AXL receptor tyrosine kinase: a possible therapeutic target in acute promyelocytic leukemia

Abstract

Background: Acute promyelocytic leukemia (APL) is a subset of acute myeloid leukemia (AML) which is characterized by the fusion of promyelocytic leukemia PML and retinoic acid receptor- alpha (RAR-alpha) genes. All-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) have resulted in durable cytogenetic and molecular remissions in most APL patients and have altered the natural history of the disease. Most APL patients treated with ATRA and/or ATO are now anticipated to have a nearly normal life expectancy. Unfortunately, relapse and resistance to the current treatment occur in APL patients and the outcome remains dismal in these refractory patients. AXL receptor tyrosine kinase (AXL-RTK) has been shown to increase tumour burden, provide resistance to therapy and is critical to maintain cancer stem cells (CSCs) in chronic myeloid leukemia (CML) by stabilizing β-catenin in the Wnt/β-catenin signalling pathway. However, the role of AXL-RTK has not been explored in PML/RARα-positive APL. This study aimed to explore the role of AXL-RTK receptor in PML/RARα-positive APL.

Methods and results: By using biochemical and pharmacological approaches, here we report that targeting of AXL-RTK is related to the down-regulation of β-catenin target genes including c-myc (p < 0.001), AXIN2 (p < 0.001), and HIF1α (p < 0.01) and induction of apoptosis in PML/RARα-positive APL cell line. Resistance to all-trans retinoic acid (ATRA) was also overcomed by targeting AXL-RTK with R428 in APL (p < 0.05).

Conclusion: Our results provide clear evidence of the involvement of AXL-RTK in leukemogenic potential of PML/RARα-positive APL and suggest targeting of AXL-RTK in the treatment of therapy resistant APL patients.

Keywords: AXL; Acute promyelocytic leukemia; All-trans retinoic acid; PML/RARα; Receptor tyrosine kinase; Retinoic acid receptor-α; Wnt/β-catenin pathway.

Fig. 1

Expression analysis of AXL-RTK in U937, K562 and NB4 cell lines. Cells were cultured in liquid medium (RPMI 1640 + 10% FBS + 1% L-glutamate and 1% penicillin/streptomycin). Total RNA was extracted from U937, K562 and NB4 cells. The expression level of AXL-RTK was analysed using qPCR. The Ct values were normalized to that of GAPDH gene and results are represented as 2^-ΔΔCt. Statistical significance was tested using student t-test (p-values < 0.05 are considered statistically significant). The means +/− SD of triplicates for one representative experiment out of three performed are given

Fig. 2

Effect of R428 and ATRA on the proliferation potential of PML/RARα-positive NB4 cells. Cells were cultured in RPMI 1640 medium + 10% FBS + 1% L-Glutamate and 1% Pencillin and Streptomycin. MTT assay was performed for (A) U937, (B) K562 and (C) NB4 cells upon exposure to 0.01% DMSO and indicated concentrations of R428 and ATRA. (D) Comparison of R428 and ATRA in the inhibition of NB4 cells after 72 h. The mean +/− SD of triplicates from one representative experiment out of three performed is given

Fig. 3

Effect of single (R428 or ATRA) and combine treatment (R428 + ATRA) on the proliferation potential of PML/RARα positive NB4 cells. Cells were cultured in RPMI medium + 10% FBS + 1% L-Glutamate and 1% Penicillin and Streptomycin to determine the proliferation potential of NB4 cells in the presence of 0.01% DMSO and indicated concentrations of R428 and ATRA. Cell proliferation was assessed through MTT assay after 72 h. (p-values < 0.05 are statistically significant). Bars show mean +/− SD

Fig. 4

Effect of R428 on ATRA resistant NB4 cells. Cells were cultured in RPMI medium + 10% FBS + 1% L-Glutamate and 1% Penicillin and Streptomycin in the presence of DMSO, 1 μM, 2μΜ and 3 μM of ATRA for first, second and third months respectively (A) Methodology for development of ATRA resistant NB4 cell line. (B) MTT assay performed for the first, second and third month for development of ATRA resistant NB4 cell line in the presence of 0.01% DMSO and indicated concentrations of ATRA. (C) q-PCR for the expression of AXL-RTK in ATRA sensitive and resistant NB4 cells. (D) MTT assay for R-NB4 cells treated with indicated concentration of ATRA. (E) MTT assay for R-NB4 cells treated with indicated concentration of R428 (p-values < 0.05 are statistically significant). Bars show mean +/− SD

Fig. 5

Effect of targeting AXL-RTK on the expression of Wnt/ β-catenin target genes. Cells were cultured in liquid medium (RPMI + 20% FBS + 1% L-Glutamate and 1% Pen/strep) in the presence of 0.01% DMSO and indicated concentrations of R428. Expression analysis was done through qPCR. (A) Expression pattern of AXIN2. (B). Expression pattern of c-myc (C). Expression pattern of HIF1α. (p-values < 0.05 are statistically significant). Bars show mean +/− SD