The Src family kinase LCK cooperates with oncogenic FLT3/ITD in cellular transformation

The non-receptor tyrosine kinase LCK belongs to the SRC family of kinases. SRC family kinases are proto-oncogenes that have long been known to play key roles in cell proliferation, motility, morphology and survival. Here we show that LCK regulates the function of the type III receptor tyrosine kinase FLT3 in murine pro-B cells. We observed that expression of LCK significantly enhances the colony forming capacity of the constitutively active FLT3 mutant FLT3-ITD (internal tandem duplication). Furthermore, cells expressing LCK developed tumor earlier compared to cells transfected with empty control vector. Staining of the tissues from mouse xenografts showed higher Ki67 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.

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 malignancies 1,2 . 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 internal tandem duplication (ITD) in the juxtamembrane domain of the receptor. Although the wild-type receptor needs its ligand, FLT3 ligand (FL), to trigger downstream signaling, FLT3-ITD is constitutively active and can activate downstream signaling cascade in the absence of ligand stimulation. The downstream signaling is tightly controlled by associating proteins, which directly or indirectly interact with the activated receptor. Associating proteins include protein kinases, protein phosphatases, ubiquitin ligases and adaptor proteins [5][6][7][8][9][10][11][12] . Protein kinase, such as SYK 6 and FYN 13 , cooperate with oncogenic FLT3-ITD, while CSK 14 and ABL2 15 partially block mitogenic signaling. The protein tyrosine phosphatase DEP1 negatively regulates FLT3-ITD-mediated colony formation 16 and loss of STS1/STS2 function results in hyperactivation of FLT3 11 . In contrast, association of another phosphatase, SHP2, seems to be essential for FLT3-ITD-mediated cellular transformation 17 . 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 SOCS2 18 , SOCS6 19 , SLAP 20 and SLAP2 9 accelerate ubiquitination-directed degradation of FLT3, signaling molecules play diverse roles in regulating mitogenic signaling. For instance, SLAP depletion partially blocked activation of FLT3 downstream signaling cascades 20 while depletion of SOCS6 accelerated mitogenesis 19 . 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 kinases 21 . LCK has important functions in T cell development, homeostasis and activation 22 . LCK knockout mice display a strong decline in the CD4 and CD8 positive thymocyte population and carry only a few peripheral T cells 23 . Although LCK under normal physiological conditions primarily is expressed in T cells and in some subpopulations of B cells 24 , it is highly expressed both in B and T cell leukemia 25,26 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 activation 27,28 . In B-cell leukemia, cells with hyperphosphorylated FLT3 also display high levels of LCK phosphorylation 29 suggesting a possible role of FLT3 in LCK activation or vice versa.
Apart from cells of hematopoietic origin, LCK is also aberrantly expressed in a number of other cancer types, including breast cancer, colon cancer and small cell lung carcinoma [30][31][32] suggesting that it has general cancer promoting activities. Several studies have reported high levels of LCK expression in acute myeloid leukemia. Early studies indicated high expression of LCK in leukemic cells from patients with less differentiated AML, i. e. AML-0 and AML-1 33 . However, a role of LCK in FLT3-dependent AML has not yet been defined. In this report we show, using the proB cell line Ba/F3 that lacks endogenous LCK, that LCK expression is not essential for wild-type FLT3 signaling but plays an important role in oncogenic FLT3-ITD-mediated cellular transformation.

