Receptor tyrosine kinase Axl is required for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia

Abstract

In acute myeloid leukemia (AML), about 25–30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by a FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. The presence of FLT3-ITD correlates with poor prognosis in AML and it makes FLT3 an attractive therapeutic target in AML. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to FLT3 inhibitors detected in blood or bone marrow. In this study, we investigated whether the RTK Axl is responsible for resistance of FLT3-ITD⁺ AML cells to PKC412 and AC220, FLT3 inhibitors currently under clinical trials for FLT3-ITD⁺ AML patients. Upon treatment with PKC412 or AC220, phosphorylation of Axl was significantly enhanced in the FLT3-ITD⁺ MV4-11 AML cell line and in primary blasts from a FLT3-ITD⁺ AML patient. Consistently, a PKC412-resistant AML cell line and PKC412-resistant primary blasts from FLT3-ITD⁺ AML patients had significantly higher levels of constitutively phosphorylated Axl and total Axl when compared with a PKC412-sensitive AML cell line and PKC412-sensitive primary blasts from FLT3-ITD⁺ AML patients. We also found that resistance of AML cells against the FLT3 inhibitor PKC412 and AC220 was substantially diminished by the inhibition of Axl via a small-molecule inhibitor TP-0903, a soluble receptor Axl fusion protein Axl-Fc or knockdown of Axl gene expression by shRNA. Collectively, our study suggests that Axl is required for resistance of FLT3-ITD⁺ AML cells against the FLT3 inhibitor PKC412 and AC220, and that inhibition of Axl activation may overcome resistance to FLT3-targeted therapy in FLT3-ITD⁺ AML.

INTRODUCTION

In acute myeloid leukemia (AML), about 25–30% of patients harbor a constitutively active receptor tyrosine kinase, FLT3, encoded by the FLT3 allele with an activating mutation, the internal tandem duplication (FLT3-ITD).^1–3 Clinical studies,⁴ including our own,⁵ have shown that the presence of a FLT3-ITD mutation translates into poor disease-free and overall survival. FLT3 is thus an attractive therapeutic target in AML, and several tyrosine kinase inhibitors (TKIs) have been developed and are being tested clinically.^2,6–8 Unfortunately, to date, these small-molecule inhibitors, in spite of promising preclinical results, have provided only partial and transient responses in the FLT3-ITD⁺ AML patients treated on clinical trials.² In addition, leukemic blasts showing resistance against FLT3 inhibitors were detected in AML patients,⁸ likely indicative of intrinsic mechanism(s) of resistance in these leukemic cells and imposing a serious obstacle to improving the clinical outcome of FLT3-ITD⁺ AML patients. Thus, understanding the molecular basis of the mechanism(s) of resistance may ultimately help to optimize FLT3-targeting treatment strategy and in turn achieve long-term survival of these AML patients.

Axl is a member of the receptor tyrosine kinase TAM family, which comprises Axl, Tyro3 (or Sky) and Mer.^9,10 Axl is activated by two highly homologous ligands, growth arrest-specific gene 6 (Gas6) and protein S,^9,11,12 and has been shown to be involved in a variety of biological functions, such as cell proliferation, apoptosis, migration, lymphoid development^13,14 and inhibition of immune responses.^10,12,15,16 Axl is overexpressed and/or aberrantly activated in many types of cancers.^17–22 Our previous study revealed that Axl is overexpressed and constitutively active in AML cell lines and primary AML patient blasts.²⁰ Previously, we have shown that inhibition of Axl activation impeded the growth of FLT3-ITD⁺ AML cells in vitro and in vivo.²⁰ We also found that Axl activation is crucial for constitutive activation of FLT3 in FLT3-ITD⁺ AML cells.²⁰ These results suggest that Axl may be involved in AML leukemogenesis through regulating FLT3 activation, and can be a potential therapeutic target for the treatment of AML. In other cancers, it has been shown that activation of Axl may cause resistance of cancer cells against TKIs. Bearss et al. reported that gastrointestinal tumors acquired resistance against the BCR-ABL TKI imatinib by overexpressing Axl,²³ and in HER2-positive breast cancer cells, activation of Axl was suggested to be responsible for the resistance to the HER2 TKI lapatinib.²⁴ Zhang et al.²⁵ also showed that activation of Axl causes resistance to epidermal growth factor receptor (EGFR)-targeted therapy in lung cancer. Increased expression of Axl and Gas6 were found in EGFR-mutant lung cancers from patients with acquired resistance to the TKI erlotinib that targets EGFR.²⁵ Furthermore, in breast cancer, an increased fraction of mutant alleles in AXL and GAS6 genes following treatment of lapatinib was associated with emergence of therapy resistance.²⁶ In this study, we investigated whether Axl is responsible for resistance of leukemic cells to FLT3-selective TKI PKC412 and AC220 in FLT3-ITD⁺ AML.

