The Dual SRC/ABL Kinase Inhibitor BMS-354825 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.

BMS-354825 is a dual SRC and ABL inhibitor, which has been shown effective in imatinib-resistant BCR-ABL+ cells, and it is in phase I trials for patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src v. 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 BMS-354825 with PP1, a SRC kinase inhibitor with in vitro IC50 at sub-micromolar concentrations. We sought to correlate growth inhibition with SRC or ABL inhibition. In these myeloid cell lines, LYN is the predominant SRC kinase.

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 BMS-354825 and PP1 caused similar growth inhibition IC50 at 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at 1 nM concentrations of both compounds. Protein expression of ABL was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for BMS-354825 was 1–10 uM (grown in IL-3) and 0.01 uM (without IL-3). The IC50 for PP1 was 1–10 uM (grown in IL-3) and 0.1 uM (without IL-3). Inhibition of phospho-SRC occurred at 10 nM. In Mo7e cells, grown in the presence of GM-CSF, the IC50 was 5 uM for BMS-354825 v. 10 uM for PP1 by MTT assay. By western blotting, inhibition of phospho-SRC 416 occurred at 1 nM for both BMS-354825 and PP1. To determine specific contribution of LYN to Mo7e growth, we treated Mo7e cells with LYN siRNA. With 70% knock-down of LYN, there was 50% growth inhibition. 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 5 uM for BMS-354825 vs. 10 uM for PP1 by MTT assay. In western blotting, inhibition of phospho-Src 416 was detected at 10 nM BMS-354825. ABL was present in Ba/F3GR cells, but no phospho-ABL was demonstrated (K562 cells served as positive control).

Conclusions: BMS-354825 is more potent than PP1 in causing growth inhibition and SRC kinase inhibition in Mo7e and Ba/F3GR cells that serve as models for acute myeloid leukemia. It is unlikely that ABL is the drug target, because MV4-11 cells do not express ABL and phospho-ABL was not found in Mo7e or Ba/F3 cells. These results suggest that inhibition of SRC tyrosine kinases contributes predominantly to growth inhibition caused by the dual SRC/ABL kinase inhibitor BMS-354825 in myeloid cell lines expressing Flt3-ITD and cytokine-driven proliferation and survival via the IL-3/GM-CSF Receptor or G-CSF Receptor.