DNA methylation analysis using RUNX1-mutated cells reveals association of FLI1 to familial platelet disorder with associated myeloid malignancies caused by a mutation in the transactivation domain of RUNX1

Background Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1 . It is characterized by thrombocytopenia with platelet dysfunction and a high risk of hematological malignancy development. Although FPDMM is a precursor condition for diseases involving abnormal DNA methylation, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the DNA methylation status of FPDMM remains unknown due to a lack of animal models and difficulty in obtaining patient-derived samples. Results Using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. The established FPDMM-mimicking iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. HPCs differentiated from FPDMM-mimicking iPSCs showed DNA methylation patterns distinct from those of wild-type HPCs. Binding motif-enrichment analysis showed the enrichment of ETS transcription factor (TF) motifs in hypermethylated regions, in contrast to the RUNX1 motif. We found that the expression of FLI1 , an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a mutation in the transactivation domain (TAD) of RUNX1. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 in FPDMM-mimicking HPC lines with a RUNX1 TAD mutation restored their Mk differentiation efficiency and hypermethylation status. Conclusion These results suggested that FLI1 is a putative causative TF responsible for differential DNA methylation and defective Mk differentiation in FPDMM-mimicking HPCs in the presence of a mutation in the TAD of RUNX1. Thus, this study provided insights into a part of pathogenesis of FPDMM.