Tyrosine Phosphoproteomics of FLT3 Signaling.

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Recently, FLT3 has been implicated in the pathogenesis of infant and childhood ALL. FLT3 inhibition has been shown to kill ALL cells with high levels of FLT3 expression. Using immunoaffinity purification of phospho-tyrosine peptides followed by tandem mass spectrometry, we not only identified two new AML cell lines with activated FLT3, but also generated phosphotyrosine profiles for five cell lines that express either WT or ITD mutation of FLT3. We identified six novel tyrosine phosphorylation sites in FLT3. To further investigate the FLT3 pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in FLT3 inhibited versus uninhibited cell lines. Quantitative SILAC experiment identified over 100 proteins, whose tyrosine phosphorylation status are regulated by FLT3. Furthermore, it reveals common and distinct signaling pathways activated by either WT FLT3 in B-ALL or FLT3-ITD in AML. Our study represents a breakthrough in our understanding of FLT3 signaling network. It could have important implications for the design of novel therapeutic approaches and the identification of biomarkers for leukemia with activated FLT3.