mTOR signaling is activated by FLT3 kinase and promotes survival of FLT3-mutated acute myeloid leukemia cells

Activating mutations of the FLT3 gene mediate leukemogenesis, at least in part, through activation of PI3K/AKT. The mammalian target of rapamycin (mTOR)-Raptor signaling pathway is known to act downstream of AKT. Here we show that the mTOR effectors, 4EBP1, p70S6K and rpS6, are highly activated in cultured and primary FLT3-mutated acute myeloid leukemia (AML) cells. Introduction of FLT3-ITD expressing constitutively activated FLT3 kinase further activates mTOR and its downstream effectors in BaF3 cells. We also found that mTOR signaling contributes to tumor cell survival, as demonstrated by pharmacologic inhibition of PI3K/AKT/mTOR, or total silencing of the mTOR gene. Furthermore, inhibition of FLT3 kinase results in downregulation of mTOR signaling associated with decreased survival of FLT3-mutated AML cells. These findings suggest that mTOR signaling operates downstream of activated FLT3 kinase thus contributing to tumor cell survival, and may represent a promising therapeutic target for AML patients with mutated-FLT3.


Background
The FMS-like tyrosine kinase-3 (FLT3) receptor, also known as CD135, is a tyrosine kinase type III normally involved in hematopoietic progenitor cell proliferation, survival, and differentiation. Previous studies have shown that constitutive activation of FLT-3 is involved in leukemogenesis, partially through phosphorylation/ activation of the serine-threonine kinase AKT, which occurs in a substantial subset of acute myeloid leukemia (AML) cases [1,2]. A frequent mechanism of FLT3 activation is mutation of the FLT3 gene, either internal tandem duplication (ITD) within the juxtamembrane domain (FLT3-ITD), or point mutations within the activation loop of the second tyrosine kinase domain (FLT3-D835/836). FLT3 gene mutations are found in approximately one third of AML patients, and are associated with inferior prognosis [3].
Recent studies have shown that rapamycin, and its analogs, have substantial antitumor activity in hematologic malignancies, including AML [7,8]. However, the significance of FLT3 gene mutation in the activation of the mTOR pathway is not clear. In this study we hypothezised that the mTOR signaling pathway has an oncogenic role in FLT3-mutated AML cells. We show that mTOR signaling is highly activated in FLT3-mutated AML cell lines and primary cells. We also demonstrate that total inhibition of mTOR signaling results in cell death, specifically of FLT3-mutated AML cells, whereas inhibition of the FLT3 kinase results in downregulation of mTOR signaling. Our findings suggest that mTOR signaling operates downstream of mutated FLT3 kinase and that AML patients harboring FLT-3-mutations may benefit from experimental therapies that target mTOR signaling.

Results and Discussion
The mTOR signaling pathway is activated in FLT3mutated AML cells Initially, we investigated the mTOR pathway activation status by immunohistochemistry and Western blot analysis of bone marrow samples and primary peripheral blood AML cells, respectively, in patients harboring FLT3 mutations. Imunohistochemical analysis showed that 10/12 (83%) p-mTOR, 10/13 (77%) p4E-BP1, 13/14 (93%) p-p70S6K, and 11/12 (83%) p-rpS6 indicating mTOR activation. All AML tumors (n = 6) with dual mutations (FLT3-ITD and tyrosine kinase point mutations) were positive for p-mTOR, p-p70S6K, and p-rpS6. Also, all AML tumors (n = 15) expressed high levels of eIF4E ( Figure 1A). This is the first immunohistochemistry study of mTOR signaling proteins in AML. We also showed activated mTOR signaling by Western blot analysis ( Figure 1), in agreement with others [9]. To further investigate a possible causal association between FLT3 mutation and the mTOR activation status, we used stably transfected FLT3-mutant BaF3 cells. Introduction of FLT3-ITD in BaF3 cells resulted in activation of mTOR signaling ( Figure 1).

mTOR Signaling contributes to FLT3-mutated AML cell survival
We assessed the role of mTOR signaling in the regulation of cell growth and survival of AML cell lines. Most previous studies investigating this issue were based on rapamycin treatment of AML cells resulting frequently in conflicting conclusions [10,11]. However, recently it was realized that rapamycin not only can indirectly and unpredictably inhibit the rapamycin-insensitive mTORC2 after prolonged treatment, but also frequently inhibits partially and not totally mTORC1, which is regarded as the specific target of rapamycin. For this reason, we employed the pharmacologic PI3K inhibitor LY294002, known to inhibit both the PI3K and mTOR kinase activity with the same kinetics, or total silencing of the mTOR gene using an mTOR-specific siRNA [12,13].
As shown in the Figure 1, treatment with LY294002 resulted in a dose-dependent decrease in phosphorylation of AKT, mTOR, 4E-BP1, p70S6K, and rpS6, associated with reduced cell viability due to increased apoptosis (additional file 1, Figure S1). Although changes in the levels of mTOR signaling proteins are similar in both FLT3-mutated AML cell lines, it seems that MV4-11 cell viability depends more on mTOR signaling (Figure 1, Dright, and additional file 1, Figure S1) and this difference is also observed after combined inhibition of mTOR with rapamycin and FLT3 with GTP14564 (additional file 2, Figure S2). The biologic explanation for this finding is uncertain. The latter biologic effects were linked to downregulation of anti-apoptotic proteins involved in both extrinsic and intrinsic (mitochondrial) apoptotic pathways (additional file 1, Figure S1). In addition, transient transfection with mTOR-specific siRNA resulted in decreased phosphorylation/activation of 4EBP1, p70S6K, and rpS6 in a concentration-dependent manner ( Figure 1). Of note, mTOR silencing was associated with decreased levels of p-AKT indicating that mTOR/Rictor complex may contribute to AKT activation in this cell system. Silencing of mTOR also resulted in decreased viability of the AML cell lines, MV4-11 and MOLM13 (both mutated-FLT3) as opposed to Wt-FLT3 U937 cells which were unresponsive ( Figure 1).
Taken together, our findings from mTOR gene silencing and pharmacologic studies suggest that mTOR signaling may contribute to the survival of AML cells harboring FLT3 mutations, and are concordant with the results of other studies [8,14,15].

