Bis(1H-indol-2yl)methanones Are Effective Inhibitors of Mutated FLT3 Tyrosine Kinase, Overcome Resistance to PKC412A In Vitro and Show Synergy with Chemotherapy.

Abstract

Inhibition of mutated FLT3 receptor tyrosine kinase has become a promising therapeutic strategy in AML. Currently, a phase III trial testing the tyrosine kinase inhibitor (TKI) PKC412A in combination with induction chemotherapy is under way. Development of resistance to PKC412A mediated by secondary FLT3 mutations has been described recently (Heidel et al., Blood 2006). This observation may have an increasing impact on duration of response in future clinical trials. Herein we investigated two novel TKIs (methanones) to determine their efficacy on FLT3-ITD transfected 32D cells as well as on primary ITD positive AML blasts.The bis(1H-indol-2-yl)methanone compounds Cpd.98 and Cpd.102 (Mahboobi et al. J. Med. Chem. 2006) that have recently shown efficacy in proliferation assays using FLT3-ITD transfected cells were characterized. Western Blotting experiments of murine 32D-FLT3-ITD cells confirmed dose dependent dephosphorylation of FLT3-ITD and its downstream target STAT5 upon incubation with Cpd.98 and Cpd.102, respectively. To define a non-myelotoxic range of these inhibitors, bone-marrow (BM) cells of healthy donors were tested with different methanone concentrations to determine the rate of apoptosis (sub G1 fraction in cell cycle analysis). No significant increase in apoptosis was seen up to 3μM of inhibitor concentration for either compound.Additionally, colony assays of healthy BM-cells also showed no significant reduction of colony formation up to 3μM. To determine the efficacy of these compounds in PKC412A-resistant cells, transfected 32D cells containing the previously described resistance mediating mutation (32D-FLT3-ITD-N676K) were also tested at different concentrations for induction of apoptosis. Resistance in these cells could be overcome by 300nM (Cpd.102) and 1μM (Cpd.98), respectively.As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both compounds in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Both compounds showed synergistic effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays.Primary AML blasts harbouring the FLT3-ITD length mutation were also incubated with both compounds to determine the efficacy in patient material. These experiments revealed apoptosis rates of up to 30% upon incubation for 72 hours. In conclusion bis(1H-indol-2-yl)methanone compounds overcome resistance mediated by the previously described FLT3-ITD mutation N676K in vitro and show efficacy on FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Experiments evaluating toxicity in murine models are currently planned to explore a possible clinical application.