Kinome Profiling in Acute Myeloid Leukemia as a New Approach for Target Discovery.

Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by a multitude of genetic events. Activating mutations in receptor tyrosine kinases have been identified in 50% of AML primary blasts, and deregulation of one or more signal transduction pathways including the JAK/STAT, RAS/Raf/MEK/ERK, and PI3K/AKT pathways is common (Kornblau et al., 2006). High-throughput procedures which generate comprehensive descriptions of cellular signaling without a priori assumptions in each sample, would enable us to directly assess a broader range of targets for future treatment strategies. In the present study we identified kinase activity profiles in 22 pediatric leukemia samples (6 acute myeloid leukemia, 9 acute lymphoid leukemia, 3 Philadelphia positive acute lymphoid leukemia and 5 chronic myeloid leukemia) and in various normal tissues such as colon and kidney. Peptide phosphorylation profiles were determined using the Pamchip® tyrosine kinase micro array system (Pamgene International B.V., ‘s Hertogenbosch, the Netherlands). This array consists of 144 peptides representing key phosphorylation sites of proteins known to be involved in signal transduction processes. We generated a comprehensive description of the phosphotyrosine proteome of all patient samples. Within the variety of profiles in the various leukemia samples, peptide corresponding with phosphorylation consensus sequences for MAP kinases showed remarkable high levels of phosphorylation, whereas in various normal tissue types substantial lower activity was observed on these substrates. These results imply activated MAPK signaling to be a prominent characteristic of leukemia as already described (Towatari et al., 1997). A differential phosphorylation pattern was seen for the RON peptide (macrophage stimulating 1 receptor (c-met-related tyrosine kinase)), only phosphorylated in one of the AML samples, 4 out of 5 CML samples and one Ph+ ALL sample. According to Phospho-ELM and the literature (Follenzi et al., 2000) this tyrosine kinase residue can be phosphorylated via autophosphorylation by RON kinase or transphosphorylation by MET kinase (met proto-oncogene (hepatocyte growth factor receptor)).To verify the role of MET kinase in AML, a cell survival assay was performed with the selective MET kinase inhibitor PHA 665752. Dose dependent decrease in cell survival was observed in three primary AML samples tested with LC50 concentrations ranging from 2 μM to 5 μM. In conclusion, this study describes a new high throughput technique to generate tyrosine phospho-proteomes or even kinome profiles of various leukemic samples in the future. With this technique we found MET to be a new therapeutic target in AML, and it demonstrates the importance of MAP kinase signaling in various leukemic samples. In the era of a rapidly increasing number of small molecule inhibitors this technique will enable us to rapidly identify new potential targets in different kinds of leukemia.