MATERIALS AND METHODS

Cell culture, AML patient samples and reagents

Human AML cell line MV4-11 was obtained from the ATCC (Manassas, VA, USA). PKC412-sensitive (MOLM13) and -resistant human AML cell lines (MOLM13-R-PKC412) were generously provided by Dr James D Griffin (Dana Farber Cancer Institute, Boston, MA, USA).²⁷ All cell lines were cultured and maintained in RPMI1640 medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics at 37 °C and 5% CO₂. Cryopreserved primary leukemic blasts were obtained from AML patients who provided written consent and with the approval from The Ohio State University Institutional Review Board. The clinical data of AML patient cases used in this study are summarized in Supplementary Table 1. After thawing, human AML patient samples were maintained in RPMI medium containing 20% fetal bovine serum, stem cell factor (100 ng/ml) and interleukin-3 (10 ng/ml). Human Control-Fc (Ctrl-Fc) and Axl-Fc chimeric proteins were purchased from R&D Systems (Minneapolis, MN, USA) and were treated at a final concentration of 1 μg/ml. FLT3 inhibitor PKC412 and AC220 were purchased from LC Laboratories (Woburn, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. Axl inhibitor TP-0903 was provided by Tolero Pharmaceuticals (Lehi, UT, USA). The MEK/ERK inhibitor U0126, the PI3K inhibitor LY294002 and the JAK2 inhibitor hexacyclohexane were purchased from Sigma-Aldrich (St Louis, MO, USA).

Immunoblotting

Cells were treated as described in the figure legends. Cells were then harvested by centrifugation, lysed and subjected to SDS-polyacrylamide gel electrophoresis, which was followed by transfer to nitrocellulose membrane. The nitrocellulose membrane was then incubated with primary antibodies against proteins mentioned above (all antibodies from Cell Signaling (Danvers, MA, USA), except anti-phospho-Axl (R&D Systems)). Nitrocellulose membrane was then incubated with secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). An enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK) was used for detection of proteins.

Intracellular staining of phospho-Axl

Harvested cells were fixated by being incubated with 2% formaldehyde at 37 °C for 10 min, which was followed by chilling on ice for 1 min. Cells were then permeabilized by adding 100% methanol and incubating for 30 min on ice. Cells were stained with anti-phospho-Axl rabbit antibody (R&D Systems) for 1 h at room temperature, which was followed by washing and staining with anti-rabbit donkey secondary antibody conjugated with Alexa Fluor 647 (Invitrogen) for 30 min at room temperature. After washing, stained cells were analyzed by flow cytometry.

Measurement of cell growth and apoptosis

Cell viability was measured using cell proliferation assay (MTS) kit from Promega (Madison, WI, USA) as instructed in the manufacturer’s manual. Detection of apoptosis was performed using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA). After incubating the cells with FITC-conjugated Annexin V in the binding buffer for 15 min at room temperature, cells were analyzed using a flow cytometer FACSCalibur (BD Biosciences).

