RUNX1 mutations enhance self-renewal and block granulocytic differentiation in human in vitro models and primary AMLs.

To unravel molecular mechanisms by which Runt-related transcription factor 1 (RUNX1) mutations contribute to leukemic transformation, we introduced the RUNX1-S291fs300X mutation in human CD34 stem/progenitor cells and in human induced pluripotent stem cells (iPSCs). In both models, RUNX1mut overexpression strongly impaired myeloid commitment. Instead, self-renewal was enhanced, as shown, by increased long-term culture-initiating cell frequencies and enhanced colony-forming cell replating capacity. Long-term suspension cultures with RUNX1mut-transduced cord blood (CB) CD34 cells continued for more than 100 days, during which the cells displayed an immature granulocyte-macrophage progenitor-like CD34/CD123/CD45RA phenotype. The CD34/CD38 hematopoietic stem cell (HSC) population most likely acted as cell of origin, as HSCs provided the best long-term proliferative potential on overexpression of RUNX1mut. expression was reduced in RUNX1mut cells, and reexpression of partly restored differentiation. RNA-seq analysis on CB/iPSC systems and on primary patient samples confirmed that RUNX1 mutations induce a myeloid differentiation block, and that a common set of RUNX1mut-upregulated target genes was strongly enriched for gene ontology terms associated with nucleosome assembly and chromatin structure. Interestingly, in comparison with AML1-ETO binding in acute myeloid leukemias (AMLs), we found significantly distinct genomic distribution and differential expression for RUNX1mut of genes such as , , and that may potentially contribute to the underlying difference in clinical outcomes between RUNX1mut and AML1-ETO patients. In conclusion, RUNX1mut appears to induce a specific transcriptional program that contributes to leukemic transformation.