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Therapeutic effect and mechanism of homoharringtonine in AML
TET1. Collectively, our data reveal a previously unrecog- nized signal pathway involving HHT, SP1 and TET1, and identify a novel mechanism by which HHT inhibits TET1 expression and thereby reduces global 5hmC modification in AML cells.
TET1 is a functionally important target of homoharringtonine that mediates homoharringtonine effects in acute myeloid leukemia
We next investigated whether knockdown of TET1 can mimic the effects of HHT (TET1 inhibition) and whether the treatment efficacy of HHT is dependent on its inhibi- tion on TET1 expression in AML. As expected, depletion of
TET1 expression by shRNA (Figure 4A) could largely mimic the effects of HHT treatment in AML cells, includ- ing inhibiting cell growth, inducing apoptosis, blocking cell cycle, and promoting myeloid differentiation (Figure 4B-G, and Online Supplementary Figure S4A-C). Furthermore, knockdown of TET1 dramatically reduced the sensitivity of AML cells to HHT treatment, with IC50 values increased to more than 2-fold (Figure 4H), suggesting that TET1 is a critical target of HHT that mediates the effects of HHT treatment in AML cells. Together, our data indicate that HHT induced cell growth inhibition, cell apoptosis, cell cycle arrest, and cell differentiation largely owing to its inhibition on TET1 expression/function in AML.
Figure 5. FLT3 is a critical target of the homoharringtonine (HHT) SP1/TET1/5hmC axis. (A) Scheme of identification of response targets of the HHT⊣SP1/TET1/5hmC axis by 5hmC-sequencing (5hmC-seq) and RNA-seq of MA9.3 RAS and MA9.3ITD acute myeloid leukemia (AML) cells treated with phos- phate-buffered saline (PBS) or HHT (10 ng/mL for 5hmC-seq samples) for 48 hours (h). Responsive targets refer to genes with downregulation in both 5hmC abun- dance and RNA level upon HHT treatment. (B) Potential HHT⊣SP1/TET1/5hmC targets found by overlap analysis of the responsive targets and putative TET1 targets (top panel). Top ten target genes were shown (bottom panel). (C) The view of 5hmC abundance across FLT3 genomic locus in MA9.3ITD cells with PBS or HHT (10 ng/mL) treatment. (D) The verification of decreased 5hmC abundance on FLT3 via Chromatin immune-precipitation (ChIP)-qPCR analysis with different primers cov- ering corresponding 5hmC peaks shown in boxes in (C). (E) ChIP-qPCR analysis of the binding of TET1, as well as MLL-fusion proteins, to the loci of FLT3 in MONOMAC 6 cells. Green bar represents the CpG island and purple bar represents exons of FLT3. Both MLL and H3K79me2 were used as positive controls. (F and G) The effects of knockdown of Tet1 on expression of Flt3 in MLL-AF9 transformed colony cells (F) and in leukemic bone marrow (BM) blast cells of bone marrow transplan- tation (BMT) recipient mice that developed MLL-AF9-induced AML (G). *P<0.05; **P<0.01; ***P<0.001; t-test. Error bar, mean±Standard Deviation.
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