Internal tandem duplication (ITD) mutations in the FLT3 tyrosine kinase have been detected in ∼20% of acute myeloid leukemia (AML) patients. Patients harboring FLT3/ITD mutations have a relatively poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival. Previous studies have shown that FLT3/ITD activates the signal transducers and activators of transcription 5 (STAT5), p42/p44 mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase (ERK) 1/2], and phosphatidylinositol 3-kinase/Akt pathways. We herein provide biochemical and biological evidence that ribosomal S6 kinase 1 (RSK1) and protein kinase A (PKA) are the two principal kinases that mediate the antiapoptotic function of FLT3/ITD via phosphorylation of BAD at Ser112. Inhibiting both MAPK kinase (MEK)/ERK and PKA pathways by a combination of U0126 (10 μmol/L) and H-89 (5 μmol/L) reduced most of BAD phosphorylation at Ser112 and induced apoptosis to a level comparable with that induced by FLT3 inhibitor AG1296 (5 μmol/L) in BaF3/FLT3/ITD cells. RNA interference of RSK1 or PKA catalytic subunit reduced BAD phosphorylation and induced apoptosis. The MEK inhibitor U0126 and/or the PKA inhibitor H-89 greatly enhanced the efficacy of the FLT3 inhibitor AG1296, suggesting that combining FLT3/ITD downstream pathway inhibition with FLT3 inhibitors may be a viable therapeutic strategy for AML caused by a FLT3/ITD mutation.
Certain mutations in receptor tyrosine kinases (RTK) cause constitutive activation of these enzymes, potentially resulting in malignancy (1). Among these RTKs, FLT3 (fms-like tyrosine kinase) has recently been found to be involved in the pathogenesis of acute myeloid leukemia (AML). Two types of FLT3 mutations have recently been detected in patients with AML: (a) internal tandem duplications (ITD; inserted repeat sequences spanning from <7 to >30 amino acids) in the juxtamembrane domain in ∼20% (2–4) and (b) substitution mutations in the activation loop (usually D835Y) in ∼7% (5, 6). Patients harboring FLT3/ITD mutations have a relatively poor prognosis (7–9), especially when the other allele is mutated and/or lost (10). Both FLT3/ITD and FLT3/D835 mutations result in autophosphorylation and activation of the receptor kinase (11–14). Syngeneic mice injected with 32D cells carrying FLT3/ITDs develop myeloproliferative disease (14, 15). Similarly, transplantation of FLT3/ITD-transduced bone marrow cells causes myeloproliferative disease in another mouse model (16).
Normal hematopoietic cells depend on growth factors, such as interleukin-3 (IL-3), for survival and proliferation, whereas leukemic cell lines and primary leukemic cells often become partially or completely factor independent. IL-3-dependent survival in BaF3 cells is mediated by various signaling pathways, including the MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK; refs. 17–19), phosphatidylinositol 3-kinase (PI3K; refs. 20–23), protein kinase A (PKA; ref. 24), and signal transducers and activators of transcription 5 (STAT5; ref. 25) pathways.
The Bcl-2 family members are downstream of antiapoptotic signals, such IL-3. Such proteins include both proapoptotic (e.g., BAD) and antiapoptotic (e.g., Bcl-2 and Bcl-XL) species (26–28). The BAD function is inactivated by phosphorylation in response to survival factors, such as IL-3, nerve growth factor, and insulin-like growth factor (18, 20, 24, 29–31). Unphosphorylated BAD forms a heterodimer with the Bcl-2 homologue Bcl-XL, thereby inhibiting its antiapoptotic function (31–33). When BAD is phosphorylated at Ser112 and/or Ser136, it forms a complex with the 14-3-3 protein in the cytosol preventing binding to Bcl-2 or Bcl-XL on the mitochondrial membrane (31, 34, 35). IL-3-mediated activation of the MEK (17, 18), PI3K (20), and PKA (24) pathways results in BAD phosphorylation. IL-3-mediated activation of STAT5 up-regulates the expression of Bcl-XL and promotes cell survival (25). Constitutive activation of STAT5 leads to expression of a set of its target genes, including Bcl-XL, and confers IL-3 independence (36). Ribosomal S6 kinase 1 (RSK1) mediates the MEK/ERK pathway cell survival signal via BAD phosphorylation at Ser112 (18). PKA also have been shown to mediate IL-3-induced BAD phosphorylation at Ser112 (24). Akt, in the PI3K pathway, has been implicated in the phosphorylation of BAD at Ser136 (20, 29), but the importance of the role of Akt in BAD phosphorylation has been questioned (24).
