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

Abstract

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.