We and others have previously shown that BCR ABL expression promotes FoxO3a BMS-599626 EGFR inhibitor phosphorylation at Aktconsensus sites leading to its persistent localization in the cytoplasm and evasion of apoptosis. The expression of a FoxO3a triple mutant, in which all three Akt phosphorylation sites have been mutated, results in constitutive activity of FoxO3a and promotes the death of BCRABL transformed cells. Further, it has been demonstrated that silencing of FoxO3a  transformed cells prevents apoptosis induced by imatinib, thereby providing additional evidence towards the significance of FoxO3a inhibition in BCR ABL transformation.
Here, we test the hypothesis that BCR ABL stimulates the proteasome dependent inhibition of members of the Forkhead family of tumor suppressors. Consequently, as FoxO proteins and several other downstream mediators of BCR ABL are regulated by the proteasome degradation pathway, we investigate whether the inhibition of the proteasome pathway, using bortezomib, causes regression of leukemia. Overall, our results provide novel evidence towards the involvement of the proteasome pathway in the inhibition of FOXO tumor suppressors in the context of leukemogenesis, and demonstrate for the first time using an in vivo model, that the proteasome pathway plays a role in BCR ABL mediated leukemogenesis. Our results also further indicate the potential for proteasome inhibition therapy in the context of imatinib resistant BCR ABL mutations.
MATERIALS AND METHODS Plasmids and Cell lines pMSCV IRES GFP and pMSCV BCR ABL IRES GFP, have been described in our previous work. BaF3 cells containing either the control vector pMSCV neomycin resistance, or pMSCV BCR ABL neomycin resistance were provided by Dr. David Baltimore. BaF3/BCR ABL T315I cells were provided by Drs. Azam Mohammad and George Daley. Reagents Imatinib mesylate and bortezomib were purchased from the Beth Israel Deaconess Medical Center Pharmacy approved for research purposes only. Antibodies include FKHRL 1, 4G10 phosphotyrosine, HSP 90, actin, phospho FKHR / FKHRL1, phospho AKT, c AKT, c ABL, BIM. Additional antibodies used for immunohistochemistry are TRAIL, BIM, and myeloperoxidase. Bone marrow transduction, transplantation and bortezomib treatment BMT was carried out according to standard protocols.
10 days post BMT, treatments via tail vein injection with either vehicle control or bortezomib was done twiceweekly. Blood was obtained from the tail vein and blood smear slides were prepared with Wright Giemsa stain solution HEMA QUIK II according to manufacturer,s instructions. Subcutaneous xenograft tumor model and treatment BaF3 BCR ABL or BaF3 BCR ABL cells were mixed with matrigel at 1:1 v/v, and 100 l of the mixture containing 5?106 BaF3/p210 or 5?106 BaF3/ p210 cells was injected subcutaneously into the right flank of NU/NU mice. When tumor volumes reached 150 200 mm3, mice were randomized to obtain 12 mice into each treatment group. Imatinib was dissolved in distilled water and delivered at 100 mg/kg in 100 l by gavage twice a day. Bortezomib was dissolved in 0.9% saline and delivered at 0.8 mg/kg in 100 l by tail vein injection twice weekly.