Syk Is a New Target for AML Differentiation.

One of the challenges of phenotype-based small molecule screening has been the difficulty of protein target identification for newly discovered compounds. For example, we previously performed a gene expression-based small molecule library screen, which identified gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, as an inducer of acute myeloid leukemia (AML) differentiation in cell lines and primary patient cells. Neither EGFR transcript nor protein is expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. However, because multiple EGFR inhibitors induce this phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized a proteomics method: peptide immunoprecipitation-HPLC-mass spectrometry. This approach enriches for phospho-tyrosine peptides with immunoprecipitation (IP) after enzyme digestion. In contrast to IP at the protein level, the final mixture following peptide-IP contains primarily phospho-tyrosine-containing peptides. We treated the AML cell line HL-60 with gefitinib versus vehicle and then identified peptide sites with loss of phosphorylation with gefitinib treatment. Spleen tyrosine kinase (Syk) was identified as one of the only kinases with loss of phosphorylation post treatment. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in malignancies such as myelodysplastic syndrome and lymphoma. We first confirmed with IP western immunoblotting the inhibition of Syk phosphorylation with gefitinib treatment in HL-60 cells. We next confirmed with both pharmacological inhibition and genetic loss of Syk the induction of differentiation in the AML cell lines HL-60 and U937. Numerous determinants of myeloid maturation were tested: a complex differentiation gene expression signature, cellular morphology, cell surface proteins CD11b and CD14 expression, and nitro-blue tetrazolium (NBT) reduction, a functional assay for myeloid differentiation. Two reported pharmacological inhibitors of Syk scored positive on all measurements of differentiation. Furthermore, the shRNA construct inducing the most complete loss of Syk also scored the highest on all measurements of differentiation. The most convincing data that Syk is the true target of these inhibitors would be the identification of an inhibitor resistant Syk mutant, analogous to BCR-ABL or c-Kit mutants that confer imatinib resistance. To this end, a Syk random mutagenesis screen is ongoing. In summary, these data identify Syk as a strong candidate target of gefitinib, demonstrate that inhibition of Syk can induce AML differentiation, and identify Syk as a potential target for AML differentiation therapy.