PF-114, a Novel Inhibitor of Bcr-Abl Chimeric Tyrosine Kinase, Attenuates Intracellular CrkL Phosphorylation and Kills Chronic Myeloid Leukemia Cells

Abstract

Background & Aims. The chimeric tyrosine kinase Bcr-Abl triggers malignant transformation of myeloid cells via phosphorylation of a number of substrates including the CrkL adaptor protein. Pharmacological inhibition of Bcr-Abl mediated signaling is a major strategy in treatment of patients with chronic myeloid leukemia (CML). A new specific Bcr-Abl inhibitor (PF-114) was designed using a molecular modeling approach. The paper defines the cytotoxicity of PF-114 against CML cells and its effect on the CrkL phosphorylation. Methods. The cytotoxicity was determined using the MTT assay. The total intracellular CrKL pool (phosphorylated and non-phosphorylated forms) was determined by means of flow cytometry. Results. Exposure of Bcr-Abl-positive, K562 cell line to PF-114 blocked intracellular CrkL phosphorylation and caused cell death. In contrast, virtually no phosphorylated CrkL was detectable in Bcr-Abl-negative HL60, U937 and Jurkat leukemia cell lines. Conclusion. Absence of phosphorylation in Bcr-Abl-negative cells (HL60, U937 and Jurkat) and death of HL60 cells under the effect of PF-114 at concentrations exceeding those required to kill K562 cells supports the emergence of PF-114 as a promising drug candidate for CML.

Keywords

• chronic myeloid leukemia
• Bcr-Abl tyrosine kinase
• protein phosphorylation
• flow cytometry
• cytotoxicity