Evi1 knockdown (KD) in MLL-AF9 cell line 4166 leads to reduced growth in vitro and in vivo. Transduction of 4166 with lentiviral vectors containing shRNA against Evi1 leads to reduced (A) Evi1 mRNA and (D top) protein expression. (B) Reduced Evi1 levels were accompanied with a significant reduction of colony formation. Fold change of 4 experiments (mean + SD) is shown for panels A and B. (C) Evi1 shRNA-mediated inhibition of cell growth was attributed to (bottom panel) an induction of apoptosis, (top panel) with no changes in cell-cycle profile. (D) Western blotting shows an increase of (pro)apoptotic markers after knockdown of Evi1. HSP90 and actin were used to show equal cell viability and loading of protein lysates. SHC indicates short hairpin control. (E) Survival curve of lethally irradiated mice transplanted with 1 × 105 4166 cells, transduced with either Evi1 shRNA or control virus supplemented with 5 × 105 normal mBM cells. As a control group, untreated 4166 cells were transplanted to monitor offset of normal 4166-mediated MLL-AF9 tumorigenesis. All 3 groups contained 9 mice each. By posttransplantation day 170, the majority of the mice in the untreated (8 of 9) or control virus (8 of 9) transduced group had succumbed to leukemia, while all mice receiving Evi1 shRNA-transduced cells were still alive. Leukemia development was significantly delayed in animals receiving Evi1 shRNA-transduced 4166 cells (log-rank P < .001) with 44% of the mice being long-term survivors.