LCK expression is dispensable for FLT3-ITD dependent cell viability and survival. It is long been
known that SRC family kinases (SFKs) play important roles in mitogenic signaling. SFKs act as an intermediate mediator of various receptor tyrosine kinases. We have shown that SRC 34,35 and FYN 13 bind to FLT3 and cooperate with FLT3-ITD in cellular transformation. To understand the role of another SFK, LCK, we generated Ba/ F3 cells stably expressing FLT3-ITD and either empty vector or LCK. FLT3 surface expression was analyzed by flow cytometry (Fig. 1A) and the total protein expression was measured by Western blotting (Fig. 1B). FLT3 surface and total expression appeared to be the same for both LCK and empty vector expressing cells, making it a suitable model for studying the impact of LCK on FLT3-ITD mediated biological events. To investigate whether LCK has any effect on cell growth, we examined the cell viability using PrestoBlue assay. Forty-eight hours post seeding of cells we observed, in comparison to the control, no alteration caused by LCK expression on the number of viable cells, regardless of FLT3 ligand stimulation (Fig. 1C). Furthermore, LCK expression neither increased nor decreased the fraction of apoptotic cells in an annexin V/7-AAD assay (Fig. 1D). Thus, we suggest that LCK expression is not essential for FLT3-ITD induced cell viability or survival in vitro.
LCK expression cooperates with FLT3-ITD in colony formation and tumor formation. Since we did not see any effect of LCK on FLT3-ITD-mediated in vitro cell survival, we asked whether it affects (D) Cells were washed to remove IL3 and seeded in a 12-well plate in IL3 free medium. After 48 h cells were stained with phycoerythrin (PE)-labeled annexin V and 7-aminoactinomycin D (7-AAD). Cells were then analyzed using flow cytometry. Cells positive for annexin V and 7-aminoactinomycin D (7-AAD), or only for annexin V, were counted as apoptotic cells. Not significant, ns.
FLT3-ITD-induced in vitro 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 in FLT3-ITD-mediated cellular transformation. To further verify the in vitro 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 suggest that LCK accelerates the FLT3-ITD-mediated transformation potential in vivo.
LCK expression increases FLT3-ITD-mediated STAT5 phosphorylation. In contrast to the constitutively active oncogenic mutant FLT3-ITD, wild-type FLT3 is dependent on FL stimulation for activation of the PI3K/AKT, RAS/ERK and p38 pathways 36 . In order to study how LCK regulates downstream signaling of FLT3, we therefore generated Ba/F3 cell lines expressing wild-type FLT3 with LCK or empty vector. Similar to the FLT3-ITD experiments, we used flow cytometry and Western blotting to verify equal surface expression (Fig. 3A) and total expression (Fig. 3B) of wild-type FLT3 in cells expressing LCK or empty vector. Ba/F3 cell line expressing wild-type FLT3 requires IL-3 for proliferation and survival, and LCK does not rescue that phenotype (Fig. 3C). LCK also does not influence the apoptotic rate of Ba/F3 cells in response to FL stimulation, while unstimulated cells expressing LCK displayed significant lower level of apoptotic cells (Fig. 3D). To gain insight into the mediators involved in downstream signaling, we looked at the known FLT3 downstream signaling pathways (Fig. 4A). We observed no change in phosphorylation of AKT (Fig. 4B), ERK (Fig. 4C) or p38 (Fig. 4D). However, STAT5 phosphorylation was significantly increased in Ba/F3 FLT3-ITD expressing LCK compared to the empty vector control (Fig. 4E), explaining the increased proliferation rate of the LCK tumors. However, we did not see any change in total FLT3 tyrosine phosphorylation in the absence or presence of LCK expression (Fig. 4F,G). Finally, we showed that FLT3-ITD was associated with LCK (Fig. 4H).

