Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells

Abstract

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKI-resistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML.

Figure 1. AXL is overexpressed in TKI-resistant CML cell lines and protects them against TKI-induced apoptosis

(A) IM-S, IM-R and PD-R cells were incubated for 48h with 1μM IM. Cell metabolism was measured using the XTT assay (top panel). Protein extracts were prepared, and 50μg of proteins was subjected to SDS-PAGE followed by immunoblot analysis (bottom panel). (B) Cell extracts were separated into cytosol, mitochondria and nuclear-enriched fractions and AXL location was assessed by Western blot in the different fractions. HSP60 and tubulin served as both loading control and validation of the membrane and cytoplasmic fractions, respectively. (C) Cell extracts were prepared and AXL was immuno-precipitated with an anti-AXL antibody. AXL phosphorylation was analyzed by western blot using the 4G10 anti-phosphotyrosine antibody. (D) Cells were transfected with control or AXL siRNAs. 48h after transfection, cells were treated with IM (1μM) for 48h. Cell metabolism was measured using the XTT assay and AXL expression was analyzed by Western blot. (E and F) Cells were transfected with control or AXL siRNAs. After 48h, IM (1μM) was added to cell lines growing in semi-solid methylcellulose medium (E). (F) Results are expressed as the percentage of colony forming cells after drug treatment in comparison with the untreated control cells.

Figure 2. AXL extinction sensitizes TKI-resistant CML cells to IM

Cells were transfected with control or AXL siRNA. 48h after siRNA transfection cells were treated with IM (1μM) for 48h. (A) Cells were stained with the PI/ annexin-V-fluos staining kit according to the manufacturer's indications. Histograms show both annexin-V⁺/PI⁻ cells (open bars) and annexin-V⁺/PI⁺ cells (filled bars). (B, C) Cells were lysed in caspase buffer and caspase-3 (B) and -9 activities (C) were evaluated in quadruplicate using 0.2mM Ac-DEVD-AMC or Ac-LEDH-AMC as substrates. Results expressed as arbitrary units (A.U.) /mg of protein.min and are means ± Standard deviation of 4 independent experiments made in quadruplicate. Error bars 95% confidence intervals. (D) AXL expression and cleavage of PARP was analyzed by Western blot.

Figure 3. Inhibition of PKCα/β signaling sensitizes TKI-resistant cells to IM-induced apoptosis

Cells were transfected with control siRNA or a dual PKCα/β siRNA. 48h after siRNA transfection cells were treated with IM (1μM) for 48 h. (A) PKCα, β and γ expression was analyzed by western blot. (B) Cell metabolism was measured using the XTT assay. (C) Cells were stained with the PI/annexin-V-fluos staining kit and analyzed as described in Fig 2A. (D) Caspase 3 activity was assessed as described in Fig 2B.

Figure 4. Overexpression of PKCα/β in IM-S CML cells confers protection against apoptosis

PKCα and/or PKCβ plasmids were transfected in IM-S cells. 48h after transfection cells were treated with IM (1 μM) for 48 h. (A) AXL, PKCα or β expression was analyzed by western blot. (B) Cell metabolism was measured using the XTT assay. (C) Cells were stained with the PI/annexin-V-fluos staining kit as described in Fig 2A. (D) Caspase 3 activity was assessed as described in Fig 2B.

Figure 5. Over-expression of AXL renders IM-S cells resistant to IM

AXL-WT or AXL-DN plasmids were transfected in IM-S (A-D) or IM-R cells (E-H), respectively. 48 h after transfection, cells were treated with IM (1μM) for 48h. (A and E), cells were lysed, immuno-precipitated with an AXL antibody and phosphorylation of AXL analyzed by Western blot using a phospho-tyrosine antibody (4G10). (B and F) Cell metabolism was measured using the XTT assay. (C and G) Cells were stained with the PI/annexin-V-fluos staining kit for evaluation of dead and apoptotic cells. (D and H) Caspase 3 activity was assessed as described in Fig 2.

Figure 6. U0126 sensitizes TKI-resistant CML cell lines to IM via inhibition of PKCα and β translocation

(A-D) Cells were treated with U0126 (10μM). 48h after, cells were treated with Imatinib (1μM) for 48h. (A) AXL, ERK expression and Phospho-ERK status were analyzed by Western blot. (B) Cell metabolism was measured using the XTT assay. (C) Cells were stained with the PI/annexin-V-fluos staining kit and analyzed as described in Figure 2A. (D) Caspase 3 activity was assessed as described in Fig 2B. (E-F) Cells were treated with U0126 (10μM) for 4h. (E) PKCα, PKCβ expression and Phospho-PKCα/β status were analyzed by Western blot. (F) Cell extracts were separated into cytosol and membrane fractions and PKCα, PKCβ and Phospho-PKCα/β location was assessed by Western blot in the different fractions. Lamp2 served as loading control and validation of the microsomal fraction.

Figure 7. Transcriptional regulation of AXL in TKI-resistant CML cells

Cells were transfected with luciferase reporter plasmids (pAXL-WT or pAXL-DN muted). (A) 24h after transfection, cells were lysed and luciferase activity was measured to determine AXL promoter activity. (B, D) 24 h after transfection, cells were treated with 10μM U0126 for 24h (B). Cells were then lysed and luciferase activity was measured. (C, D) 24 h after transfection, cells were transfected with a dual PKC α/β siRNA (C) or with PKC α or/and PKC β plasmid (D) for 24h. Cells were then lysed and luciferase activity measured. (A to E) Results are expressed as arbitrary units (A.U.) / mg of protein. min and are means ± standard deviation of 3 independent experiments made in triplicate. Error bars 95% confidence intervals.