A Potent Src/Abl Kinase Inhibitor (Ap23464) Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by Flt3-Itd Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an Abl-Independent Mechanism.

Abstract

AP23464 is a potent Src and Abl inhibitor (in vitro IC50 < 1 nM), which has been shown to effectively inhibit growth of imatinib-resistant Bcr-Abl+ cells, and thus, is a promising compound for treating patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src versus Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared AP23464 with the PP1, a previously described Src kinase inhibitor (IC50 < 1 uM). We sought to correlate growth inhibition with Src or Abl inhibition.Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, Src or Abl kinase inhibition was analyzed by blotting with a polyclonal phospho-Src (Tyr416) antibody or a polyclonal phospho-Abl (Tyr245) antibody.Results: In MV4-11 cells AP23464 was more potent than PP1 in causing growth inhibition with IC50 at <1 uM vs 2.5 to 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at the lowest dose of AP23464 and PP1 studied (0.1 nM and 1 nM, respectively). Abl was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for AP23464 was 1 uM (grown in IL-3) and 0.01–0.1 uM (grown without IL-3). In Ba/F3-ITD cells, IC50 for phosphoSrc was 0.1 uM for both AP23464 and PP1. Abl was present in Ba/F3-ITD cells, but no phospho-Abl was detected. In Mo7e cells grown in the presence of GM-CSF, the IC50 was 1 uM for AP23464 v. 10 uM for PP1. In Mo7e cells treated with Lyn siRNA, there was >50% growth inhibition with 70% knock-down of Lyn. The IC50 for phosphoSrc was 1 nM for both AP23464 and PP1. Abl was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 1 uM for AP23464 vs. 10 uM for PP1. In western blotting, the IC50 for phospho-Src 416 was detected at 10 nM AP23464. Abl was present in Ba/F3GR cells, but no phosphoAbl was demonstrated (K562 cells served as positive control).Conclusions: AP23464 is more potent than PP1 in causing growth inhibition and Src kinase inhibition in these cell lines that serve as models for acute myeloid leukemia. It is unlikely that Abl is the drug target, because MV4-11 cells do not express detectable Abl, and phospho-Abl was not detected in Mo7e or Ba/F3 cells. These results suggest that inhibition of Src tyrosine kinases contributes predominantly to growth inhibition caused by the Src/Abl kinase inhibitor AP23464 in these cell types.