Simultaneous evaluation of apoptosis and phosphorylation of Axl Simultaneous detection of both apoptosis and phospho-Axl in the same cell was done as described previously.²⁸ Briefly, cells were first incubated with Annexin V-FITC in the binding buffer (both from BD Biosciences) for 15 min at 4 °C, which was followed by fixation and permeabilization. After washing with the binding buffer containing 1% bovine serum albumin, cells were incubated with anti-phospho-Axl rabbit antibody (R&D Systems) followed by anti-rabbit donkey antibody conjugated with Alexa Fluor 647 (Invitrogen). After staining, cells were washed with binding buffer and analyzed using the FACSCalibur (BD Biosciences).

Axl expression knockdown by lentivirus-encoding shRNA targeting Axl

After cells were resuspended with culture medium containing polybrene (5 μg/ml) (Sigma-Aldrich), control (Ctrl) or Axl shRNA lentivirus particles (Santa Cruz Biotechnology) were added and incubated for 24 h. Then culture medium was changed into fresh one without polybrene and the cells were cultured for another 24 h. Stably transfected cells were selected using puromycin (2 μg/ml), and resulting puromycin-resistant cells were tested for reduction in the level of Axl expression by immunoblot.

In vivo mouse study

All animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) and according to the procedures of the University Laboratory Animal Resources (ULAR) at the Ohio State University. All animals were accommodated and maintained in a clean, sterile animal care facility. NOD/SCID/IL-2Rγ^−/− (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and mice (about 8 weeks old) were injected intravenously with PKC412-resistant MOLM13-R-PKC412 AML cell line (2 × 10⁶ cells per mouse). After 2 weeks, treatment of drugs began and randomly divided groups of mice were daily administered with vehicle control, PKC412 (25 mg/kg/day), TP-0903 (50 mg/kg/day) or PKC412 plus TP-0903. All drugs were administered by oral gavages and Kolliphor solution (1% Tween 80, 10% Kolliphor RH40 (Sigma-Aldrich) and 89% water with the pH adjusted to 5.0) was used as a vehicle control. In order to check the engraftment of leukemic cells, bone marrow cell suspensions were obtained from each mouse as previously reported²⁹ and analyzed in a blind manner by flow cytometry to detect leukemic cells using an anti-human CD45 antibody. Mice were euthanized when they became moribund.

Statistical analyses

Two-sample t-tests or paired t-tests were used for two group comparisons depending on whether the data were independent or correlated. For independent multiple group comparisons, analysis of variance was used. For correlated data, such as the cell viability experiment using cells from the same subject treated with different drugs, linear mixed-effects models were used to take account of the correlation among observations from the same subject. Spearman’s correlation was used to test the association between cell viability and the level of Axl/phospho-Axl expression. Holm’s procedure was used to control for multiple comparisons. A P-value < 0.05 is considered as significant. All analyses were performed using SAS9.2 (SAS Institute Inc., Cary, NC, USA).

RESULTS

Increased phosphorylation of Axl by the FLT3 inhibitors PKC412 and AC220 in FLT3-ITD⁺ AML cells

Previous studies have shown that Axl phosphorylation was increased in AML cells under adverse conditions, such as serum starvation³⁰ and chemotherapy.^30,31 We thus tested whether Axl phosphorylation could be also induced by treatment of FLT3-ITD⁺ AML cells with FLT3 inhibitors. As shown in Figure 1a (immunoblot) and Figure 1b (intracellular staining followed by flow cytometry), phosphorylation of Axl was enhanced by treatment with the FLT3 inhibitor, PKC412, in the FLT3-ITD⁺ MV4-11 AML cell line and in primary blasts from an FLT3-ITD⁺ AML patient (Figure 1). Axl phosphorylation was also increased in FLT3-ITD⁺ AML cells following treatment with a second FLT3 inhibitor, AC220 (Figure 1c). Treatment with PKC412 also led to enhanced activation of intracellular signaling molecules, such as ERK, AKT and STAT5. AC220 increased the level of phospho-ERK and phospho-AKT, but not phospho-STAT5 (Supplementary Figure S1). We thus tested which signaling pathway(s) may be involved in increasing Axl activation upon treatment with PKC412. As shown in Figure 1d, the MEK/ERK inhibitor U0126 and, more strongly, the PI3K inhibitor LY294002, inhibited Axl activation following PKC412 treatment. However, the JAK2 inhibitor, hexacyclohexane, showed no effect. These data suggest that increased Axl activation by PKC412 in FLT3-ITD⁺ AML cells may be mediated by the MEK/ERK and, particularly, PI3K/AKT pathways.

