Evi1 Promotes Leukemogenesis by Anti-Apoptotic Rather Than Differentiation-Blocking Effects in Murine MLL-AF9 Leukemia
Authors: Kumar Ashish , Li Quanzhi , Hudson Wendy A , Sam Thien N , Chen Weili , Kersey John H 
Published: April 2020

Abstract
In a search for molecular targets in MLL -leukemias, we found that transformed hematopoietic stem cells in knock-in MLL-AF9 mice expressed high levels of Evi1 compared to their wild type counterparts (Chen et al, Cancer Cell, 2008). About 50% of human AMLs with MLL rearrangements also express Evi1 and Evi1-expressing human leukemias are associated with an aggressive disease. The current experiments were designed to study mechanisms in which Evi1 is important for the pathogenesis of leukemia. To investigate its role in MLL-AF9 leukemia, we studied the effect of Evi1 knock-down in a cell line derived from a leukemic MLL-AF9 knock-in mouse. This cell line (4166) displays a myelo-monocytic phenotype, similar to human MLL-AF9 leukemias and induces leukemia with high efficiency when transplanted in wild type mice. To knock down Evi1, we screened multiple shRNA constructs cloned into a lentivirus expression vector (The RNAi Consortium/Open Biosystems). Of five shRNAs screened, two clones (E95 and E97) consistently inhibited cell growth in a dose dependent manner at multiplicities of infection (MOI) of 10 or greater. In one representative experiment, at 5 days post transduction at an MOI of 100, growth of E95 transduced 4166 cells was decreased by &gt;80% compared to control-virus transduced cells. Evi1 mRNA was decreased in E95 transduced cells by &gt;70% compared to controls at 48 hours. When cultured in semi-solid methylcellulose media for 7 days, E95 transduced cells formed significantly fewer colonies than control virus transduced cells (457.8 ± 24.7 and 847.8 ± 92.9 colonies per 1000 cells respectively, p&lt;0.02). Surprisingly however, there was no change in the proportions of dense vs. loose colonies indicating that loss of Evi1 did not result in differentiation. Morphologic analysis also showed no change in morphology of E95 transduced cells. Further confirming a lack of differentiation-inhibiting effects of Evi1, quantitative RT-PCR assays showed no change in expression of genes associated with myeloid differentiation (integrin alpha-L, integrin beta-5, lysozyme, Csf-1). In Evi1 knock-down cells, analysis of DNA content by flow cytometry at 48 hours showed no change in the proportions of cells in the G1/G0, S and G2/M phases of the cell cycle. In contrast to lack of an effect on differentiation, there was an increase in apoptosis with Evi1 knock-down as evidenced by an increase in the proportion of cells stained with Annexin V and Propidium iodide (51% vs. 29%) or a pan activated-caspase-marker and Propidium iodide (41% vs. 15%, E95 vs. control virus transduced cells respectively). To investigate the role of Evi1 in leukemia in vivo, 105 cells transduced with either E95 or control virus were injected into irradiated wild type mice. Establishment of leukemia was confirmed by leukocytosis, splenomegaly and evidence of malignant infiltrates in the spleen by morphology and FACS. Eight of nine mice that received control virus transduced cells died of leukemia by day 170 while all those given E95 transduced cells were alive. Leukemia development was significantly delayed in the animals receiving E95 transduced cells (p&lt;0.001, log rank test) with 44% of the animals being long term survivors. Overall, our results suggest that contrary to the view that leukemic-oncogenes induce self-renewal coupled to a block in differentiation, in murine MLL-AF9 leukemia Evi1 inhibits pro-apoptotic pathways and promotes cell-survival without a differentiation-block. Additionally, given the critical requirement of this transcription factor in the survival of leukemia cells, targeting Evi1 may have therapeutic potential to treat leukemia.