Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-JUN N-terminal kinase inhibition

Abstract

Background: The Philadelphia chromosome (Ph), which leads to the creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) have shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph⁺ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph⁺ B-ALL.

Methods: Activation of the JNK signaling pathway in human and mouse BCR-ABL⁺ B-ALL cells with or without dasatinib treatment was analyzed by Western blotting. JNK was inhibited either by RNA interference or chemical inhibitors, such as JNK-IN-8. The effect of JNK inhibition with or without BCR-ABL TKI dasatinib on BCR-ABL⁺ B-ALL cells was analyzed by the CellTiter-Glo® Luminescent Cell Viability Assay. The in vivo effects of JNK-IN-8 and dasatinib alone or in combination were tested using a BCR-ABL induced B-ALL mouse model.

Results: We found that the c-JUN N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL⁺ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph⁺ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph⁺ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone.

Conclusions: Our findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph⁺ B-ALL.

Keywords: Combination target therapy; Dasatinib; JNK; Ph+ B-ALL.

Fig. 1

JNK kinases remain abnormally activated upon the treatment of dasatinib in Ph⁺ B-ALL. a Human Ph⁺ B-ALL cell line SUP-B15 cells were treated with dasatinib at various concentrations for 6 hours (h) and subsequently examined by western blot using indicated antibodies against key effectors in BCR-ABL and MAPK signaling pathways: phosphorylated (p) and total BCR/ABL, Stat5, AKT, ERK, p38 and JNK, respectively. Actin was used as a loading control. b SUP-B15 cells were treated with dasatinib at various concentrations as indicated for 24 h and subsequently examined by western blot using indicated antibodies against phosphorylated and total BCR/ABL, Stat5, AKT, ERK, p38, and JNK, respectively. Actin was used as a loading control. c Western blot analysis of lysates of CD19⁺ B lymphocytes isolated from bone marrows of normal and BCR/ABL^p190 bone marrow transduction and transplantation mice using antibodies described in (a) plus western blot analysis using antibodies against phosphorylated and total c-JUN. d CD19⁺ B lymphocytes from BCR/ABL^p190 bone marrow transduction and transplantation mice were treated with 10 nmol/L dasatinib or vehicle for 12 h, followed by western blot analysis with antibodies against phosphorylated and total BCR-ABL and JNK

Fig. 2

JNK inhibition reduces viability of Ph⁺ B-ALL cells. a Levels of phosphorylated and total JNK and c-JUN in SUP-B15 cells transduced with lentivirus vectors containing control shRNA (ShNC), shRNA targeting JNK (ShJNK#1), or ShJNK#2 were detected by western blot analysis with antibodies indicated. Actin was used as a loading control. b Proliferation of SUP-B15 cells expressing control and JNK shRNA were plotted by counting viable cells over a period of 3 days. c SUP-B15 cells were treated with JNK inhibitor JNK-IN-8 or SP600125 for 12 h, followed by western blot analysis using antibodies against phosphorylated and total c-JUN. d, e Viability of SUP-B15 and a CML blast crisis cell line K562 cells treated with various concentrations of JNK-IN-8 (d) or SP600125 (e) for 48 h was measured by the CellTiter Glo assay. Normalized cell proliferation was presented and compound’s IC50 value calculated. f, g Viability of CD19⁺ B lymphocytes isolated from normal and BCR/ABL^p190 bone marrow transduction and transplantation mice treated with various concentrations of JNK-IN-8 (f) or SP600125 (g) for 48 h was measured by the CellTiter Glo assay. Normalized cell proliferation was presented and compound’s IC50 value calculated. h The nucleated bone marrow cells (NBMC) from a healthy donor and primary BM cells isolated from 6 patients with Ph⁺ B-ALL were treated with different concentrations of JNK-IN-8 for 48 h. Cell viability was measured by the CellTiter Glo assay. Data are presented as mean ± SD, and P values were calculated using Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

JNK inhibition cooperates with dasatinib in killing Ph⁺ B-ALL cells. a SUP-B15 cells expressing ShNC, ShJNK#1, or ShJNK#2 were treated with different concentrations of dasatinb for 24 h. Viability of these cells was then measured by the CellTiter Glo assay. b SUP-B15 cells were treated with indicated concentrations of dasatinib combined with or without JNK-IN-8 (6 μmol/L and 8 μmol/L) for 24 h. Viability of these cells was measured by the CellTiter Glo assay. c “Combination index” (CI) values were calculated at each concentration of JNK-IN-8 based on data in b and plotted, Fractional inhibition abbreviated as Fa. Data are presented as mean ± SD

Fig. 4

JNK-IN-8 cooperates with dasatinib in killing primary Ph⁺ B-ALL cells. a, c, e, g, i, and k Primary bone marrow cells isolated from 6 Ph⁺ B-ALL patients were treated with dasatinib (at the concentrations of 0, 0.125, 0.25, 0.5, 1, and 2 μmol/L), JNK-IN-8 (2.5, 5 μmol/L ) or both in combination for 48 h. Cell viability was measured by the CellTiter Glo assay. Data are presented as mean ± SD; P values were calculated by the comparison between the combination group and JNK-IN-8 or dasatinib alone group. *P < 0.05, **P < 0.01. b, d, f, h, j, and l CI values were calculated based on data from a, c, e, g, i, and k, respectively, and plotted. Fractional inhibition abbreviated as Fa

Fig. 5

The combination treatment with JNK-IN-8 and dasatinib significantly prolongs the life of BCR/ABL⁺ B-ALL mice. a Percentage of GFP-positive CD19⁺ B lymphocytes in peripheral blood (PB) from BCR/ABL^p190 bone marrow transduction and transplantation mice treated with vehicle, dasatinib (2 mg/kg), JNK-IN-8 (20 mg/kg) alone, or both in combination QD, respectively, for 3 days. b Percentage of GFP-positive CD19⁺ B lymphocytes in PB from BCR/ABL^p190 bone marrow transduction and transplantation mice treated with compounds as described in a for 8 days. c Hematoxylin and eosin staining of spleens from BCR/ABL^p190 bone marrow transduction and transplantation mice treated with compounds as described in a for 10 days. Scale bars in the upper and lower panel are 200 μm and 50 μm, respectively. d Flow cytometry analysis of bone marrow cells from mice as described in C. e The Kaplan-Meier survival curves of BCR/ABL^p190 bone marrow transduction and transplantation mice continuously administrated with vehicle, dasatinib (2 mg/kg), JNK-IN-8 (20 mg/kg) alone, or both drugs in combination QD for 18 days. P values were calculated by log-rank test and shown