In vivo bortezomib treatment of Mll^PTD/wt:Flt3^ITD/wtdKI mice. Whole spleen cells isolated from secondary transplants of leukemic Ly5.2⁺ dKI mice were intravenously injected into sublethally irradiated Ly5.1⁺ syngeneic C57Bl/6 mice. Upon development of leukemia, as measured by WBC counts >11 K/µL, treatment was initiated as the following: 1 mg/kg IV twice per week for 2 weeks followed by 2.5 mg/kg IV twice per week for 2 weeks. (A) Kaplan-Meier survival curve. n = 5 for each group. Median survival times: 34 days vehicle, 45 days free bortezomib, not reached with liposomal bortezomib. At 90 days posttransplant, remaining liposomal bortezomib–treated mice were euthanized. (B) Spleen images and weights of mice treated with vehicle, empty liposomes, free bortezomib, or liposomal bortezomib at time of euthanization. n = 5 for each group. (C) Bone marrow, spleen, and liver were stained with hematoxylin and eosin to show normal morphology and lack of infiltrating leukemic blasts in the liposomal bortezomib–treated mice. Slides from 3 individual mice from each treatment group were read in a blinded manner by a pathologist. A representative example is shown for each group. Empty liposomes or free bortezomib–treated mice showed significant blast infiltration in all tissues and loss of normal morphology. Bars represent 50 µm for BM, 500 µm for spleen, and 100 µm for liver. (D-E) RNA was isolated from whole bone marrow (D) or blood (E) taken 0 (n = 3) or 24 (n = 3) hours after a single 1 mg/kg dose of bortezomib on leukemic mice. After complementary DNA preparation, real-time RT-PCR was performed for pri-miR-29b-2.