independent experiments. In the stromalculturing system, leukemic spleen cells were treated with DMSO or in combination of imatinib and everolimus for 5 days. Cells were stained with CD34, CD38, human CD45, propidium iodide, and Annexin V. Cells were gated for human CD45t and CD34/ CD38, and cell viabilities in CD34t38 population are shown. Panels show a representative analysis. AT9283 Bcr-Abl inhibitor Everolimus overcomes resistance in quiescent Pht leukemia cells Y Kuwatsuka et al 4 humanized NOG mouse. Percentage of CD19t leukemic cells in peripheral blood was lowest in the imatinibplus everolimus treated group, compared with the vehicle or imatinib alone. Overall tumor burden, as assessed by spleen weight and the total number of splenic human CD19t leukemic cells, was observed to be lowest in the imatinib plus everolimus treated group.
Immunohistochemistry showed that the combination Ki16425 355025-24-0 of imatinib plus everolimus K562 IIM p BCR ABL p BCR ABL BCR ABL BCR ABL ABL ABL 4 hrs 12 hrs p S6K MCL 1 BCL 2 Tubulliin MCL 1 BCL 2 Tubulliin p mTOR p S6K p 4EBP1 Tubulliin CD34 CD34 IIM IIM IIM IIM IM/ Eve IM/ Eve Eve Eve Eve Eve Eve Eve p210 p190 p210 p190 CD34 CD34 CD34CD34CD38 CD34CD38 a b c Figure 3 Western blot analysis of ex vivo treated leukemic cells. CD34t cells were separated with MACS column and cells were treated with and without imatinib or everolimus for 4 h. Each sample was lysed and western blotting analysis was performed with each antibody. MACS separated cells were treated with or without everolimus 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. CD34t38 and CD34t38t 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. NOG SP NOD/SCID 50 100 HE CD34 80 60 40 20 40 30 20 10 0 Vehicle Vehicle 1.4 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.2 1 0.8 0.6 0.4 0.2 0 IM IM Eve Eve IMEve Vehicle IM Eve IMEve Vehicle IM Eve IMEve Vehicle IM Eve IMEve IMEve Vehicle control, IM 100mg/kg, Eve 5mg/kg, combination p.o, q.d. x 10 days Dissection 4w i.v. Day 0 IR 2Gy a f b c d e Figure 4 In vivo effects of everolimus on leukemic spleen cells in combination with imatinib. Non obese diabetic/severe combined immunodeficiency, and leukemic spleen cells from NOG were injected.
Experiments were performed twice with three in each treatment group. Control vehicle, imatinib 100 mg/kg, everolimus 5 mg/kg or a combination of both were administered for 10 days, and mice were dissected 24 h after the last administration on day 28 following the tumor injection. Percentages of CD19t cells in peripheral blood and bone marrow are shown, respectively. Spleen weight was relatively compared with the average of control mice in each experiment. Bars indicate average of spleen weight in each study group. CD19t leukemic spleen cell numbers were relatively compared with the average of control. Hematoxylin and eosin staining and immunohistochemical analysis with CD34 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. Everolimus overcomes resistance in quiescent Pht leukemia cells Y Kuwatsuka et al 5 decreased the infiltrated CD34t human leukem