The non-receptor tyrosine kinase LCK is one of the SRC family of kinases

The non-receptor tyrosine kinase LCK is one of the SRC family of kinases. staining in cells expressing LCK suggesting that expression of LCK enhances the FLT3-ITD-mediated proliferative capacity. LCK expression did not affect either FLT3-WT or FLT3-ITD -induced AKT, ERK1/2 or p38 phosphorylation. However, LCK expression significantly enhanced FLT3-ITD-mediated STAT5 phosphorylation. Taken together, our data suggest that LCK cooperates with oncogenic FLT3-ITD in cellular transformation. Introduction Oncogenic mutations or overexpression of tyrosine kinases are very common in a wide range of cancers. Several members of type III receptor tyrosine kinases including FLT3, KIT and CSF1R have been implicated in hematopoietic malignancies1,2. FLT3 was found to be mutated in as high as 35% of?acute myeloid leukemia (AML) and in a small portion of acute lymphoblastic leukemia (ALL)3,4. One of the most common FLT3 mutations includes the inner tandem duplication (ITD) in the juxtamembrane site from the receptor. Even though the wild-type receptor requirements its ligand, FLT3 ligand (FL), to result in downstream signaling, FLT3-ITD is dynamic and may activate downstream signaling cascade in the absence constitutively?of ligand stimulation. The downstream signaling can be managed by associating proteins, which or indirectly connect to the turned on receptor directly. Associating proteins consist of proteins kinases, proteins phosphatases, ubiquitin ligases and adaptor protein5C12. Proteins kinase, such C3orf13 as for example FYN13 and SYK6, cooperate with oncogenic FLT3-ITD, while CSK14 and ABL215 stop mitogenic signaling partially. The proteins tyrosine phosphatase DEP1 adversely regulates FLT3-ITD-mediated colony formation16 and lack of STS1/STS2 function leads to hyperactivation of FLT311. On the other hand, association of another phosphatase, SHP2, appears to be needed for FLT3-ITD-mediated mobile transformation17. These findings suggest that?the role of protein kinases or phosphatases cannot be simplified and specific kinase or phosphatase can act as negative or positive regulators of FLT3 signaling. Furthermore, although several E3 ubiquitin ligases such as SOCS218, SOCS619, SLAP20 and SLAP29 accelerate ubiquitination-directed degradation of BTZ043 FLT3, signaling molecules play diverse roles in regulating mitogenic signaling. For instance, SLAP depletion partially blocked activation of FLT3 downstream signaling cascades20 while depletion of SOCS6 accelerated mitogenesis19. Therefore, knowledge of individual FLT3 interacting proteins is required in order to understand how FLT3 downstream signaling is regulated. The lymphocyte-specific protein tyrosine kinase, LCK, is a member of the SRC family of kinases (SFKs). SFKs are a family of 11 non-receptor tyrosine kinases21. LCK has important functions in T cell development, homeostasis and activation22. LCK knockout mice display a strong decline in the CD4 and CD8 positive thymocyte population and carry only a few peripheral T cells23. Although LCK under normal physiological conditions primarily is expressed in T cells and in some subpopulations of B cells24, it is highly expressed both in B and T cell leukemia25,26 and contributes to the malignant phenotype. Loss of LCK expression in T-cell leukemia cells, or peripheral T lymphocytes, results in impaired T cell receptor activation27,28. In B-cell leukemia, cells with hyperphosphorylated FLT3 also display high levels of LCK phosphorylation29 suggesting a possible role BTZ043 of FLT3 in LCK activation or cell survival, we asked whether it affects FLT3-ITD-induced colony formation. We observed that the potential to form colonies in the semi-solid medium was significantly increased in cells expressing LCK when compared to cells expressing empty vector control (Fig.?2A). However, the size of the colonies remained basically unchanged compared to controls (Fig.?2B). This suggests that LCK might play a role BTZ043 in FLT3-ITD-mediated cellular transformation. To further verify the findings, NOD/SCID mice were injected subcutaneously with Ba/F3-FLT3-ITD cells transfected with LCK or empty vector. After 25 days mice were sacrificed and the total volume of the tumors was measured. We could show that LCK expression significantly increased the tumor size in xenografted mice (Fig.?2C). To investigate whether the increased tumor size of LCK mice was due to an increase in proliferation, we stained tumor tissues for Ki67 and observed that tumors expressing LCK showed higher Ki67 staining, indicative of a higher proliferative potential (Fig.?2D). Therefore, we claim that LCK accelerates the FLT3-ITD-mediated change tumor and potential development cell viability, but improved colony formation capability, recommending that LCK regulates specific signaling pathway downstream of FLT3. That is backed by the info that STAT5 phosphorylation also, BTZ043 however, not AKT, ERK1/2 and p38 phosphorylation, was improved in the current BTZ043 presence of LCK. That is similar from what has been referred to for PCP-ALL cells, in which a PAX5 fusion proteins drives overexpression of LCK. In those cells, there can be an LCK-dependent hyperphosphorylation of STAT542. Just like colony development data, mice injected with cells expressing FLT3-ITD and LCK developed tumors faster than cells lacking LCK expression. Collectively, our data claim that LCK enhances the FLT3-ITD mediated change potential by cooperating.