Figure 3.
RUNX1A synergizes with Gata1s in leukemic transformation of murine FLCs. Cas9-knockin Ter119− FLCs were lentivirally transduced with either Gata1 (sgGata1s) or sgCtrls, as well as with RUNX1A, RUNX1B, RUNX1C, or EV control. (A) Absolute cell number of murine FLCs after combined transduction with lentiviral vectors and maintenance in liquid cultures. Data from 1 representative experiment performed in replicates are shown as mean ± SD (2-way ANOVA); ∗P < .05, ∗∗P < .001, ∗∗∗∗P < .0001. (B) Flow cytometry plots of murine sgGata1s + RUNX1A, sgGata1s + EV, and sgCtrl + RUNX1A transduced FLCs in liquid culture. The percentage of cells belonging to each immunophenotype is indicated in the corresponding gates. Data from 3 replicates ± SD are shown. (C-D) Bar graphs showing NESs of significantly upregulated or downregulated gene sets associated with differentiation and oncogenic cellular programs, in Gata1s- (sgGata1s + EV, orange), RUNX1A- (sgCtrl + RUNX1A, blue), and Gata1s- and RUNX1A- (sgGATA1s + RUNX1A, red) murine FLCs compared with sgCtrl + EV control FLCs. This analysis highlights the unique and synergistic functional features of Gata1s and RUNX1A. ∗False discovery rate (FDR) q < .25; ∗∗FDR q < .05. (E) Bar graph showing NESs of significantly upregulated or downregulated gene sets in (red) Gata1s + RUNX1A– and (green) Gata1s + RUNX1C–expressing FLCs compared with control sgCtrl + EV FLCs. This analysis highlights the contrasting functional features of RUNX1A and RUNX1C on a Gata1s background. ∗FDR q < .25; ∗∗FDR q < .05. (F-H) Analysis of mice transplanted with RUNX1A- or EV-transduced Gata1s-FLCs (n = 10 per group), including comparisons of Kaplan-Meier survival curves (log-rank test) (F), spleen weights (unpaired Student t test) (G), and flow cytometry on bone marrow cells from the diseased mice (2-way ANOVA) (H). (I) Heat map representation of RNA-seq data, showing unsupervised hierarchical clustering on the 2824 most variable genes (SD > 1) across 5 bone marrow samples from leukemic mice and fluorescence-activated cell sorter–sorted murine fetal liver HSCs [LSK (Lin−Sca1+cKit+), common myeloid progenitors (CMPs) (Lin−Sca1−cKit+CD34+ CD16/32med), granulocyte-monocyte progenitors (GMPs) (Lin−Sca1−cKit+CD34+ CD16/32+), and megakaryocyte-erythroid progenitors (MEPs) (Lin−Sca1−cKit+CD34− CD16/32low)]. The sample types are color coded on the bottom of the heat map and z scores are indicated by the legend at the top of the panel. (J) Bar graph showing NESs of significantly upregulated or downregulated gene sets in the murine leukemia samples compared with all progenitor populations. ∗FDR q < .25; ∗∗FDR q < .05. LT-HSCs, long-term hematopoietic stem cells.

RUNX1A synergizes with Gata1s in leukemic transformation of murine FLCs. Cas9-knockin Ter119− FLCs were lentivirally transduced with either Gata1 (sgGata1s) or sgCtrls, as well as with RUNX1A, RUNX1B, RUNX1C, or EV control. (A) Absolute cell number of murine FLCs after combined transduction with lentiviral vectors and maintenance in liquid cultures. Data from 1 representative experiment performed in replicates are shown as mean ± SD (2-way ANOVA); ∗P < .05, ∗∗P < .001, ∗∗∗∗P < .0001. (B) Flow cytometry plots of murine sgGata1s + RUNX1A, sgGata1s + EV, and sgCtrl + RUNX1A transduced FLCs in liquid culture. The percentage of cells belonging to each immunophenotype is indicated in the corresponding gates. Data from 3 replicates ± SD are shown. (C-D) Bar graphs showing NESs of significantly upregulated or downregulated gene sets associated with differentiation and oncogenic cellular programs, in Gata1s- (sgGata1s + EV, orange), RUNX1A- (sgCtrl + RUNX1A, blue), and Gata1s- and RUNX1A- (sgGATA1s + RUNX1A, red) murine FLCs compared with sgCtrl + EV control FLCs. This analysis highlights the unique and synergistic functional features of Gata1s and RUNX1A. ∗False discovery rate (FDR) q < .25; ∗∗FDR q < .05. (E) Bar graph showing NESs of significantly upregulated or downregulated gene sets in (red) Gata1s + RUNX1A– and (green) Gata1s + RUNX1C–expressing FLCs compared with control sgCtrl + EV FLCs. This analysis highlights the contrasting functional features of RUNX1A and RUNX1C on a Gata1s background. ∗FDR q < .25; ∗∗FDR q < .05. (F-H) Analysis of mice transplanted with RUNX1A- or EV-transduced Gata1s-FLCs (n = 10 per group), including comparisons of Kaplan-Meier survival curves (log-rank test) (F), spleen weights (unpaired Student t test) (G), and flow cytometry on bone marrow cells from the diseased mice (2-way ANOVA) (H). (I) Heat map representation of RNA-seq data, showing unsupervised hierarchical clustering on the 2824 most variable genes (SD > 1) across 5 bone marrow samples from leukemic mice and fluorescence-activated cell sorter–sorted murine fetal liver HSCs [LSK (Lin−Sca1+cKit+), common myeloid progenitors (CMPs) (Lin−Sca1−cKit+CD34+ CD16/32med), granulocyte-monocyte progenitors (GMPs) (Lin−Sca1−cKit+CD34+ CD16/32+), and megakaryocyte-erythroid progenitors (MEPs) (Lin−Sca1−cKit+CD34− CD16/32low)]. The sample types are color coded on the bottom of the heat map and z scores are indicated by the legend at the top of the panel. (J) Bar graph showing NESs of significantly upregulated or downregulated gene sets in the murine leukemia samples compared with all progenitor populations. ∗FDR q < .25; ∗∗FDR q < .05. LT-HSCs, long-term hematopoietic stem cells.

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