Novel Flt-3/KDR inhibitor is effective against activated Flt-3 and is cytotoxic against Flt-3-driven leukemias in vitro and in vivo

5629 Flt-3 is a hematopoietic progenitor cell receptor that promotes stem cell proliferation, development and survival. Flt-3 also plays a role in leukemogenesis, being both highly expressed and frequently mutated in adult and pediatric acute leukemia. Mutations in Flt-3 result in a constitutive activation of the receptor tyrosine kinase, conferring ligand independence for growth and for activation of Flt-3 signaling pathways. The most frequently identified mutation in Flt-3 is an internal tandem duplication (ITD), an in-frame insertion of 2-60 amino acids which duplicates a sequence within the juxtamembrane domain of Flt-3. Additionally, point mutations or a single-residue deletion in the activation loop of the tyrosine kinase domain have been identified. The presence of these mutations correlates with an increased risk for relapse and is an indicator of poor prognosis for response to currently available therapy. The transforming potential of Flt-3 was shown to be dependent on its kinase activity, a finding that has led several companies to develop small molecule kinase inhibitors for treating Flt-3-driven leukemias. To provide proof-of-concept support for a leukemic indication for Flt-3 inhibitors, we cloned the most prevalent Flt-3 mutations and established cell-based assays to evaluate their sensitivity to inhibitors. Compounds in the indolyl quinolinone series were identified as potent inhibitors of wild-type as well as of mutant Flt-3 proteins in biochemical (IC 50 range = 9-24 nM) and cellular phosphorylation assays (IC 50 = 1-76 nM). Inhibition of receptor phosphorylation resulted in dose-dependent anti-proliferative effects, including G1 arrest and apoptosis in cells dependent on Flt-3 for growth. In vivo studies in nude mice bearing MV4;11 (ITD-Flt-3) human xenografts demonstrated that chronic treatment with the inhibitor led to inhibition of tumor Flt-3 activity and caused tumor regressions. These observations provided support for a clinical trial of a novel orally bioavailable Flt-3 kinase inhibitor in AML patients. Pharmacodynamic responses were observed in bone marrow and peripheral blood from leukemic patients with an estimated potency of 61 nM against constitutively activated Flt-3.