This analysis indicated that BCR ABL mice express phosphorylated FoxO3a and FoxO1, albeit at low levels, whereas similar or slightly higher levels were observed in control mice. Consistent with our in vitro findings, these results suggest that the lower level of FoxO3a that is present in BCR ABL mice is predominantly phosphorylated and is therefore inactive. We also observed LY2886721 inhibitor FoxO3a targets, observed TRAIL and BIM were greatly diminished in leukemic BCR ABL mice, in contrast to vector control mice. Importantly, bortezomib treated BCR ABL transduced mice indicated a clear restoration of expression of these proteins to the levels found in control mice. Bortezomib restores expression of FoxO3a in imatinib resistant T315I cells The T315I BCR ABL mutation frequently occurs in CML patients that are resistant to imatinib, and even remains refractory to the more potent second generation kinase inhibitors.
Bortezomib treatment of imatinib resistant BCRABL T315I cells signifcantly reduced cell survival and induced apoptosis, as measured by the MTT assay and Annexin V PE/7 AAD staining, respectively. By 48h of bortezomib treatment, both BaF3/ BCR ABL and BaF3/BCR ABL T315I cells showed similar percentages of predominantly late apoptotic cells. Similar to imatinib sensitive BCR ABL cells, FoxO3a protein expression was also restored in T315I cells upon treatment with bortezomib. As an indicator of downstream apoptotic events in response to bortezomib treatment, the protein expression of BIM splice variant was increased upon bortezomib treatment.
These results indicate that FoxO3a is repressed in imatinib resistant T315I cells, and that proteasome inhibition restores FoxO3a and induces apoptosis. FoxO3a depletion via siRNAs resulted in a partial rescue against bortezomib sensitivity in BCR ABL T315I cells, suggesting that FoxO3a activity in part contributes to the effects of bortezomib. We then examined the effect of bortezomib on BCR ABL expressing tumors in vivo. BaF3 BCR ABL or BaF3 BCR ABL cells were utilized to generate xenograft tumor in nude mice by subcutaneous injection. Either imatinib or bortzomib treatment resulted in inhibition of wild type BCR ABL expressing tumors as early as 1 week. 20 days of imatinib or bortezomib treatment caused significant inhibition of tumor growth compared with vehicle group.
In the BaF3 BCR ABL tumors, bortezomib treatment started to show tumor inhibition as early as 1 week after treatment, while imatinib did not show any effect. By 20 days after treatment, bortezomib caused significant inhibition on tumor growth compared with vehicle group and imatinib group. These results show that while BaF3 BCR ABL xenograft tumors are resistant to imatinib, their growth can be significantly inhibited by bortezomib. FoxO3a expression is suppressed in Philadelphia chromosome positive CML and ALL patients In order to investigate whether FoxO3a protein expression is also lost in Ph leukemia patients, we analyzed the peripheral blood samples of several CML and ALL patients. Peripheral blood was obtained from newly diagnosed CML patients who had not yet initiated any therapy. We also obtained peripheral blood from an ALL patient who was failing the standard therapy for ALL. Fluorescent in situ hybridization confirmed that thes