In association with factor-independent survival and growth in stably transduced 32D and BaF3 cells, FLT3/ITD has been shown previously to activate STAT5, p42/p44 mitogen-activated protein kinase (MAPK), and PI3K/Akt pathways (13, 14). Constitutive STAT5 phosphorylation has been observed widely in AML patients, including that caused by FLT3 phosphorylation (37). Inhibiting FLT3/ITD reduced the phosphorylation of STAT5a and expression of Bcl-XL in FLT3/ITD transduced cells (38). FLT3/ITD inactivates the proapoptotic BAD by phosphorylation. However, inhibiting both MEK/ERK and PI3K pathways is not sufficient for inducing apoptosis in FLT3/ITD-transduced BaF3 cells (38).
This study provides biochemical and biological evidence that p90RSK (RSK1) in the MEK/ERK pathway and PKA are the two principal kinases that mediate the antiapoptotic function of FLT3/ITD via phosphorylation of BAD at Ser112. Pharmaceutical inhibition of FLT3/ITD, MEK, and/or PKA reduced BAD phosphorylation at Ser112 and induced apoptosis. Combined inhibition of MEK and PKA essentially eliminated the phosphorylation of BAD at Ser112 and synergistically induced apoptosis. Inhibition of RSK1 or PKA with small interfering RNAs (siRNA) supports the conclusion made from pharmaceutical inhibition. These findings provide an augmented understanding of FLT3/ITD-induced leukemogenesis and suggest potential therapeutic targets for AML patients. Moreover, inhibition of FLT3/ITD and its downstream MEK/ERK or/and PKA pathways greatly improves the efficacy of FLT3 inhibitor drugs in inducing apoptosis of BaF3/FLT3/ITD cells.
Materials and Methods
Antibodies. Anti-human FLT3 rabbit polyclonal antibody, anti–PKA catalytic subunit (PKAc) rabbit polyclonal antibody, anti-RSK1 rabbit polyclonal antibody, anti-BAD rabbit polyclonal antibody, and anti-Bcl-2 and Bcl-XL monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-BAD (total) monoclonal antibody was obtained from Upstate, Inc. (Waltham, MA). The following antibodies were from Cell Signaling Technologies, Inc. (Boston, MA): anti-phospho-FLT3 (Tyr591) polyclonal antibody, anti-phospho-BAD (Ser112) monoclonal antibody, phospho-BAD (Ser136) polyclonal antibody, anti-BAD polyclonal antibody (does not recognize phospho-Ser112-BAD), anti-phospho-MEK1/2 (Ser217/Ser221) rabbit polyclonal antibody, anti-MEK1/2 rabbit polyclonal antibody, anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204) rabbit polyclonal antibody, anti-p42/MAPK 3A7 monoclonal antibody, and anti-phospho-RSK1 (Ser381) rabbit polyclonal antibody. All the phosphosites refer to those on human proteins. Anti-RSK1 and anti-PKAc monoclonal antibodies and phycoerythrin (PE)– or FITC-conjugated goat anti-mouse or goat anti-rabbit antibodies were from BD PharMingen (San Diego, CA).
Inhibitors. The FLT3 inhibitor AG1296, the MEK inhibitor UO126, and the PKA inhibitor H-89 were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA) and Cell Signaling Technologies.