Figure 1.

Axl phosphorylation is increased by treatment with the FLT3 inhibitor PKC412 and AC220 in AML cells. (a and c) MV4-11 AML cell line (left) and primary blasts from FLT3-ITD⁺ AML patient (right) were treated with PKC412 (10 nm) (a) or AC220 at the indicated concentrations (c) for 72 h. Cells were then subject to immunoblot to detect phospho-Axl and Axl. Actin was used as a loading control. This blot is representative of three experiments with similar results. (b) MV4-11 AML cell line was treated with PKC412 (1 or 10 nm) for 72 h, which was followed by fixation and permeabilization. Cells were stained with rabbit anti-phospho-Axl antibody and anti-rabbit secondary antibody conjugated with Alexa Fluor 647. Stained cells were analyzed by flow cytometry. This is the representative histogram of two separate experiments. (d) (Left) MV4-11 cells were treated with PKC412 (10 nm) for 72 h and the cells were then subject to immunoblot to detect phospho-ERK, phospho-AKT and phospho-STAT5. (Right) MV4-11 was treated with PKC412 (10 nm) for 72 h in the presence of MEK/ERK inhibitor U0126 (U) (10 μm), PI3K inhibitor LY294002 (LY) (20 μm) or JAK2 inhibitor hexacyclohexane (Hex) (50 μm). Cells were subject to immunoblot to detect phospho-Axl and Axl.

Previous studies by our group and others have demonstrated that activation of Axl confers a growth advantage to AML cells.^20,30 As shown in Figure 2, when primary blasts from FLT3-ITD⁺ AML patients were treated with PKC412, the majority of AML cells undergoing apoptosis (Annexin V-positive) harbored little phosphorylated Axl (lower right quadrant in Figure 2a). On the other hand, most AML cells resistant to PKC412 (Annexin V negative) possessed a significantly higher level of phosphorylated Axl (upper left quadrant in Figure 2a). These results indicate that increased Axl phosphorylation following treatment with PKC412 is associated with a subset of FLT3-ITD⁺ AML cells that survive against the FLT3 inhibitor PKC412. We therefore hypothesized that activated Axl may be involved in the molecular mechanism responsible for resistance of FLT3-ITD⁺ AML cells to FLT3-targeted therapy.

Figure 2.

Increased Axl phosphorylation confers an advantage in survival to AML cells treated with the FLT3 inhibitor PKC412. Primary blasts from FLT3-ITD⁺ AML patients were treated with PKC412 (10 nm) for 72 h. For simultaneous detection of apoptosis and Axl phosphorylation, cells were then stained with Annexin V-FITC, which was followed by fixation, permeabilization and staining with anti-phospho-Axl antibody. (a) Representative fluorescence-activated cell sorting plot (left) and histogram (right) are shown. (b) The graph summarizes the data from three different FLT3-ITD⁺ AML patients. **P < 0.01, paired t-test.

