Figure 3.
EVI1 regulates a common core of transcriptional targets in human and murine AML. (A) Schematic outline of the workflow to investigate RUNX1/EVI1-dependent transcriptional programs. For in vivo studies, GFP-positive leukemia cells from primary recipient animals driven by NrasG12D and regulatable (TET-OFF model) or constitutive RUNX1/EVI1 (Dox control model) were transplanted into secondary recipients. After initial engraftment, mice were treated with Dox (4 mg/mL) for 3 days or kept untreated. GFP+CD45.2+cKit+Mac1− cells were sorted, and gene expression was analyzed by RNA-seq (n ≥ 3). For in vitro studies, BM-derived leukemia cells driven by constitutive RUNX1/EVI1, NrasG12D and expressing rtTA3 (TET-ON model) were cultivated and biological triplicates of cells were transduced with inducible shRNA vectors targeting RUNX1/EVI1 (shEVI1.2658) or nontargeting (shRen.713) control. Gene expression was analyzed upon purifying cells by fluorescence-activated cell sorter 3 days post Dox-mediated (0.5 μg/mL) GFP/shRNA induction. (B) Scatter plot showing log2-FCs (LFCs) of significantly deregulated (adjusted P < .05) genes (gray dots) in leukemia cells 3 days upon Dox-mediated RUNX1/EVI1 knockdown in vitro compared with nontargeting control (x-axis) vs Dox-mediated RUNX1/EVI1 repression in vivo (y-axis). Red dots represent commonly upregulated genes. Blue dots represent commonly downregulated genes. (C) Venn diagram illustrating significant (adjusted P < .05) transcriptional changes 3 days upon shRNA-mediated RUNX1/EVI1 knockdown in vitro and Dox-mediated RUNX1/EVI1 repression in vivo in murine leukemia cells. (D) GO term enrichment analyses of gene sets deregulated upon EVI1 or RUNX1/EVI1 shutdown in human and murine leukemia in vitro or in vivo. (E) Heatmap of the 68 commonly upregulated and downregulated genes of human HNT-34 cells upon EVI1 knockdown and BM-derived murine RUNX1/EVI1-driven leukemia cells upon Dox-mediated fusion protein repression (adjusted P < .05). (F) Gene set enrichment analysis illustrating the enrichment of the genes that are downregulated upon loss of EVI1 in human patients with AML with EVI1 rearrangements. IFN-γ, interferon gamma; NES, normal enrichment score; NF, nuclear factor; TGFβ, transforming growth factor β; TNF, tumor necrosis factor.

EVI1 regulates a common core of transcriptional targets in human and murine AML. (A) Schematic outline of the workflow to investigate RUNX1/EVI1-dependent transcriptional programs. For in vivo studies, GFP-positive leukemia cells from primary recipient animals driven by NrasG12D and regulatable (TET-OFF model) or constitutive RUNX1/EVI1 (Dox control model) were transplanted into secondary recipients. After initial engraftment, mice were treated with Dox (4 mg/mL) for 3 days or kept untreated. GFP+CD45.2+cKit+Mac1− cells were sorted, and gene expression was analyzed by RNA-seq (n ≥ 3). For in vitro studies, BM-derived leukemia cells driven by constitutive RUNX1/EVI1, NrasG12D and expressing rtTA3 (TET-ON model) were cultivated and biological triplicates of cells were transduced with inducible shRNA vectors targeting RUNX1/EVI1 (shEVI1.2658) or nontargeting (shRen.713) control. Gene expression was analyzed upon purifying cells by fluorescence-activated cell sorter 3 days post Dox-mediated (0.5 μg/mL) GFP/shRNA induction. (B) Scatter plot showing log2-FCs (LFCs) of significantly deregulated (adjusted P < .05) genes (gray dots) in leukemia cells 3 days upon Dox-mediated RUNX1/EVI1 knockdown in vitro compared with nontargeting control (x-axis) vs Dox-mediated RUNX1/EVI1 repression in vivo (y-axis). Red dots represent commonly upregulated genes. Blue dots represent commonly downregulated genes. (C) Venn diagram illustrating significant (adjusted P < .05) transcriptional changes 3 days upon shRNA-mediated RUNX1/EVI1 knockdown in vitro and Dox-mediated RUNX1/EVI1 repression in vivo in murine leukemia cells. (D) GO term enrichment analyses of gene sets deregulated upon EVI1 or RUNX1/EVI1 shutdown in human and murine leukemia in vitro or in vivo. (E) Heatmap of the 68 commonly upregulated and downregulated genes of human HNT-34 cells upon EVI1 knockdown and BM-derived murine RUNX1/EVI1-driven leukemia cells upon Dox-mediated fusion protein repression (adjusted P < .05). (F) Gene set enrichment analysis illustrating the enrichment of the genes that are downregulated upon loss of EVI1 in human patients with AML with EVI1 rearrangements. IFN-γ, interferon gamma; NES, normal enrichment score; NF, nuclear factor; TGFβ, transforming growth factor β; TNF, tumor necrosis factor.

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