FLT3^ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

The FLT3 Internal Tandem Duplication (FLT3^ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3^ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3^ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3^ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3^ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3^ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3^ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3^ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3^ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3^ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3^ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3^ITD target enhancers in either or adult HPCs. The data show that FLT3^ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3^ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3^ITD effectors.