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.

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.

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.

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.

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.

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.