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C. Li et al.
FLT3 is a direct target of TET1 and is suppressed by the homoharringtonine⊣TET1/5hmC/ axis
We next performed 5hmC-seq and RNA-seq of AML cells with or without HHT treatment to identify down- stream targets that are regulated by the HHT⊣TET1/5hmC axis. As TET1 has been reported to positively regulate expression of many target genes through a 5hmC-dependent mechanism,17,37 we sought to
identify responsive targets that exhibit reduced 5hmC level and downregulation in expression upon HHT treat- ment (Figure 5A). By overlapping such responsive targets with the list of TET1 potential direct targets as detected by ChIP-on-chip or ChIP-seq in the mammalian genome,37-39 we identified 1,687 potential TET1 targets that showed significant decreases in 5hmC level and sig- nificant downregulation in expression (Figure 5B, top
Figure 6. Pathways affected by the homoharringtonine (HHT)-SP1/TET1/5hmC axis. (A) Integrative analysis of our HHT-treatment RNA-sequencing (RNA-seq) data with published Tet1 knockout RNA-seq data42 to identify pathways or gene sets that were commonly affected by both HHT treatment and Tet1 knockout. RNA-seq data from MA9.3RAS and MA9.3ITD AML cells treated with phosphate-buffered saline (PBS) or HHT (10 ng/mL) for 48 hours (h), along with RNA-seq data from mouse BM Lin–/c-Kit+/Sca1+ (LSK) and multipotent progenitor (MPP) cells with or without Tet1 knockout,42 were used in the analysis. Six gene sets were identified to be affected by both HHT treatment and Tet1 knockout in all four conditions. (B) Normalized enrichment score (NES) of the six gene sets. (C) Among the six signaling pathways, MYC targets V1, MYC targets V2, E2F targets, and G2M checkpoints gene sets were significantly suppressed upon both HHT treatment and Tet1 knockout. (D) Decreased relative expression levels of genes of the MYC targets V1/V2 gene sets in MA9.3ITD and MA9.3RAS upon HHT treatment. The dot inside represents the median expression levels of the gene sets. (E) Western blot analysis of TET1, FLT3, and MYC in PBS- or HHT-treated MA9.3ITD and MONOMAC-6 cells and in MONOMAC-6 cells with or without TET1 knockdown. ACTIN was used as an endogenous control.
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