Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with nilotinib against BCR-ABL-positive leukemia cells involves the ABL kinase domain mutation

Abstract

Imatinib, an ABL tyrosine kinase inhibitor (TKI), has shown clinical efficacy against chronic myeloid leukemia (CML). However, a substantial number of patients develop resistance to imatinib treatment due to the emergence of clones carrying mutations in the protein BCR-ABL. The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway regulates various processes, including cell proliferation, cell survival, and antiapoptosis activity. In this study, we investigated the efficacy of NVP-BEZ235, a dual PI3K and mTOR inhibitor, using BCR-ABL-positive cell lines. Treatment with NVP-BEZ235 for 48 h inhibited cell growth and induced apoptosis. The phosphorylation of the AKT kinase, eukaryotic initiation factor 4-binding protein 1 (4E-BP1), and p70 S6 kinase were decreased after NVP-BEZ235 treatment. The combination of NVP-BEZ235 with a BCR-ABL kinase inhibitor, imatinib, or nilotinib, induced a more pronounced colony growth inhibition, whereas the combination of NVP-BEZ235 and nilotinib was more effective in inducing apoptosis and reducing the phosphorylation of AKT, 4E-BP1, and S6 kinase. NVP-BEZ235 in combination with nilotinib also inhibited tumor growth in a xenograft model and inhibited the growth of primary T315I mutant cells and ponatinib-resistant cells. Taken together, these results suggest that administration of the dual PI3K and mTOR inhibitor NVP-BEZ235 may be an effective strategy against BCR-ABL mutant cells and may enhance the cytotoxic effects of nilotinib in ABL TKI-resistant BCR-ABL mutant cells.

Keywords: BCR-ABL mutation; NVP-BEZ235; PI3K; chronic myeloid leukemia; mTOR; nilotinib; ponatinib resistance.

Figure 1. Effects of NVP-BEZ235 and imatinib or nilotinib on Ba/F3 BCR-ABL random mutagenesis cells. Ba/F3 BCR-ABL random mutagenesis cells were treated with the indicated concentration of NVP-BEZ235 and/or nilotinib (A) or imatinib (B) for 14 d. The colony-formation assay was performed as described in Materials and Methods. The data are representative of 3 independent sets of experiments. Statistical analysis was also performed by using two-way ANOVA. Two-way ANOVA analysis revealed combined effects of two drugs were additive.

Figure 2. NVP-BEZ235 inhibits cell growth and induces apoptosis in Ph-positive cells. (A) Cells were cultured at a concentration of 8 × 10⁴/mL in the presence or absence of NVP-BEZ235 for 48 h. Viable cells were evaluated as described in Materials and Methods. (B) K562 cells were treated with NVP-BEZ235 at the indicated concentration for 48 h. The percentage of apoptotic cells was examined as described in Materials and Methods. (C) K562 cells were treated with NVP-BEZ235 for 24 h; total extracts were analyzed by immunoblot analysis with anti-phospho Akt, 4E-BP1, S6 kinase, MAPK, and anti-actin antibodies. The data presented here are representative of 3 separate experiments. (D) Ph-positive primary cells were cultured at a concentration of 2 × 10⁵/mL in the presence or absence of NVP-BEZ235 for 48 h. Viable cells were evaluated as indicated in Materials and Methods.

Figure 3. Effect of NVP-BEZ235 and nilotinib treatment on K562 cells. (A and B) Cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells and apoptotic cells were evaluated as described in Materials and Methods. *P < 0.05 for nilotinib and NVP-BEZ235 treatment vs. treatment with 50 nM nilotinib alone in the same cell line. (C) K562 cells were treated with NVP-BEZ235 and/or nilotinib for 24 h; total cellular lysates were immunoblotted with anti-phospho Abl, Akt, 4E-BP1, S6 kinase, BCL-XL, PARP, and actin Abs.

Figure 4. Effect of NVP-BEZ235 and nilotinib on Ba/F3 BCR-ABL random mutagenesis cells in xenograft model. (A) In vivo studies were performed as described in Materials and Methods. (B) Tumor cells treated with or without NVP-BEZ235 and nilotinib for 24 d were examined by immunohistochemical analysis as described in Materials and Methods. Original magnification: 400×. H&E, hematoxylin and eosin; TUNEL, TdT-mediated dUTP nick-end labeling. *P < 0.01, **P < 0.001 compared with control.

Figure 5. Co-treatment with NVP-BEZ235 and nilotinib inhibits cell growth and induces apoptosis of wild-type and T315I mutant BCR-ABL-positive cells. (A) Wild-type primary cells were cultured at a concentration of 2 × 10⁵/mL in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells were evaluated. (B) Ba/F3 T315I cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells and percentage of apoptotic cells was examined. (C) T315I mutant primary cells were cultured in the presence or absence of NVP-BEZ235 or nilotinib for 48 h. Viable cells and percentage of apoptotic cells was examined. (D) Ponatinib-resistant primary cells were treated with NVP-BEZ235 and/or nilotinib at the indicated concentration for 48 h. Viable cells were evaluated as described in Materials and Methods. *P < 0.05, nilotinib and NVP-BEZ235 treatment vs. treatment with 1 μM nilotinib alone in the same cell line.