Constitutively active internal tandem duplication (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (FLT3) is the most common molecular defect associated with acute myleoid leukemia. Its presence corresponds to a poor outcome in patients with acute myleoid leukemia who receive conventional chemotherapy. Therefore, FLT3-ITD has been considered to be an attractive molecular target for a novel therapeutic modality. Although several kinds of FLT3 inhibitors have been established in vitro, their activities as single agents have not been satisfactory in clinical studies. These observations encouraged us to seek for other classes of FLT3 inhibitors.

Recent studies showed that antitumor imidazoacridinone C-1311, synthesized in our Department and selected for phase II clinical trials, is highly potent inhibitor of certain receptor tyrosine kinase (RTKs), with nanomolar potency against FLT3 kinase.

The aim of this work was to investigate whether our next acridinone derivative,  triazoloacridinone C-1305, a structural analog of C-1311, exhibited inhibitory activity towards FLT3 kinase in vitro. The antitumor C-1305 is DNA-damaging agent selected for extended preclinical trials.

Here, we tested C-1305 on two human leukemic cell lines with contrasting FLT3 status; MV4;11 cells with internal tandem duplications (ITD) mutation in the FLT3 receptor versus RS4;11 cells expressing wild type FLT3. Treatment of FLT3-ITD-positive cells, MV4;11 with C-1305 for 72 h suppressed cells’ proliferation with an EC₅₀ concentration of the drug 0,2 mM. In contrast, significantly higher concentration of the drug (EC₅₀ 1,8 mM) was required to inhibit the growth of RS4;11 FLT3-WT cells. The direct effect of C-1305 on FLT3 receptor activation in MV4;11 cells was determined by analysis of its phosphorylation status. C-1305 reduced FLT3 phosphorylation in a time-dependent manner but at concentrations higher than EC₅₀ value (between 5 and 10 µM).  To determined whether the cytotoxic effect of C-1305 on MV4;11 FLT3-ITD-positive cells was due to the induction of apoptosis, we conducted annexin V binding assay as well as analysis of sub-G1 DNA fraction using flow cytometry. In both tests, the number of apoptotic cells increased after treatment with C-1305 in a time– and dose-dependent manner. The morphological examination of MV4;11 cells further confirmed the presence of apoptotic cells following C-1305 treatment, as evidenced by chromatin condensation and formation of apoptotic bodies. Importantly, apoptotic cell death was observed at concentrations of C-1305 required to block FLT3 phosphorylation suggesting that inhibition of FLT3 kinase by C-1305 may account for its cytotoxic activity in MV4;11 cells.

The overall results suggest that C-1305 is new inhibitor of FLT3 kinase in vitro which exerts potent activity towards acute leukemia cells harboring activating mutations of FLT3 tyrosine kinase.  

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