Increased level of phosphorylated Axl in FLT3 inhibitor PKC412-resistant AML cells and the effect of the Axl inhibitor TP-0903 In order to further investigate whether Axl is involved in resistance of FLT3-ITD⁺ AML cells to FLT3 inhibitors, we analyzed the FLT3 inhibitor PKC412-resistant AML cell line, MOLM13-R-PKC412, which was generated from its parental PKC412-sensitive cell line, MOLM13, by being exposed to PKC412 for a long period of time.²⁷ The PKC412-resistant MOLM13-R-PKC412 AML cell line possessed a much higher level of phospho-Axl and total Axl when compared with PKC412-sensitive MOLM13 (Figure 3a). This corroborates the data from Figures 1 and 2, which showed increased Axl phosphorylation when FLT3-ITD⁺ AML cells were treated with PKC412. In order to test whether Axl is necessary for resistance of FLT3-ITD⁺ AML cells to PKC412, we first used the Axl TKI, TP-0903, provided by Tolero Pharmaceuticals. As shown in Figure 3b, TP-0903 was able to inhibit phosphorylation of Axl in both the MOLM13 and MOLM13-R-PKC412 FLT3-ITD⁺ AML cell lines. Furthermore, when the PKC412-resistant FLT3-ITD⁺ AML cell line MOLM13-R-PKC412 was treated with the Axl inhibitor TP-0903, it was able to resensitize the cells to PKC412 (Figure 3c) as well as to the other FLT3 inhibitor, AC220 (Figure 3d). This suggests that Axl is important for resistance of FLT3-ITD⁺ AML cells against the FLT3 inhibitors PKC412 and AC220. Although a low concentration (5 nm) of TP-0903 showed an additive effect, TP-0903 alone was sufficient for almost complete inhibition of the growth of MOLM13-R-PKC412 AML cells at a higher concentration (>40 nm) (data not shown).

Figure 3.

FLT3 inhibitor PKC412-resistant AML cells harbor a higher level of phospho-Axl and Axl, and Axl inhibitor, TP-0903, can abrogate resistance to PKC412 and AC220. (a) The level of phospho-Axl and Axl was compared between FLT3 inhibitor PKC412-sensitive (MOLM13) and -resistant (MOLM13-R-PKC412) by immunoblot. Actin was used as a loading control. This blot is representative of three experiments. (b) MOLM13 and MOLM13-R-PKC412 were treated with the indicated concentrations of Axl inhibitor TP-0903 for 4 h. Cells were then subject to immunoblot to detect phospho-Axl and Axl. This blot is representative of two experiments. (c and d) MOLM13 or MOLM13-R-PKC412 AML cell line was treated with the indicated concentrations of PKC412 (c) or AC220 (d) in the absence or presence of TP-0903 (5 nM) for 72 h. Relative cell viability to vehicle control was then measured. Data represent mean ± s.e.m. from three separate experiments (each experiment with triplicates). **P < 0.01, ***P < 0.001.

Axl is required for AML resistance to the FLT3 inhibitors PKC412 and AC220

Although the data above from the experiment using Axl inhibitor TP-0903 (Figures 3c and d) is suggestive, we could not rule out the possibility that abrogation of FLT3-ITD⁺ AML resistance to FLT3 inhibitors, PKC412 and AC220, by TP-0903 is due to off-target activities of TP-0903. In fact, TP-0903 can not only target Axl but also other kinases such as aurora kinase and JAK2 (unpublished data). In order to investigate whether Axl is specifically involved in resistance of FLT3-ITD⁺ AML cells against the FLT3 inhibitors, PKC412 and AC220, we took two different experimental approaches. First, we used soluble Axl chimeric protein, called Axl-Fc, to sequester ligands for Axl, thereby blocking Axl activation.^13,20 When the PKC412-resistant MOLM13-R-PKC412 AML cells were treated with Axl-Fc, resistance of MOLM13-R-PKC412 AML cells to PKC412 was substantially diminished (Figure 4a). Second, when MOLM13-R-PKC412 AML cells were stably transfected with lentivirus-encoding shRNA targeting Axl, Axl expression was knocked down (right, Figure 4b) and the sensitivity to PKC412 (left, Figure 4b) and to AC220 (Figure 4c) was restored. Both results indicate that Axl is specifically required for resistance of FLT3-ITD⁺ AML cells against the FLT3 inhibitors, PKC412 and AC220.

Figure 4.

