Figure 6.
High RUNX1 levels are required for transformation of human AML cells. (A) Schematic representation of the experimental design is shown. FLT3-ITD–expressing MV4;11 cells were transduced with lentiviral vectors expressing RUNX1-specific or scrambled shRNA and analyzed for cell growth in vitro and tumor growth in vivo. SFFV, spleen focus-forming virus promoter. (B) Western blot analysis of RUNX1 in shRNA-transduced MV4;11 cells. (C) Representative flow cytometry analyses of shRNA-transduced and nontransduced MV4;11 in competitive in-vitro culture. The graph depicts the relative frequency of shRNA-transduced (Venus+) versus nontransduced cells over time. Shown is the mean of two independent experiments. Error bars represent SD. (D) Flow cytometry analyses of transduced MV4;11 cells before transplantation into NSG mice demonstrating efficient transduction of 95% (sh_scr) or 92% (RUNX1_sh_II). Two independent cell lines for each vector were established with similarly high transduction frequencies. (E) Combined Kaplan-Meier survival curves of the indicated NSG cohorts for two independent experiments with a total of seven mice per cohort. Mice were transplanted with 105 cells from two independently transduced MV4;11 cultures. The p-value was calculated by a Mantel-Cox log-rank test. ***, P < 0.001. (F) Relative transcript levels of CXCR4 in scrambled or RUNX1-shRNA–transduced MV4;11 cells as determined by RT-PCR. n = 3. Error bars represent SD.