RUNX1^R320∗ results in significant transcriptional dysregulation of megakaryocytic differentiation pathways and MYC targets. (A) Principal component analysis of RUNX1 wild-type (n = 3) and RUNX1^R320∗ (n = 3) RNA-seq samples following analysis using DESeq2. (B) Volcano plot showing differentially expressed genes between RUNX1 wild-type and RUNX1^R320∗ cells. Genes were considered significantly differentially expressed (red) with FDR ≤ 0.05 and fold-change ≥ ±1.5). (C) Comparison of differentially expressed genes between RUNX1^R320∗ and RUNX1 knockdown experiments. RUNX1^R320∗ cells were compared with RUNX1 wild-type controls and RUNX1 shRNA knockdown cells to shRNA control cells in triplicate. Both data sets were analyzed with DESeq2, with significance determined by FDR ≤ 0.05, and fold-change ≥ ±1.5. (D) Reactome pathway analysis of differentially expressed genes in RUNX1^R320∗ and RUNX1 knockdown cells, as described in panels (A-C). Pathways were considered significant with P-value < .05. (E-F) GSEA enrichment results between wild-type and RUNX1^R320∗ RNA-seq data sets, NES = normalized enrichment score. (G) MYC expression in RUNX1 and RUNX1^R320∗ cells via RNA-seq. Student t test was used to determine significance: ∗ P ≤ .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.