Enhanced Antitumor Activity by the Combination of Dasatinib and Selinexor in Chronic Myeloid Leukemia

Abstract

Background: Chronic myeloid leukemia is a hematological malignancy characterized by the abnormal proliferation of leukemic cells. Despite significant progress with tyrosine kinase inhibitors, such as Dasatinib, resistance remains a challenge. The aim of the present study was to investigate the potential of Selinexor, an Exportin-1 inhibitor, to improve TKI effectiveness on CML.

Methods: Human CML cell lines (LAMA84 and K562) were treated with Selinexor, Dasatinib, or their combination. Apoptosis, mitochondrial membrane potential, and mitochondrial mass were assessed using flow cytometry. Real-time RT-PCR was used to evaluate the expression of genes related to mitochondrial function. Western blot and confocal microscopy examined PINK and heme oxygenase-1 (HO-1) protein levels.

Results: Selinexor induced apoptosis and mitochondrial depolarization in CML cell lines, reducing cell viability. The Dasatinib/Selinexor combination further enhanced cytotoxicity, modified mitochondrial fitness, and downregulated HO-1 nuclear translocation, which has been associated with drug resistance in different models.

Conclusions: In conclusion, this study suggests that Dasatinib/Selinexor could be a promising therapeutic strategy for CML, providing new insights for new targeted therapies.

Keywords: Selinexor; chronic myeloid leukemia; mitochondria; tyrosine kinase inhibitors.

Figure 1

Selinexor elicits an apoptotic effect on CML cell lines. Representative flow cytometry graphs and relative quantitation of annexin V/PI assay on LAMA84 treated at 48 h (A) and 72 h (B) with dosages of 50 nM, 100 nM, 1 μM, 2 μM, and 5 μM. (C) Representative flow cytometry graphs and relative quantitation of annexin V/PI assay on K562 treated at 72 h with dosages of 50 nM, 100 nM, 1 μM, 2 μM, and 5 μM. (D) Flow cytometry analysis and relative quantitation of membrane depolarization assessment following 48 h of 50 nM, 100 nM, 1 μM, 2 μM, and 5 μM of Selinexor treatment on LAMA84. (E) Flow cytometry analysis and relative quantitation of membrane depolarization assessment following 72 h of 50 nM, 100 nM, 1 μM, 2 μM, and 5 μM of Selinexor treatment on K562. Data are reported as mean ± SD. Graphs are representative of 4 experimental replicates. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 comparing live or depolarized cells to control. ° p < 0.05; °° p < 0.01; and °°°° p < 0.0001 comparing necrotic cells to control. # p < 0.05 comparing early apoptosis cells to control. § p < 0.05 and §§ p < 0.01 comparing late apoptosis cells to control.

Figure 2

Selinexor increases Dasatinib’s effects on CML cell lines. (A) Representative flow cytometry graphs and relative quantitation of annexin V/PI assay on LAMA84 treated for 24 h with 2 nM of Dasatinib or 500 nM and 1 μM of Selinexor either alone or in combination. Representative flow cytometry graphs and relative quantitation of annexin V/PI assay on K562 treated for 24 h (B), 48 h (C), and 72 h (D) with 2 nM of Dasatinib or 500 nM and 1 μM of Selinexor either alone or in combination. (E) Flow cytometry analysis and relative quantitation of membrane depolarization assessment on LAMA84 following 48 h with 2 nM of Dasatinib or 500 nM and 1 μM of Selinexor either alone or in combination. Data are reported as mean ± SD. Graphs are representative of 4 experimental replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 comparing live or depolarized cells to control. ° p < 0.05; °° p < 0.01; °°° p < 0.001; and °°°° p < 0.0001 comparing necrotic cells to control. # p < 0.05; ## p < 0.01; ### p < 0.001; and #### p < 0.0001 comparing early apoptosis cells to control. § p < 0.05 and §§ p < 0.01 comparing late apoptosis cells to control.

Figure 3

Selinexor impairs CML cell line mitochondrial dynamics. (A) Flow cytometry gating and relative quantitation of Mitotracker Red on LAMA84 treated for 48 h with 2 nM of Dasatinib or 1 μM of Selinexor either alone or in combination. (B) Western blot analysis and relative quantitation assessing PINK5 accumulation in LAMA84 following 48 h of treatment with 2 nM of Dasatinib or 1 μM of Selinexor either alone or in combination. (C) Real-time PCR analysis assessing MNF1, MNF2, OPA, CYTB, and ATP5F1A expression on LAMA84 following 48 h of treatment with 2 nM of Dasatinib or 1 μM of Selinexor either alone or in combination. Data are reported as mean ± SD. Graphs are representative of 4 experimental replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 compared to control or Dasatinib.

Figure 4

Selinexor decreases HO-1 nuclear translocation. (A) Immunofluorescence analysis (×20) investigating HO-1 nuclear localization on LAMA84 following 48 h of treatment with 2 nM of Dasatinib or 1 μM of Selinexor either alone or in combination. (B) Western blot analysis and relative quantitation detecting HO-1 accumulation in LAMA84 following 48 h of treatment with 2 nM of Dasatinib or 1 μM of Selinexor either alone or in combination. Data are reported as mean ± SD. Graphs are representative of 4 experimental replicates. *** p < 0.001 and **** p < 0.0001 compared to control or Dasatinib.