Isolation of full-length FLT3 cDNA and construction of expression plasmids. We screened for ITDs in FLT3 in genomic DNA from AML patients' bone marrow or peripheral blood (after informed consent on DFCI protocol 01-017 was obtained) using primers flanking the ITD region (3, 7). Total RNA was isolated from Ficoll-separated mononuclear bone marrow cells from an AML patient whose blasts displayed an ITD (26 amino acids) in the juxtamembrane domain of FLT3. Full-length FLT3 cDNA clones both with or without ITD were obtained by reverse transcription-PCR. The cDNAs were sequenced from both ends to confirm the sequences. The deduced amino acid sequence in the ITD region is shown in Fig. 1A. The cDNA clone without an ITD mutation was found to have 1 bp deleted. This cDNA clone was changed to normal wild-type (WT) sequence using site-directed mutagenesis (Invitrogen Corp., Carlsbad, CA) following the manufacturer's protocol and further confirmed by sequencing. The cDNAs of the FLT3/ITD and FLT3/WT were subcloned into a murine retroviral vector MSCVneo (kindly provided by R. Hawley, University of Toronto, Toronto, Ontario, Canada) under the control of a long terminal repeat.
Cell culture and generation of stable cell lines expressing human FLT3/ITD. The murine IL-3-dependent hematopoietic cell line, BaF3, was maintained in RPMI 1640 containing 10% fetal bovine serum (FBS) and 2 ng/mL murine IL-3 (R&D Systems, Inc., Minneapolis, MN). 293T cells were maintained in DMEM with 10% FBS. The leukemic cell line MV4-11 harboring FLT3/ITD (10 amino acids) was cultured in RPMI 1640 with 20% FBS.
The FLT3/ITD and FLT3/WT cDNA constructs or the vector were cotransduced with a packaging construct, pIK69 (Genesys, Redwood City, CA), into 293T cells using Superfect (Qiagen, Valencia, CA) according to the manufacturer's instructions. Supernatants were collected 48 hours after transduction, filtered (0.45 μm), and used to transduce BaF3 cells as described previously (16). Transduced BaF3 cells were selected in methylcellulose medium (StemCell Technology, Vancouver, British Columbia, Canada) with 1 mg/mL G418 and 2 ng/mL IL-3 to obtain G418-resistant colonies. Expression of FLT3 was verified by Western blotting and by staining with PE-conjugated monoclonal anti-human FLT3 antibody and analysis with flow cytometry (data not shown).
Treatment of interleukin-3, FLT3 ligand, and inhibitors. BaF3/FLT3/WT or BaF3/FLT3/ITD cells were cultured at a starting density of 2 × 105 cells/mL in RPMI 1640 with 10% FBS with IL-3 (20 ng/mL) or FLT3 ligand (100 ng/mL) or without cytokine for 24 hours before cells were harvested for apoptosis analysis. To investigate the effects of the inhibitors of FLT3/ITD and its downstream pathways on apoptosis, the FLT3 inhibitor AG1296 (2.5 and 5 μmol/L), the MEK1/2 inhibitor U0126 (12.5 and 25 μmol/L), the PKA inhibitor (2.5 and 5 μmol/L), or a combination of these inhibitors were added to the culture medium. MV4-11 cells were cultured at a starting density of 2 × 105 cells/mL in RPMI 1640 with 10% FBS. AG1296 (2.5 μmol/L), U0126 (10 μmol/L), H-89 (10 μmol/L), or a combination of these inhibitors were added to the medium for 48 hours before harvesting for apoptosis analysis.
RNA interference. SMARTpool siRNAs were obtained from Dharmacon (Chicago, IL) and GeneSilencer siRNA Transfection Reagent from Gene Therapy Systems, Inc. (San Diego, CA). The siRNA transfection was done according to the manufacturer's instruction. siGuard RNase inhibitor (Gene Therapy Systems) was added to the culture just before transfection to protect siRNA from degradation during transfection. To increase the efficiency of RNA interference, siRNA transfection was done for a second time 24 hours after first transfection. Cells were harvested 48 hours after the second transfection.
Analysis of apoptosis and protein using flow cytometry. The cells were stained with Annexin V-FITC and propidium iodide (PI) before flow cytometry analysis using the apoptosis detection kit protocol (CN Biosciences, Inc., Boston, MA).
The analysis of protein using flow cytometry included cell fixing, permeabilization, and immunostaining. The fix buffer, permeabilization buffer, and washing/staining buffer were purchased from Santa Cruz Biotechnology and the process was done according to the manufacturer's instructions.
