The role of RUNX1 serine residue phosphorylation and CDK9 in megakaryocyte specification from megakaryocyte-erythroid progenitors. (A) RUNX1 serine/threonine residues, with schemata of wild-type, alanine-mutated, phosphorylation-resistant (4A) and aspartate-mutated phosphomimetic (4D) constructs. (B) Overexpression of RUNX1-WT in transduced MEPs results in increased megakaryocyte colony formation. (C) Phosphoserine residues pS276P and pS303P are relatively increased megakaryocyte progenitors. (D) Overexpression of RUNX1-WT and RUNX1-4D constructs, but not RUNX1-4A, in HEL cells induces increased megakaryocyte colony formation and expression signatures enriched for megakaryocyte genes. (E) Inhibition (flavopiridol) or degradation (Thal-SNS-032) of CDK9 inhibits RUNX1 pS276P and pS303P, whereas degradation (Thal-SNS-032) also inhibits megakaryocyte colony formation from MEPs. AD, activation domain; DBD, DNA binding domain; Meg, megakaryocyte; MEP, megakaryocyte-erythroid progenitor.