Axl is necessary for AML resistance to the FLT3 inhibitor PKC412 and AC220. (a) MOLM13-R-PKC412 cells were treated with the indicated concentrations of PKC412 for 72 h in the presence of control Fc (Ctrl-Fc) or Axl-Fc. Then relative cell viability to vehicle control was measured. As a control, MOLM13 was treated with PKC412 alone. Data represent mean ± s.e.m. from three separate experiments. ***P < 0.001. (b and c) MOLM13-R-PKC412 cells were stably transfected with lentivirus-encoding scrambled shRNA (Ctrl) or Axl shRNA (Axl) (box on the right), which was followed by treatment of PKC412 (b) or AC220 (c) for 72 h. Relative cell viability to vehicle control was then measured. As controls, MOLM13 and MOLM13-R-PKC412 were also treated with PKC412 alone. Data represent mean ± s.e.m. from three separate experiments. **P < 0.01, ***P < 0.001.

We next investigated whether Axl-specific inhibitor TP-0903 was able to show better efficacy against PKC412-resistant AML in vivo. At 14 days following injection of MOLM13-R-PKC412 AML cells, immunodeficient NSG mice were treated with either vehicle control, PKC412 alone, TP-0903 alone or a combination of PKC412 and TP-0903. The combination of TP-0903 and PKC412 significantly prolonged survival of mice transplanted with MOLM13-R-PKC412 AML cells when compared with PKC412 (P < 0.05) or TP-0903 alone (P < 0.01). This indicates that, even in this highly resistant human AML cell line, inhibition of Axl can sensitize AML cells to the FLT3 TKI PKC412 and significantly improve survival.

The level of phospho-Axl and Axl is higher in primary AML patient blasts intrinsically resistant to FLT3 inhibitors PKC412 and AC220 In order to investigate AML resistance to FLT3 inhibitors in primary blasts from AML patients, we first screened primary blasts from FLT3-ITD⁺ AML patients to identify those displaying intrinsic resistance or sensitivity to PKC412 (Figure 5a). As seen in Figure 5a, regardless of being sensitive or resistant to PKC412, the growth of all AML patient blasts we examined were significantly inhibited by Axl inhibitor, TP-0903. Although phospho-Axl and total Axl were easily detected in primary FLT3-ITD⁺ AML patient blasts intrinsically resistant to PKC412 (P1, P2, P5 and P7), we observed little or no phospho-Axl or total Axl in AML patient blasts sensitive to PKC412 (P3, P4 and P6) (Figures 5b and c). The results indicate that there is a significantly positive correlation between the cell viability following treatment with PKC412 (1 μM) and the level of phospho-Axl/Axl (correlation coefficient of 0.78, P = 0.03 for phospho-Axl; coefficient 0.79, P = 0.04 for Axl). Two AML patient samples showing resistance to PKC412 were incubated with AC220 and found to be resistant, whereas both samples were sensitive to TP-0903 alone (Figure 5d). In addition, bone marrow AML blasts from double knock-in mice co-expressing the partial tandem duplication (PTD) of MII (PTD/wt) and the Flt3-ITD (ITD/wt)³² were intrinsically resistant to PKC412 (Figure 5e) and to AC220 (Figure 5f), yet this was reversed with the treatment of TP-0903 alone. These data from primary human and mouse FLT3-ITD⁺ AML cells strengthen the conclusion that Axl is important for AML resistance against the FLT3 inhibitors, PKC412 and AC220.

Figure 5.

The levels of phospho-Axl and Axl were increased in primary AML blasts intrinsically resistant to the FLT3 inhibitor PKC412 and AC220. (a) Primary blasts from seven AML patients (P1–P7) were plated (triplicates) and treated with TP-0903 or PKC412 at the indicated concentrations for 72 h. Cell viability was then measured, and the viability of the cells treated with vehicle control was set as 100%. (b) The same AML blasts described in a (P1–P7) were subjected to immunoblot to detect phospho-Axl and Axl. Actin was used as a loading control. (c) Data from a and b were plotted in graphs. The band density of phospho-Axl (left) or Axl (right) (x axis) was measured by densitometry and normalized by the level of actin. Each square represents an individual AML patient sample. (d) Primary AML blasts from two patients (P1 and P2) described in a were treated with TP-0903 or AC220 (each 1 μm) for 72 h. Cell viability was then measured, and the viability of the cells treated with vehicle control was set as 100%. (e and f) AML cells obtained from bone marrow of double knock-in mice co-expressing MII (PTD/wt) and Flt3 (ITD/wt) (PTD+/ITD⁺ mouse BM) were treated with indicated concentrations of PKC412 (e), AC220 (f) or TP-0903 (e and f) for 3 days. Cell numbers were then counted and the number of cells treated with vehicle control (dimethyl sulfoxide) was set as 100%. Data represent mean ± s.e.m. from three separate experiments. ***P < 0.001.