Protein kinase A kinase assay. BaF3/FLT3/WT and BaF3/FLT3/ITD cells were placed in RPMI 1640 with 1% FBS for 8 hours, washed with PBS, and harvested with cold extraction buffer containing 25 mmol/L Tris-HCl (pH 7.4), 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 10 mmol/L β-mercaptoethanol, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF). PKA kinase activity was assessed using the Nonradioactive Cyclic AMP (cAMP)–Dependent Protein Kinase Assay kit (Promega, Madison, WI). Protein concentration of the crude lysates were quantitated, and equal mounts of protein were added to a reaction mixture (kinase buffer) containing 40 mmol/L Tris-HCl (pH 7.4), 20 mmol/L MgCl2, 0.1 mg/mL bovine albumin, 100 μmol/L Pep-Tag A1 peptide, and 0.5 mmol/L ATP with or without 1 mmol/L cAMP. Experiments were done in parallel with addition of PKAc as positive control and no addition of PKA as negative control. The phosphorylated and nonphosphorylated Pep-Tag peptides were separated by electrophoresis on 0.8% agarose gel and visualized under UV.
Immunoprecipitation and Western blotting. Cell were harvested and rinsed with ice-cold PBS. Ice-cold lysis buffer [0.5 mL; 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 μg/mL leupeptin, 1 mmol/L PMSF] was added to 1 × 107 cells and sonicated on ice four times for 5 seconds each followed by microcentrifugation for 10 minutes at 4°C. Primary antibody (10 μL) was added to 200 μL of the cell lysate and incubated with gentle rocking at 4°C for 2 hours. Protein A-agarose beads (Upstate; 20 μL of 50% beads slurry) were added and incubated with gentle rocking at 4°C for 2 hours followed by microcentrifugation for 30 seconds at 4°C, washing thrice for Western blotting analysis, or washing twice with lysis buffer and twice kinase buffer for the kinase assay.
For Western blotting, cells were lysed in Laemmli sample buffer, sonicated for 10 to 20 seconds, heated for 5 minutes, separated by electrophoresis in SDS-polyacrylamide gel, and transferred to PROTRAN membranes. The membranes were blotted with primary antibodies and then with horseradish peroxidase–conjugated secondary antibodies. The secondary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). The same blots were stripped and reprobed the blots with desired antibodies to confirm equal loading.
Immunoprecipitation kinase assay: BAD phosphorylation in vitro. Immunoprecipitation was done as described above. The agarose-protein A-immunoprecipitate complex was washed twice with kinase buffer. Beads were then suspended in 40 μL of 1× kinase buffer supplemented with 500 μmol/L ATP and 5 μg soluble BAD (Upstate). After incubation at 30°C for 30 minutes, the reaction was terminated with 20 μL SDS sample buffer. Samples were heated to 95°C to 100°C for 5 minutes and analyzed by Western blotting.
FLT3/ITD prevents apoptosis induced by interleukin-3 deprivation in BaF3 cells. To investigate the mechanism of FLT3/ITD-mediated apoptosis inhibition, a full-length human FLT3 cDNA with a 78-nucleotide ITD (encoding 26 amino acids; Fig. 1A) from an AML patient and WT human FLT3 cDNA were cloned into the retrovirus vector MSCV and transduced into BaF3 cells. BaF3 cells depend on IL-3 for their continued growth and survival (39, 40). Cells transduced with vector or FLT3/WT could not survive in the absence of IL-3, whereas cells transduced with FLT3/ITD showed no significant growth rate difference with or without IL-3. To confirm constitutive activation of FLT3/ITD in transduced BaF3 cells, anti-phospho-FLT3 (Tyr591) polyclonal antibody was used to detect the phosphorylation (activation) of FLT3/ITD. As shown in Fig. 1B (lanes 1 and 2), phospho-FLT3 (Tyr591) was detected in BaF3/FLT3/ITD cells but not in BaF3/FLT3/WT cells. As shown in Fig. 1C, BaF3/FLT3/WT cells underwent apoptosis after 24 hours of IL-3 deprivation. If FLT3 ligand was added to the culture medium, FLT3/WT cells remained viable, suggesting that FLT3/WT signaling prevents apoptosis. However, after deprivation of IL-3 or FLT3 ligand, BaF3/FLT3/ITD cells remained viable.
