Abstract
Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.