DISCUSSION

Previous studies showed that the receptor tyrosine kinase Axl is responsible for resistance to EGFR HER2 TKIs in breast cancer,^24,33 to both EGFR and PI3K TKIs in lung cancer,^25,34 to imatinib in gastrointestinal stromal tumors²³ and in chronic myeloid leukemia.³⁵ Here we have identified Axl as an important component in the mechanism of resistance to the FLT3 inhibitors, PKC412 and AC220, in FLT3-ITD⁺ AML. We demonstrated that Axl phosphorylation increased when FLT3-ITD⁺ AML cells were exposed to FLT3 TKIs, PKC412 and AC220, and that inhibition of Axl activation could reverse resistance of FLT3-ITD⁺ AML cells against the FLT3 inhibitor PKC412 and AC220. Previously other studies have shown that Axl phosphorylation is upregulated in AML cells under adverse conditions, such as serum starvation³⁰ and chemotherapy.^30,31 In fact, the level of phosphorylated Axl has been shown to be correlated with chemoresistance of AML cells.³¹ These results, together with our report herein, suggest that Axl may play a more general protective role in the survival of AML cells upon adverse conditions, including treatment with FLT3 TKIs.

In order to model acquired resistance that occurs in AML patients, we used a PKC412-resistant AML cell line, MOLM13-R-PKC412, which was established by prolonged exposure of PKC412 to the sensitive parental AML cell line, MOLM13. MOLM13-R-PKC412 showed resistance not only to PKC412 but also to AC220. Furthermore, its resistance to both FLT3 TKIs was largely reversed by inhibition of Axl. As PKC412 and AC220 inhibit FLT3 activities through distinct mechanisms,³⁶ this suggests that Axl provides a general mechanism of resistance to FLT3 inhibitors. We next provided comparable data from primary AML patient blasts that displayed intrinsic resistance to both PKC412 and AC220, thereby demonstrating the clinical relevance of our findings. Positive correlation between the resistance of primary AML patient blasts to PKC412 and the level of phospho-Axl (and total Axl) was observed (Figure 5c). This result suggests that the response of an AML patient to FLT3 TKIs, such as PKC412 and AC220, could be predicted based on the expression level of phospho-Axl (and total Axl) at least in vitro. It also suggests that Axl, as a biomarker, may be able to help to select AML patients being expected to respond to PKC412 or similar drugs. Selection of appropriate patients for the therapy should lead to more efficient and improved outcomes of AML patients and avoid unhelpful treatments for those predicted to do poorly. In the case of chemotherapy, the level of phosphorylated Axl has been shown to be correlated with chemoresistance of AML cells.³¹ In fact, Axl³⁰ and its ligand, Gas6,³⁷ were found to be independent, adverse prognostic markers for AML patients that were treated with standard chemotherapy. A prospective study of these observations should likely be considered in a cooperative group setting.

In this study, we have also shown that PKC412 and AC220 induced activation of several intracellular signaling molecules, and that increased Axl phosphorylation could be reduced by inhibition of MEK/ERK and PI3K/AKT signaling pathways. This suggests that the MEK/ERK and PI3K/AKT pathways activated by FLT3 TKI may mediate the increase in Axl phosphorylation. However, how activation of MEK/ERK and PI3K/AKT pathways leads to increased Axl phosphorylation still remains to be addressed. In fact, Axl has been shown to interact functionally and/or physically with a number of signaling molecules, including PI3K.¹⁰ It could thus be speculated that direct and/or indirect cross-talk between Axl and PI3K (and MEK/ERK) may be responsible for increased Axl activation.