AG1296, a FLT3 inhibitor (41), was used to inhibit the activation of FLT3/ITD. Exposure to 5 μmol/L AG1296 for 90 minutes essentially eliminated autophosphorylation of FLT3/ITD (Fig. 1B). BaF3/FLT3/ITD cells cultured in medium containing AG1296 (2.5 and 5 μmol/L) for 24 hours underwent apoptosis in a dose-dependent manner, which could be reversed by IL-3 addition (Fig. 1D). AG1296 (5 μmol/L) did not result in apoptosis in BaF3/FLT3/WT cells in the presence of IL-3 (Fig. 1E), suggesting that this drug specifically inhibited FLT3/ITD signaling in the absence of IL-3. These data suggest that FLT3/ITD substitutes for the antiapoptotic function of IL-3 in BaF3 cells.
FLT3/ITD induced BAD phosphorylation at serine 112 and Bcl-XL expression. Survival signals, such as IL-3, inhibit apoptosis via BAD phosphorylation at Ser136 through the PI3K/Akt and Ser112 through the MEK/ERK and PKA pathways (17, 18, 20, 24). We determined whether FLT3/ITD-mediated activation of one or more of these pathways lead to BAD phosphorylation at Ser112 and/or Ser136. To detect endogenous BAD phosphorylation in BaF3/FLT3/WT and BaF3/FLT3/ITD cell lines, we did immunoprecipitation and Western blot analysis using anti-phospho-Ser136-BAD–specific antibody, anti-phospho-Ser112-BAD–specific antibody and antibody for total BAD. As shown in Fig. 2A, when IL-3 or FLT3 ligand was present, endogenous BAD was phosphorylated at Ser112 in both BaF3/FLT3/WT and BaF3/FLT3/ITD cells. However, when IL-3 or FLT3 ligand was removed, BAD remained phosphorylated at Ser112 in BaF3/FLT3/ITD cells but not in BaF3/FLT3/WT cells (Fig. 2A). No phosphorylation of endogenous BAD phosphorylation at Ser136 was detected by immunoprecipitation/Western blotting analysis with anti-phospho-BAD (Ser136) antibody. In vitro phosphorylated BAD was used as positive control.
To confirm that the phosphorylation of BAD at Ser112 was due to the functioning of FLT3/ITD, FLT3/ITD inhibitor AG1296 was used to inhibit autoactivation of FLT3/ITD. The phosphorylation at Ser112 was eliminated by AG1296 (Fig. 2B). However, phosphorylation of BAD at Ser136 was not detected in BaF3/FLT3/ITD cells (Fig. 2B) and at a minimal level if the dephosphorylation was blocked by calyculin A (data not shown). These data confirm our conclusion that phosphorylation at Ser112 is the principal endogenous BAD phosphorylation induced by FLT3/ITD and that BAD phosphorylation at Ser112 (not Ser136) might be related to the antiapoptotic function of FLT3/ITD.
BaF3/FLT3/WT cells depended on IL-3 for Bcl-XL expression. When deprived of IL-3, Bcl-XL decreased in BaF3/FLT3/WT cells (Fig. 2C). BaF3/FLT3/ITD cells exhibited constitutive expression of Bcl-XL after IL-3 deprivation. Inhibition of FLT3/ITD by AG1296 reduced the expression of Bcl-XL (Fig. 2D). Phosphorylation of BAD leads to its binding to 14-3-3 protein and thus releases Bcl-XL and Bcl-2 from BAD/Bcl-2/Bcl-XL. Bcl-XL binds BAX and inhibits cytochrome c release from mitochondria, which is a key step in apoptosis (31–35). In FLT3/ITD cells, BAD was shown to be associated with Bcl-XL and, to a lesser degree, with Bcl-2 (Fig. 2E), suggesting the involvement of the interaction of BAD and other Bcl-2 family members during FLT3/ITD-mediated, IL-3-independent cell proliferation.
