Complete regression of MV4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Tumors from mice treated with ABT-869 showed decreased proliferation by Ki67 and increased apoptosis by TUNEL staining. We also observed that the mice treated with ABT-869 the day after injection of AML cells remained tumor-free for over 3 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. ABT-869 also suppresses the growth of Molm-13 (human AML cell line that expresses both FLT3-ITD and wt FLT3) at an IC50 between 1 and 10nM. To examine the effects of ABT-869 in vivo, we employed a murine bone marrow transplantation model. After chemical ablation of the bone marrow, SCID mice were injected with Molm-13 cells through the tail vein to allow engraftment. We observed that mice treated with an oral preparation of ABT-869 at 40 mg/kg/day prevented the engraftment of Molm-13 cells. The SCID mice that were not administered ABT-869 demonstrated clinical engraftment with hind leg paralysis and chloroma formation. Chloroma formation was confirmed by immunohistochemical staining with CD33 and CD45. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. We will also determine if there is any difference in efficacy in relation to the FLT3 status of each primary AML sample. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic at the doses studied, and provide rationale for the treatment and prevention of relapse in AML patients.