The molecular mechanism(s) by which the activation of Axl mediates resistance of cancer cells to TKIs also remains to be fully elucidated. In breast cancer cells, activation of EGFR transactivates Axl and this ligand-independent Axl activation expands the EGFR-induced signaling pathways beyond those triggered by EGFR alone. This increase in signaling pathways by Axl amplifies the EGFR signaling response and limits the response to EGFR-targeted TKI.³³ We have previously shown that Axl and FLT3 physically interact with each other in FLT3-ITD⁺ AML cells where FLT3 is constitutively activated.²⁰ It remains to be determined whether stimulation of FLT3 by FLT3 ligand (FL) is able to transactivate Axl and expand the FLT3-induced signaling pathways beyond those triggered by FLT3 alone. If that is the case, this would likely provide further details regarding the mechanism by which Axl mediates resistance to FLT3 TKI in FLT3-ITD⁺ AML. This transactivation by an alternative receptor also provides an explanation for our data, showing that simultaneous inhibition of both FLT3 and Axl was more effective in suppressing the growth of FLT3-ITD⁺ AML cells than inhibition of either molecule alone.

To our knowledge, there has not been a report describing a genetic mutation causing constitutive phosphorylation of Axl. As PKC412-resistant AML blasts harbor a higher level of total Axl as well as of phospho-Axl, a constitutively activating mutation in Axl may not be the mechanism for the propagation of resistant AML in this setting. Axl expression is negatively regulated by microRNA miR-34a. In AML, miR-34a has been reported to be aberrantly downregulated³⁸ and its ectopic overexpression induces the downregulation of both E2F1 and B-Myb oncogenes,³⁹ but expression of Axl was not investigated in that study. If Axl is indeed higher in AML cells with relatively low expression of miR-34a, these data could provide at least one mechanism by which AML cells harbor a higher level of Axl and thus are resistant to FLT3 TKI. We are investigating this as part of another study, and will test whether increased expression of miR-34a can reverse AML resistance to FLT3 TKI, PKC412. Upregulation of the Axl ligand, Gas6, may also contribute to the elevated level of Axl activation. In fact, Gas6 overexpression has been shown to be associated with TKI resistance in solid tumors.^24,25 Recently, Murtaza et al.²⁶ have shown that abundance of a splicing mutation in GAS6 increased following the treatment of breast cancer cells with the TKI lapatinib. It remains to be determined whether the same or a similar type of mutation may exist in FLT3-ITD⁺, TKI-resistant AML cells.

With our evidence showing that Axl is required for resistance to FLT3 TKI PKC412 in AML, it provides a rationale for testing an Axl inhibitor, such as TP-0903, alone or in combination with FLT3 inhibitors in clinical trials for AML patients who responded poorly to FLT3 inhibitors. Phase II clinical trials of the Axl inhibitor, foretinib (XL-880, GSK1363089), have been completed for solid tumors.^40,41 Two other selective Axl inhibitors, S49076 and R428 (BGB324), are in phase I clinical trials for solid tumors.⁴² TP-0903 used in this study is anticipated to enter a phase-I clinical trial for AML soon.

Previous studies found that multiple other mechanisms may contribute to resistance of AML cells to FLT3 TKIs.⁸ Our study identifying Axl as having a role in AML resistance to FLT3 TKI should help us understand AML heterogeneity and the molecular mechanisms of AML resistance to FLT3-targeted therapy. Axl induces activation of multiple signaling pathways, including MAPK and PI3K/AKT.⁴³ Axl also physically and/or functionally interacts with a variety of signaling molecules and pathways.^10,20 It will be important to dissect which of these signaling pathways and molecules are critical for Axl-driven resistance to FLT3 TKI in AML in order to select the best targets for a multi-drug approach to combat TKI resistance in AML, if necessary.