The mTOR inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells

Abstract

In Ph-positive (Ph(+)) leukemia, the quiescent cell state is one of the reasons for resistance to the BCR-ABL-kinase inhibitor, imatinib. In order to examine the mechanisms of resistance due to quiescence and the effect of the mammalian target of rapamycin inhibitor, everolimus, for such a resistant population, we used Ph(+) acute lymphoblastic leukemia patient cells serially xenotransplanted into NOD/SCID/IL2rγ(null) (NOG) mice. Spleen cells from leukemic mice showed a higher percentage of slow-cycling G(0) cells in the CD34(+)CD38(-) population compared with the CD34(+)CD38(+) and CD34(-) populations. After ex vivo imatinib treatment, more residual cells were observed in the CD34(+)CD38(-) population than in the other populations. Although slow-cycling G(0) cells were insensitive to imatinib in spite of BCR-ABL and CrkL dephosphorylation, combination treatment with everolimus induced substantial cell death, including that of the CD34(+)CD38(-) population, with p70-S6 K dephosphorylation and decrease of MCL-1 expression. The leukemic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse system with the in vivo combination treatment with imatinib and everolimus showed a decrease of tumor burden including CD34(+) cells. These results imply that treatment with everolimus can overcome resistance to imatinib in Ph(+) leukemia due to quiescence.

Figure 1

Ex vivo analysis of humanized mouse positive (Ph⁺) acute lymphoblastic leukemia cells. (a) Leukemic spleen cells were CD34 positively selected with MACS column. CD34⁺ cells were stained with Hoechst, PyroninY and CD38-allophycocyanin (APC). Cells including CD34⁻ population that had flowed through the column were stained with Hoechst, PyroninY and CD34-APC. (b) Leukemic spleen cells were ex vivo cultured with cytokines and treated with or without imatinib (IM) for 48 h. Human CD45⁺ propidium iodide (PI)⁻ Annexin-V⁻ viable population was analyzed for CD34 and CD38 distribution. Panels show a representative experiment. (c) After treated with IM for 48 h, CD34⁺ cells were positively selected with MACS column, and stained with Hoechst, PyroninY and CD38-APC. Cells including CD34⁻ population that had flowed through the column were stained with Hoechst, PyroninY and CD34-APC. Graphs show the number of forward scatter/side scatter gated G₀ cells in each CD34/CD38 sub-population, each relative to the untreated control. Bars indicate mean±s.d. values of three independent experiments (^*P=0.03 between control and IM 3 μ for CD34⁺ CD38⁻, and ^**P=0.02 between control and IM 3 μ for CD34⁻, by one-way analysis of variance followed by Bonferroni). (d) CD34/CD38 sorted populations were treated with or without IM 3 μ for 6 h. Expression of BCR-ABL and phosphorylation of BCR-ABL and CrkL in each population was examined by western blotting analysis.

Figure 2

Ex vivo effects of everolimus on leukemic spleen cells in combination with imatinib (IM). (a) Leukemic spleen cells were co-cultured with S-17 stromal cells for up to 35 days. Cells were counted with Trypan blue, and viable cells were maintained. (b) Cells were treated with or without everolimus (Eve, 100 n) and imatinib (1 μ) alone and in combination for 5 days on S-17 cells. DNA contents were assessed (upper panels) and Hoechst/PyroninY cell cycle analysis (lower panels) was performed. (c) Percentages of G₀ population in total acquired cells were compared with dimethylsulfoxide (DMSO) control after 5-day treatment with imatinib (1 μ), everolimus (100 n) or in combination. Graph shows the means±s.d. values of three independent experiments (^*P<0.05 by one-way analysis of variance followed by Bonferroni). (d) In the stromal-culturing system, leukemic spleen cells were treated with DMSO (upper column) or in combination of imatinib and everolimus (lower column) for 5 days. Cells were stained with CD34, CD38, human CD45, propidium iodide, and Annexin-V. Cells were gated for human CD45⁺ and CD34/CD38, and cell viabilities in CD34⁺38⁻ population are shown. Panels show a representative analysis.

Figure 3

Western blot analysis of ex vivo-treated leukemic cells. (a) CD34⁺ cells were separated with MACS column and cells were treated with and without imatinib (IM, 3 μ) or everolimus (Eve, 300 n) for 4 h. Each sample was lysed and western blotting analysis was performed with each antibody. (b) MACS-separated cells were treated with or without everolimus (100 n) for 4 h. Each sample was lysed and western blotting was performed with p-mTOR, p-S6 K, p-4EBP1 antibodies. Immunoblotting by antitubulin was performed for the control. (c) CD34⁺38⁻ and CD34⁺38⁺ sorted cells were treated with treatment drugs for 4 or 12 h. Each sample was lysed and western blotting analysis was performed with each antibody.

Figure 4

In vivo effects of everolimus on leukemic spleen cells in combination with imatinib. (a) Non-obese diabetic/severe combined immunodeficiency ( mice were irradiated (IR), and leukemic spleen (SP) cells from NOG (2 × 10⁷) were injected. Experiments were performed twice with three in each treatment group. Control vehicle (n=6), imatinib 100 mg/kg (n=6), everolimus 5 mg/kg (n=6) or a combination of both (n=6) were administered for 10 days, and mice were dissected 24 h after the last administration on day 28 following the tumor injection. (b, c) Percentages of CD19⁺ cells in peripheral blood (b) and bone marrow (c) are shown, respectively. (d) Spleen weight was relatively compared with the average of control mice in each experiment. Bars indicate average of spleen weight in each study group. (e) CD19⁺ leukemic spleen cell numbers were relatively compared with the average of control (b–e: ^*P<0.001, ^**P<0.005 and ^***P<0.05 by one-way analysis of variance followed by Bonferroni). (f) Hematoxylin and eosin (HE) staining (left panels) and immunohistochemical analysis with CD34 (right panels) of the spleen from control mice and imatinib and/or everolimus-treated mice were performed. A charge-coupled device camera provided images at approximately × 100 of the original magnification.