Discussion
Receptor tyrosine kinases transduce signals predominantly through interacting proteins. Therefore, understanding of the function of individual interacting partners of a specific receptor is important for understanding the regulation of downstream signaling cascades. Using an SH2 domain array we have recently shown that several SH2 domain-containing proteins such as ABL2, CRK, FYN, ABL1, RASA1, CRKL, LCK, SOCS6, BLK, TNS1, BRK etc. associate with several phosphotyrosine residues in FLT3 15 . We and others have characterized several of those proteins and demonstrated important roles in FLT3 signaling. The role of SRC family kinases, including SRC, LYN, FYN and HCK, has been outlined. Activation of FLT3 results in elevated tyrosine phosphorylation of LYN and SRC, and inhibitors targeting the SRC family kinases significantly reduced cell viability in FLT3-ITD-dependent AML cell lines, suggesting that the function of SRC family kinases is required for FLT3-induced cell survival 37,38 . While SRC and LYN displayed a role in cell survival, FYN appeared to be involved in FLT3-ITD-mediated cell transformation 13 . A recent report suggests that HCK is involved in FLT3-ITD mediated CDK6 expression and thereby supports cell survival and transformation 39 . Therefore, it is likely that, despite the high structural The blots were cropped to focus upon the specific proteins indicated. (C) Cells were washed to remove IL3 and seeded in a 96-well plate. PrestoBlue cell viability assay was used to measure viable cells. (D) Cells were washed to remove IL3 and seeded in a 12-well plate in IL3-free medium. After 48 h cells were stained with phycoerythrin (PE)-labeled annexin V and 7-aminoactinomycin D (7-AAD). Cells were then analyzed using flow cytometry. Cells positive for annexin V and 7-aminoactinomycin D (7-AAD), or only for annexin V, were counted as apoptotic cells. Not significant, ns; *p < 0.05. similarity between SRC family kinases, individual members play distinct roles in FLT3 downstream signaling. In this report, we define the role of LCK in FLT3 signaling.
The function of LCK has mainly been studied in lymphocytes due to the abundant expression in the lymphoid lineage, in particular in T cells. It is highly expressed in several chronic lymphocytic leukemia's of both B cell and T cell lineages 28,29 . In addition, the myeloid cell line 32D and several non-lymphoid human tumor cell lines also show LCK expression 30,31,34 . Thus, LCK function may not only be restricted to the lymphoid lineage. Several studies have demonstrated a role of LCK in acute myeloid leukemia. Early studies 33 demonstrated higher expression of LCK in less differentiated cases of AML. In a recent proteomics study the role of individual kinases in AML was investigated and a correlation between high expression of LCK correlated with good response to a PI3K/ mTOR-specific inhibitor 40 . Using a bioinformatics approach aiming at identifying relevant therapeutic targets in AML 41 , several transcripts were identified that were differentially expressed between normal bone marrow samples and AML samples. Based on these data, they constructed a protein-protein interaction network and identified, among other proteins, LCK as one of the proteins of the hub nodes. Additionally, activation of FLT3 in AML samples resulted in abundant phosphorylation in the activation loop of LCK 29 .
Taken together, these data collectively suggest that LCK might play a role in FLT3-ITD-mediated AML. Using Ba/F3 cells, lacking endogenous LCK expression, as a model system, we could show that LCK expression did not contribute to overall tyrosine phosphorylation of FLT3 suggesting that LCK does not have a role in the cells expressing either wild-type FLT3 or FLT3-ITD and either LCK or empty vector (EV) were washed to remove IL-3 and starved four hours before FL stimulation. Total cell lysates were subjected to Western blotting analysis using phosphospecific antibodies against AKT, ERK1/2, p38 and STAT5. (B-E) Blots from three independent experiments were quantified. Signals of phosphorylated proteins were normalized against total protein. (F) A fraction of the lysate was used for immunoprecipitation using an anti-FLT3 antibody. The blots were cropped to focus upon the specific proteins indicated. (G) Blots from three independent experiments from experiment F were quantified. Signals of phosphorylated FLT3 were normalized against total FLT3. (H) COS-1 cells were transfected with FLT3-ITD and LCK-FLAG or empty vector. Cells were lysed and lysates were subjected to anti-FLAG antibody immunoprecipitation. Not significant, ns; *p < 0.05. FLT3 activation process. Furthermore, LCK did not contribute to FLT3-ITD-induced in vitro cell viability, but enhanced colony formation capacity, suggesting that LCK regulates distinct signaling pathway downstream of FLT3. This is also supported by the data that STAT5 phosphorylation, but not AKT, ERK1/2 and p38 phosphorylation, was enhanced in the presence of LCK. This is similar to what has been described for PCP-ALL cells, where a PAX5 fusion protein drives overexpression of LCK. In those cells, there is an LCK-dependent hyperphosphorylation of STAT5 42 . Similar to in vitro colony formation data, mice injected with cells expressing LCK and FLT3-ITD developed tumors quicker than cells lacking LCK expression. Collectively, our data suggest that LCK enhances the FLT3-ITD mediated transformation potential by cooperating with STAT5 pathway activation. Thus, LCK is a potential target for the development of selective SRC family kinase inhibitors that could potentially be used together with FLT3 inhibitors to treat patients with FLT3-ITD positive AML.
Cell proliferation, apoptosis, and colony formation assay. Cells were washed three times to remove cytokine before all experiments. annexin V and 7-aminoactinomycin D (7-AAD) apoptosis kit (BD Biosciences) was used to measure apoptosis in cytokine-depleted cells. Cells positive for annexin V and both annexin V/7-AAD were counted as apoptotic cells. To measure cell proliferation, 10,000 cells were seeded into each well of a 96-well plate and incubated for 48 h. PrestoBlue (Thermo Fisher Scientific) was used to measure cell viability. Semi-solid methylcellulose medium (Stem Cell Technologies) was used for colony formation assay. Around 500 cells were seeded and cultured for seven days before counting colonies.
Animal work. NOD/SCID female mice were purchased from Charles River laboratories. 1 × 10 6 control or LCK expressing Ba/F3 FLT3-ITD cells were injected subcutaneously into 7 mice in each group. The tumor progression was monitored for 25 days. On the day of sacrifice, the tumors were measured and stored in 4% PFA for 24 h followed by the standard protocol of fixation. The experiment was performed under ethical permit from the Swedish Animal Welfare Authority following approved guideline.

Statistical analysis.
Where required Western blots from three independent experiments were quantified.
All statistical analyses were performed using the unpaired, two-tailed Student's t-test.