FLT3/ITD activates RSK1 via MEK/ERK pathway, and RSK1 phosphorylates BAD at serine 112. The MEK/ERK pathway mediates the antiapoptotic function of survival factors, such as IL-3 (17, 18). RSK1 has been shown to mediate BAD phosphorylation at Ser112 as a downstream effector of the MEK/ERK pathway after IL-3 stimulation (18). The MEK/ERK pathway has been reported previously to be activated by FLT3/ITD in the absence of IL-3 (13, 14). We have confirmed that BaF3/FLT3/WT cells depend on IL-3 or FLT3 ligand for the activation of the MEK/ERK pathway, although this pathway is constitutively activated in BaF3/FLT3/ITD cells (Fig. 3A). Inhibition of FLT3/ITD by AG1296 abolished phosphorylation of MEK1/2 (Ser217/Ser221), ERK1/2 (Thr202/Tyr204), and RSK1 (Ser381; Fig. 3B). When BaF3/FLT3/ITD cells were treated with the MEK inhibitor U0126, phosphorylation on ERK1/2 (Thr202/Tyr204) and RSK1 (Ser381) was eliminated (Fig. 3C), confirming that FLT3/ITD induced activation of RSK1 via the MEK/ERK pathway. At the concentration that eliminates phosphorylation of ERK1/2, U0126 reduced but did not eliminate the BAD phosphorylation at Ser112 (Fig. 3C), suggesting the involvement of other pathway(s) in FLT3/ITD-induced BAD phosphorylation at Ser112.
To determine if RSK1 mediates BAD phosphorylation in BaF3/FLT3/ITD cells, we conducted an immunoprecipitation/kinase assay. RSK1 was immunoprecipitated from BaF3/FLT3/ITD cells with anti-RSK1 polyclonal antibody and used to phosphorylate BAD in vitro. RSK1 from BaF3/FLT3/ITD cells had the ability to phosphorylate BAD at Ser112 (Fig. 3D), suggesting that RSK1 is a BAD kinase that is activated by FLT3/ITD via the MEK/ERK pathway as has been shown in Fig. 3B and C. When BAD protein was not included in the reaction mixture, no BAD phosphorylation was noted, suggesting that BAD was phosphorylated by RSK1 in the in vitro kinase reaction rather than having been immunoprecipitated with RSK1.
Our study excluded the role of MEK/ERK pathway in Bcl-XL expression, which was constitutively induced by FLT3/ITD. Inhibition of MEK by U0126 did not reduce the protein level of Bcl-XL (Fig. 3E). We concluded that the MEK/ERK pathway might contribute to FLT3/ITD's antiapoptotic signaling at least partially through BAD phosphorylation by RSK1. Inhibition of the MEK/ERK/RSK1 pathway by U0126 induced apoptosis in BaF3/FLT3/ITD cells in a dose-dependent manner (Fig. 3F). Compared with the FLT3 inhibitor AG1296 (Fig. 1D), U0126 had much less effect on inducing apoptosis, indicating that other pathways are involved in FLT3/ITD's antiapoptotic function.
FLT3/ITD activates PKA and PKA phosphorylates BAD at Ser112. Because the MEK/ERK pathway only partially mediated FLT3/ITD-induced BAD phosphorylation at Ser112 (Fig. 3D), we attempted to find other pathways that might lead to BAD phosphorylation at Ser112. PKA was activated in BaF3/FLT3/ITD cells but not in BaF3/FLT3/WT cells after IL-3 deprivation (Fig. 4A). The PKA inhibitor H-89 (2.5, 5, and 10 μmol/L) reduced the phosphorylation level of BAD at Ser112 (Fig. 4B). To confirm that PKA mediated BAD phosphorylation in BaF3/FLT3/ITD cells, we showed that immunoprecipitated PKA from BaF3/FLT3/ITD cells had the ability to phosphorylate BAD at Ser112 with a BAD absent control, indicating that the phosphorylated BAD was not from immunoprecipitation (Fig. 4C). We also excluded the role of PKA in Bcl-XL expression because the PKA inhibitor H-89 (5 μmol/L) did not affect the protein level of Bcl-XL (Fig. 4D). As expected, the PKA inhibitor H-89 (2.5 and 5 μmol/L) induced apoptosis in BaF3/FLT3/ITD cells in a dose-dependent manner (Fig. 4E).