mTOR signaling acts downstream of mutated-FLT3 Kinase
To further test this hypothesis, we evaluated the selective FLT3 inhibitor, GTP-14564, on mTOR signaling in AML cells. No changes in activation of mTOR signaling proteins, as well cell proliferation or death were observed in Wt-FLT3 U937 cells after treatment ( Figure 2). However, treatment of the FLT3-mutated AML cell lines MV4-11 and MOLM13 resulted in a concentration-dependent decrease of p-mTOR, p-p70S6K, p-rpS6 and p-4EBP1. These changes were slightly more pronounced in the homozygously mutated MV4-11 compared with the heterozygously mutated MOML13 ( Figure 2). Unexpectedly, AKT (p-AKT) activation was affected minimally or not at all (Figure 2). The biologic explanation for this finding is uncertain, however, the possibility that activation of the mTOR signaling pathway induced by mutated FLT3 kinase may partly involve AKT-independent mechanisms cannot be excluded. These changes were accompanied by a proportional decrease in cell proliferation, and increase in cell death (Figure 2). These biologic effects were further augmented when combined treatment with GTP-14564 and Rapamycin was used (additional file 2, Figure  S2). Downregulation of mTOR signaling accompanied by similar biologic effects were also observed after GTP-14564 treatment of FLT3-mutated primary AML cells  ( Figure 2). These data provide evidence that mTOR signaling is an important downstream mediator of the oncogenic pathway activated by mutated FLT3 kinase (Figure 3), and that direct inhibition of mTOR may possibly augment the therapeutic potential of FLT3 inhibitors [16]. Our findings are in agreement with previous observations that rapamycin in combination with the FLT3 inhibitor, PKC-412, markedly inhibits cell proliferation in murine BaF-FLT3-ITD cells and AML samples bearing FLT3 gene dual mutations of ITD and point mutation types, and that rapamycin inhibits cell proliferation in murine 32D-FLT3-ITD cells [2,8,17].

Conclusions
In conclusion, our results taken together suggest that FLT3 mutations may lead to activation of mTOR signaling pathway and thus contribute to tumor cell survival and growth in AML (Figure 3). Therefore, selective inhibition of mTOR signaling pathway seems to be a promising therapeutic target for patients with FLT3mutated AML.

Immunohistochemistry
Fifteen formalin-fixed, paraffin-embedded, AML bone marrow samples (9 harboring FLT3-ITD mutation, and 6 harboring dual, FLT3-ITD and point mutations) were used for the construction of a tissue microarray. Immunohistochemistry was performed using histologic sections of the tissue microarray or cell blocks, and antibodies specific for p-mTOR, p-p70S6K, p-rpS6, p-4E-BP1, and eIF4E (Cell Signaling Technology) as previously described [19]. AML cases were considered immunohistochemically positive for any of the proteins analyzed if at least 10% of tumor cells showed expression after examination of 10 representative high-power fields (magnification ×400). The study was approved by The University of Texas M.D. Anderson Cancer Center institutional review board.

Cell Death, Apoptosis and Proliferation Studies
Cell viability was evaluated by trypan blue exclusion cell assay in triplicate. Annexin-V staining (BD Biosciences Pharmingen, San Diego, CA), detected by flow cytometry, was used to assess apoptosis, as previously described [20].

Statistical analysis
Data for cell viability, growth and apoptosis are shown in diagrams as mean +/-SD and their differences between different cell lines and concentrations were evaluated using Student t-test. p-values < 0.05 were considered statistically significant.

Additional material
Additional file 1: Inhibition of the AKT-mTOR signaling pathway results in apoptotic cell death of AML cells harboring mutated FLT-3 ( Figure S1). A. Pharmacologic inhibition of the AKT-mTOR signaling pathway by using 10 μg/ml LY249002 resulted in decreased cell viability and apoptotic cell death by 32% and 31% respectively in MOLM13 cells harboring heterozygously mutated FLT3 and by 48% and 75% respectively in MV4-11 cells harboring homozygously mutated FLT3, 48 hours after treatment (p < 0.05) ( Figure S1A). B. Western blot analysis showed that LY249002-mediated cell death was accompanied by downregulation of the inhibitor of the extrinsic apoptotic pathway FLIP S/ L and the inhibitor of the intrinsic apoptotic pathway BCL-XL in both MOLM13 and MV4-11 cells ( Figure S1B).
Additional file 2: Combined treatment of AML cells with a FLT-3 inhibitor and a small dose of rapamycin results in enhanced cytotoxicity specifically in AML cells harboring mutated FLT-3 ( Figure S2). Combination of 5 nM GTP14564 with 1 nM rapamycin resulted in enhanced apoptotic cell death, by 28% and 63%, in MOLM13 and MV4-11 cells harboring heterozygously and homozygously mutated FLT3 respectively (p < 0.05) ( Figure S2), as compared to 19% and 15% induced by 5 nM GTP14564 alone ( Figure 2). No effect was observed in U937 cells harboring wt-FLT3.