Deregulated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including AML 
. However, tyrosine phosphorylation represents less than 2% of total protein phosphorylation 
, making the study of phosphorylated tyrosine residue challenging. For the past decade, our knowledge of FLT3 signaling pathways has been painstakingly accumulated, mainly through the study of individual molecules in certain pathways. In our study, using PhosphoScan®, a phosphotyrosine profiling proteomics approach we profiled 11 leukemia cell lines (8 AML and 3 B-cell ALL), and six primary AML patients. We identified over 1000 tyrosine phosphorylated sites from about 800 proteins. Our study significantly expands the inventory of phosphorylated proteins associated with FLT3 signaling in human AML and B-ALL. Our data thus provide a unique insight into in vivo FLT3 signaling components and their relevant regulatory sites. This global, unbiased view of the in vivo phosphoproteome revealed a large number of cellular proteins phosphorylated, and identified an important subset of proteins and their phosphorylation sites regulated in response to FLT3 inhibitor.
Recent Studies done by Choudhary et al. and Zhang et al., which represent the first two large-scale Phospho-profiling of mutant FLT3 signaling, provide broad view of oncogenic FLT3 signaling to date. However, these studies were performed with overexpression of mutant FLT3 in murine systems. In contrast to the study done by Choudhary et al., which was performed with PKC412 for 12 hours, our study was done with MLN518 for 2 hours, which reflects early changes in protein phosphorylation. We observed FLT3 regulates tyrosine phosphorylation in protein kinases, adaptor/scaffold proteins, and phosphatase. In addition, some FLT3 regulated sites are tied to many previously unknown cellular systems, as demonstrated here by identification of regulated sites on cell surface proteins, cytoskeletal proteins, RNA processing proteins and other important protein classes. The diversity of protein classes found to be regulated by FLT3 inhibitor suggests that FLT3 affects a broad spectrum of signaling pathways and cell functions not limited to proliferation and survival. For example, Y399 of DNMT1 is a drug sensitive site, raising the possibility that that FLT3-ITD regulates the activity of DNMT1, and affects global DNA methylation patterns in FLT3-ITD leukemic cells. Not only does FLT3 activate tyrosine signaling pathways, but it also regulates epigenetic events through phosphorylation of transcription factors on serine/threonine residues. Future studies should be aimed at connecting these data directly to downstream gene-expression changes as measured by microarray technology.
Some of the FLT3 regulated proteins contained more than one regulated site, such as Y67, 88, and 139 of cofilin1 in MV(4;11) cells, as well as Y266 and 476 of GAB2 in Molm 14 cells. Our data also showed that phosphorylation of different sites within the same protein can also be differentially regulated. For example, Y383 and Y598, but not Y208 on GIT1 (G protein-coupled receptor kinase interactor 1) are responsive to MLN518 In MV(4;11). A similar observation was made for tyrosine sites on ENO1 and HCK. This highlights the importance of measuring the degree of site-specific phosphorylation rather than phosphorylation the protein as a whole in order to obtain an accurate picture of a protein's function.
In SEM cells, we observed multiple FLT3 regulated sites on key elements of B-cell receptor signaling pathways such as BCAP, CD19, LAB, DOK1, GAB1, and GAB2. In addition, we confirmed phosphorylation of BCAP is regulated by FLT3. BCAP binds PI3K and plays an essential role in B cell development 
. However, immunoprecipitation with FLT3 antibody did not pull down CD19, BCAP and LAB (data not shown). Thus, FLT3 might indirectly regulate tyrosine phosphorylation of these proteins. Since B cell receptor signaling is involved in B cell development, activation and differentiation 
, oncogenic FLT3 might provides proliferative and survival signal for B cells by regulating components of B cell receptor signaling. It is plausible that oncogenic FLT3 may cooperate with other oncogenic events such as MLL fusions to promote leukemogenesis of B-ALL 
In this study, we identified five FLT3 tyrosine phosphorylation sites in primary leukemia cell lines, adding new information on how phosphorylation may affect the activity of wild-type FLT3 and FLT3-ITD. FLT3 has been implicated in the pathogenesis of both AML and B-ALL. We observed that in both AML and B-ALL cell lines, FLT3 activates many of the similar signaling pathways, such as JAK-STAT pathway, RAS-MAPK pathway, and phospholipase C-gamma ( and ). Yet, a key difference is the apparent recruitment of the BCR complex by FLT3 in B-ALL. Our data is therefore consistent with the observation that FLT3 inhibitors have therapeutic effects in B cell acute lymphoblastic leukemia with high level of FLT3 expression 
. This suggests that FLT3 pathogenesis in B-ALL could depend on elements of that pathway which could be additionally targeted by tailored therapies.
When we compared phosphotyrosine profile between two FLT3-ITD cell lines (MV(4;11) and Molm 14) and three FLT3-ITD AML patients, we noticed that 77% (130 out of 168) of phosphoproteins identified in AML patients were also present in these two cell lines, suggesting that FLT3-ITD cell lines are good models to understand FLT3-ITD signaling for primary patients (Figure S3
). On the other hand, there are a large number of phosphoproteins identified in FLT3-ITD cell lines not present in primary patients. It could be due to the lengthy period required to collect bone marrow and lyse red blood cells, during which a significant amount of tyrosine phosphorylation events were lost. Nonetheless, tyrosine phosphorylated proteins, like FLT3, ESYT1/2 (FAM62A/B), HGS, HSP90AA, INPPL1(SHIP-2), LYN, SF3B4, SHC1, STAT5A, and VASP, are not only enriched in Molm14 and MV(4;11) cell lines with statistical significance (FDR<0.05) and regulated by FLT3 inhibitor, but also present in FLT3-ITD AML patients. While some of these proteins are known components of FLT3 signaling pathways, the function of other proteins remain to be determined.
Several small molecule FLT3 inhibitors are in phase I/II clinical trials. However, the fundamental relationship between FLT3 inhibition, its consequences on downstream signaling, and the onset of cytotoxicity remains poorly understood 
. A recent study showed that cytotoxic responses to CEP-701 and PKC412 were highly heterogeneous and were only weakly associated with FLT3 expression level and mutation status 
. Clearly, if FLT3 small molecule inhibitors were to have an impact on AML treatment, a better understanding of how FLT3 signaling contributes to the disease, and how FLT3 drugs work at the cellular level, would be necessary. This study not only provides a comprehensive view of FLT3 regulated signaling pathways in human AML cell lines, but also gives us an initial glance onto the activation of these pathways in primary AML patient samples. We showed that oncogenic FLT3 affects multiple signaling pathways in human AML patients and regulates proteins from different cellular compartment and diverse cellular processes. Targeting pathways of Fc epsilon RI-mediated signaling, MAPK, BCR, and CD40 signaling may offer new hope to treat FLT3-ITD AML. This study opens the door to a deeper understanding of FLT3 signaling networks, to identify novel and better biomarkers to trace clinical response to FLT3 inhibitors. In addition, this study provides a valuable resource for the scientific community to further investigate oncogenic FLT3 signaling in primary AML patients.