Asxl1 mutation enhances CSF3R-driven differentiation. (A) Representative flow cytometric plots of CD11b and GR1 48 hours after estrogen withdrawal in HoxB8 cells transduced with plasmids containing Asxl1^MT2-GFP and/or CSF3R^T618I-mCherry and/or their corresponding empty controls producing all following 4 groups: control, Asxl1^MT2, CSF3R^T618I, and double mutant or Asxl1^MT2/CSF3R^T618I. Quantification of transduced mCherry/GFP–positive cells (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with a Tukey multiple comparison test. (B) RNA sequencing was performed 24 hours after estrogen withdrawal with all 4 groups (n = 3 per group). Heat maps represent the normalized, row-scaled (gene-based z-score scaled) counts of the most significantly differentially expressed genes (Padj < .05) between the control and double-mutant cells, and genes are clustered by similar expression patterns. Each cluster has a corresponding Gene Ontology (GO) tree of the top 15 most significantly enriched GO terms and their associated number of gene hits and adjusted P values. (C) Gene Set Enrichment Analysis (GSEA) between Asxl1^MT2/CSF3R^T618I and control cells. (D) GSEA between Asxl1^MT2/CSF3R^T618I and Asxl1^MT2 cells. (E) GSEA between Asxl1^MT2/CSF3R^T618I and CSF3R^T618I cells. Gene ranks from GSEA are represented as line charts for the top 5 most significant hallmark gene sets from the Molecular Signatures Database (MSigDB). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. NES, normalized enrichment score; Padj, adjusted P value.