Fig. 2: MiR-139 upregulation and repression of miR-139 targets eliminates MLL-AF9 AML in vitro and inhibits leukemogenesis in mice.

A Expression levels of miR-139-5p and miR-139-3p in MLL-AF9-i139 clones (n = 3), treated with DOX (5 µg/mL, +) relative to snRNA U6 and mock-treated (−) cells, as determined by miRNA qPCR in triplicate, are shown. The two-tailed unpaired student’s t test was used for the statistical analysis. B The number of colony-forming units (CFU) of MLL-AF9-ieGFP and MLL-AF9-i139 (n = 3) HSPC clones treated with doxycycline (DOX; 5 µg/mL, (+) or mock-treated (−) as control per 8000 cells plated are shown. Data represent the average of three independent experiments. The two-tailed paired student’s t test was used for the statistical analysis. C Representative flow cytometry plot of MLL-AF9-i139 cells with or without DOX stained with propidium iodide and Annexin-V. Data are representative of three independent clones. D Relative number of colony-forming units (CFU) of MLL-AF9-ieGFP and MLL-AF9-i139-1 cells in triplicate, treated with Doxycycline (DOX; 5 µg/mL, (+)) or mock-treated (−) as control per 8000 cells plated are shown of the first plating and the subsequent replating. The cells were cultured with IL-3, IL-6, GM-CSF, and SCF (left panel) or with IL-3, IL-6, and SCF (right panel). The two-tailed paired student’s t test was used for the statistical analysis. E Kaplan–Meier plot showing the survival of mice, transplanted with MLL-AF9-GFP-TetR-KRAB-i139 LSK cells and treated with DOX (4 mg/kg) (red, n = 11) or mock-treated (black, n = 11). The Mantel–Cox test was used for the statistical analysis. F Expression levels of miR-139-5p and miR-139-3p in leukemia cells from mice treated with DOX (n = 4) in E relative to mock-treated and snRNA U6 are shown. Tissues were analyzed from mice when moribund. G Fold change of Eif4g2 (Padj: 0.0002), Hpgd (Padj: 0.0006), and Ptprt (Padj: 0.005) expression in DOX-treated (+DOX) MLL-AF9-i139 clones (n = 3) relative to mock-treated (−DOX) clones (n = 3) as determined by RNA-seq is shown. The Wald’s test with Benjamini–Hochberg correction was used for the statistical analysis. H Boxplots showing the reads (n + 1) of the total six sgRNAs targeting Men1, Dot1l, Mllt3, Eed, Rbbp4, Eif4g2, Hpgd, and Ptprt in MLL-AF9 cells 24 h or 14 days post transduction. Per gene, the order of the sgRNAs in the 24 h sample corresponds to the same sgRNAs in the 14d sample. Blue circles indicate a loss of >5 fold, while red circles indicate a loss of <5 fold of the sgRNA. All graphs depict mean ± SEM.