RNA interference for RSK1 or PKAc reduced BAD phosphorylation. To confirm our conclusion that RSK1 and PKA contribute to FLT3/ITD-induced BAD phosphorylation at Ser112, we used siRNA SMARTpool to partially shutdown the expression of RSK1 and PKAc and checked the phospho-BAD (Ser112) level and apoptosis in MV4-11, a cell line harboring FLT3/ITD. As shown in Fig. 5, RNA interference of RSK1 or PKAc reduced BAD phosphorylation (Ser112) and induced apoptosis, thereby confirming the results noted in BaF3/FLT3/ITD cells when inhibitors of these pathways were used (Figs. 3 and 4).
U0126 and H-89 synergistically induced apoptosis in BaF3/FLT3/ITD cells. As shown above, FLT3/ITD induced BAD phosphorylation predominantly at Ser112 (Fig. 2). The MEK/ERK pathway mediated BAD phosphorylation at Ser112 via RSK1 (Fig. 3) and PKA also mediated phosphorylation at the same site (Fig. 4). Inhibition of either MEK (Fig. 3) or PKA (Fig. 4) induced a lesser degree of apoptosis compared with that induced by the FLT3 inhibitor AG1296 (Fig. 1D). To determine if MEK/ERK and PKA pathways are the principal pathways responsible for antiapoptotic function of FLT3/ITD, we blocked the two pathways at the same time. The MEK inhibitor U0126 and the PKA inhibitor H-89 synergistically induced apoptosis to a level comparable with that induced by AG1296 (5 μmol/L; Fig. 6A and C), suggesting that MEK/ERK and PKA pathways are required for the antiapoptotic function of FLT3/ITD. U0126 or H-89, alone or in combination, did not reduce the expression of Bcl-XL (Figs. 3, 4, and 6B); yet, the combination of these two drugs reduced most of the BAD phosphorylation at Ser112 (Fig. 6B).
U0126 and/or H-89 enhanced the efficacy of FLT3 inhibitor AG1296 on inducing apoptosis in BaF3/FLT3/ITD cells. We tested the inhibition of FLT3/ITD in combination with inhibition of its downstream MEK/ERK or/and PKA pathways in BaF3/FLT3/ITD cells. The FLT3 inhibitor AG1296 (2.5 and 5 μmol/L) was combined with U0126 (10 and 20 μmol/L) or H-89 (5 μmol/L) in the treatment of BaF3/FLT3/ITD cells for 24 hours. Apoptosis, BAD phosphorylation, and Bcl-XL expression were each compared among different combinations. As shown in Fig. 6A, combination of the FLT3 inhibitor AG1296 (2.5 μmol/L) with the MEK inhibitor U0126 (10 μmol/L) or/and the PKA inhibitor H-89 (5 μmol/L) greatly enhanced the efficacy of these drugs on inducing apoptosis in BaF3/FLT3/ITD cells, especially when all three drugs were combined. As we have described above, the combination of U0126 with H-89 reduced most of the BAD phosphorylation at Ser112 (Fig. 6B) and synergistically induced apoptosis (Fig. 6A and C). U0126 (10 μmol/L) or/and H-89 (5 μmol/L) combined with AG1296 (2.5 μmol/L) further reduced BAD phosphorylation but to a lesser degree (Fig. 6B). However, AG1296 (2.5 μmol/L) significantly reduced the protein level of Bcl-XL (Fig. 6B), suggesting that the synergistic effects of U0126 or/and H-89 with AG1296 might be due to the inhibition of BAD phosphorylation and/or Bcl-XL expression. To show that coinhibition of the PKA and MEK pathways was at least additive in other systems, we did a similar experiment in a FLT3/ITD-bearing human leukemia cell line, MV4-11 (Fig. 6D), and found similar results to those obtained in the Baf3/FLT3/ITD cells (Fig. 6A).
