Expression of RUNX1::RUNX1T1 deregulates the proteome, induces C/EBPβ suppression, and blocks myeloid differentiation

Expression of RUNX1::RUNX1T1 (also known as RUNX1::ETO) is frequently observed in acute myeloid leukemia (AML) and has been shown to block myeloid development. Although several studies have shown RUNX1::RUNX1T1 transcriptional deregulation, the proteome in human hematopoietic stem and progenitor cells (HSPCs) is poorly characterized. Using mass spectrometry, we show that expression of RUNX1::RUNX1T1 in human HSPCs was linked to differential expression of 257 proteins, including CCAAT/enhancer-binding protein beta (C/EBPβ) downregulation. Consistent with this observation, although CEBPB mRNA is generally overexpressed in AML, patients with t(8;21) have comparatively low CEBPB expression. We show in human HSPCs that ectopic expression of C/EBPβ can promote proliferation of myeloid cells. Conversely, short hairpin RNA-mediated knockdown of C/EBPβ inhibited the growth of normal human myeloid cells; however, it promoted the growth of cells expressing RUNX1::RUNX1T1. C/EBPβ also influenced differentiation, with ectopic expression promoting monocyte development, whereas reduced expression favored granulocyte differentiation, which in turn suggests that C/EBPβ expression may act as a lineage discriminator for these myeloid cell types. In summary, our data reveal how RUNX1::RUNX1T1 drives deregulation of the HSPC proteome and suggest a mechanism by which RUNX1::RUNX1T1 expression contributes to leukemia development, including C/EBPβ, which itself affects normal myeloid development.