Development of resistance to dasatinib in Bcr/Abl-positive acute lymphoblastic leukemia

Abstract

Dasatinib is a potent dual Abl/Src inhibitor approved for treatment of Philadelphia chromosome-positive (Ph-positive) leukemias. At a once-daily dose and a relatively short half-life of 3-5 h, tyrosine kinase inhibition is not sustained. However, transient inhibition of K562 leukemia cells with a high-dose pulse of dasatinib or long-term treatment with a lower dose was reported to irreversibly induce apoptosis. Here, the effect of dasatinib on treatment of Bcr/Abl-positive acute lymphoblastic leukemia (ALL) cells was evaluated in the presence of stromal support. Dasatinib eradicated Bcr/Abl ALL cells, caused significant apoptosis and eliminated tyrosine phosphorylation on Bcr/Abl, Src, Crkl and Stat-5. However, treatment of mouse ALL cells with lower doses of dasatinib over an extended period of time allowed the emergence of viable drug-resistant cells. Interestingly, dasatinib treatment increased cell-surface expression of CXCR4, which is important for survival of B-lineage cells, but this did not promote survival. Combined treatment of cells with dasatinib and a CXCR4 inhibitor resulted in enhanced cell death. These results do not support the concept that long-term treatment with low-dose dasatinib monotherapy will be effective in causing irreversible apoptosis in Ph-positive ALL, but suggest that combined treatment with dasatinib and drugs such as AMD3100 may be effective.

Figure 1

Dasatinib has significant activity against human and mouse Bcr/Abl lymphoblastic leukemia cells grown with stromal support. (a). 8093, Bin2 and TXL2 lymphoblastic leukemia cells (1×10⁶/well) were grown in the presence of irradiated MEFs or OP9 cells. Viable cell counts were performed in triplicate over a 1-4 day period as indicated. Note: scale on X-axis is not linear. (b). 8093, Bin2 and TXL2 cells were treated with different concentrations of dasatinib, as indicated, for 48 hrs. The percentage apoptotic cells was determined by FACS analysis. Error bars indicate SD. Results shown are one of two independently performed experiments with similar results. (*, p < 0.05; **, p < 0.01). (c). 8093 and (d) TXL2 cells were treated with different concentrations of dasatinib for 72 hrs. Cell cycle distribution was determined by flow cytometry. The percentage of cells in the different phases of the cell cycle (sub-G1, G0/G1, S and G2/M) after treatment with dasatinib is indicated.

Figure 2

Dasatinib inhibits Bcr/Abl and Src kinase activities. 8093 cells were treated with indicated concentrations of dasatinib for 4 hrs. Western blot analysis was done on total lysates with the antibodies indicated to the right of the panels. Blots were stripped and reprobed with Bcr (N-20), Src and β-actin antibodies to ensure equal loading and transfer of protein. The Crkl panel is from a different set of samples of an experiment with similar outcome. Arrows point to the tyrosine-phosphorylated, more slowly migrating and non-tyrosine phosphorylated, more rapidly migrating Crkl isoforms.

Figure 3

Resistance to dasatinib is facilitated by stroma. (a). Leukemia cells as indicated (3×10⁶/well) were treated with DMSO (■) or with 0.5 nM (8093) or 1.0 nM (Bin2) dasatinib (▲) with or without MEFs as indicated. (b). 8093 cells grown on MEFs that had become resistant to 0.5 nM dasatinib (8093-R) on day 8 were cultured in the presence or absence of MEFs, as indicated, for an additional 8 days. Fresh dasatinib was added every second day with a complete medium change. Viability was assessed by the Trypan Blue exclusion method and is expressed as percentage. Each point represents the average of triplicate values ± SD.

Figure 4

Re-emergence of tyrosine kinase activity after 8093 cells develop resistance to dasatinib. 8093 cells were grown in the presence of MEFs with 0.5 nM dasatinib. Fresh dasatinib was added every second day with the change of medium. 8093 cell lysates were prepared at different time points as indicated. The levels of PY20, p-Src Tyr416, Crkl, p-Stat5 and p-ERK1/2 were determined by Western blot analysis. Blots were stripped and re-probed with Bcr (N-20), Src and GAPDH antibodies to ensure equal loading and transfer of protein.

Figure 5

Dasatinib increases cell-surface expression of CXCR4. 8093 cells were treated with dasatinib and AMD3100 at the concentrations indicated below the figures in the presence of irradiated MEFs. (a) Percentage of cells expressing CXCR4 on the surface from 4-48 hrs of the total number of live cells determined by flow cytometry. (b) Viability as assessed by Trypan Blue exclusion. Results shown are from one of three independent experiments with similar results. Error bars, mean values ±SD of triplicate samples. *, p < 0.05; **, p< 0.001 compared to control group at the same time point.