Activating mutations in FLT3 due to an ITD in the juxtamembrane domain or a point mutation at D835 in the activation loop result in constitutive activation of this RTK (2–6). Constitutively activated FLT3 leads to IL-3-independent growth and activates MAPK, PI3K, and STAT5 in 32D and BaF3 cells (12–14). The inhibition of apoptosis by IL-3 is mediated by the MEK/ERK, PI3K, and PKA pathways (17–24). BAD phosphorylation plays an important role in the antiapoptotic signaling mediated by IL-3 (17, 18, 20, 21, 24). However, the mechanisms involved in the antiapoptotic functions of FLT3/ITD are still largely unknown. In this study, we introduced a human FLT3 gene with an ITD mutation into BaF3 cells and investigated the effects of the ITD mutation on cell survival and the mechanism thereof. Our results show that FLT3/ITD leads to constitutive activation of MEK/ERK and PKA pathways and that these pathways are required for IL-3-independent survival. FLT3/ITD induces BAD phosphorylation at Ser112 and constitutive expression of Bcl-XL. RSK1 and PKA are responsible for the endogenous BAD phosphorylation at Ser112, which is the predominant phosphorylation site induced by FLT3/ITD. Therefore, inhibition of both MEK/ERK/RSK1 and PKA pathways synergistically induces apoptosis (Fig. 6). The proposed mechanism of FLT3/ITD-mediated activation of the MEK/ERK/RSK1 and PKA pathways to phosphorylate BAD and inhibit apoptosis is summarized in Fig. 7.
A recent study (38) shows that FLT3/ITD induces phosphorylation of BAD at Ser136 via the PI3K pathway when BAD is overexpressed. In this study, we have detected endogenous phosphorylation of BAD predominantly at Ser112 and much lower level of endogenous phosphorylation of BAD at Ser136 even when the dephosphorylation of BAD was inhibited. Experiments that rely on overexpressed BAD may not reflect the situation in the cell, because overexpression may artificially increase the chance of BAD to interact with kinases, such as Akt. Nevertheless, the PI3K inhibitor LY294002 potentiates the ability of the MEK inhibitor U0126 to induce apoptosis in BaF3/FLT3/ITD cells (38), suggesting that the PI3K/Akt pathway has some role in the antiapoptotic function of FLT3/ITD but not necessarily via BAD phosphorylation. Our studies detected a low level of BAD phosphorylation at Ser136 only after BAD phosphatase was inhibited, indicating PI3K pathway was not the main pathway in BAD phosphorylation.
Our results suggest that the MEK/ERK and PKA pathways are the two principal pathways for BAD phosphorylation and play important roles in antiapoptotic function of FLT3/ITD but do not exclude the involvement of other pathways. For example, when the MEK inhibitor U0126 (10 μmol/L) and PKA inhibitor H-89 (5 μmol/L) were combined, only 60% of FLT3/ITD cells underwent apoptosis (Fig. 6A). As shown in Fig. 2, AG1296 inhibition of FLT3/ITD reduced the expression of Bcl-XL. However, U0126 or H-89 did not reduce the expression of Bcl-XL (Figs. 3 and 4). The interaction of these pathways is diagramed in Fig. 7.
Our data strongly support that FLT3/ITD might play a crucial role in leukemogenesis by constitutive signaling through both MEK/ERK and PKA pathways that in turn suppress apoptosis and promote cell survival. Specific FLT3 inhibitors are currently under development as therapeutic agents for AML, which could potentially benefit those patients whose blasts show activating mutations in FLT3. Understanding the downstream signaling pathways of FLT3/ITD could help to dissect the pathogenesis of AML as well as to develop new antileukemic agents. Inhibitors currently used as clinical drugs, such as PKC412 (42), CT53518 (43), and SU5614 (44), have been shown to inhibit autophosphorylation of FLT3/ITD. Blocking downstream signaling could be an effective way to enhance the efficacy of these tyrosine kinase inhibitors, even after drug resistance begins to develop. Our results show that the combination of the FLT3 inhibitor AG1296 with the MEK inhibitor U0126 and/or the PKA inhibitor H-89 greatly enhanced the efficacy of AG1296 on inducing apoptosis. This strategy may represent a novel approach to treat AML patients with FLT3/ITD mutations.
Grant support: 2PO1CA66996-06.
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