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Therapeutic effect and mechanism of homoharringtonine in AML
panel, and Online Supplementary Table S3). Across the genomic locus of these potential targets, the top ten tar- geted genes with the most significant decreases in 5hmC levels were listed (Figure 5B, bottom panel). Notably, FLT3, a well-recognized oncogene related to leukemogen- esis,7,9 is in the top list, associated with substantially decreased 5hmC abundance and significant downregula- tion in expression after HHT treatment (Figure 5C and Online Supplementary Figure S5A). Our ChIP-qPCR further confirmed the decreased 5hmC abundance on FLT3 gene locus upon HHT treatment in AML cells (Figure 5D).
The ChIP-seq data reported previously38 showed that Flt3 is a direct target gene of Tet1 in mouse embryonic stem cells (Online Supplementary Figure S5B). To validate whether FLT3 is also a direct target of TET1 in human AML cells, we performed ChIP-qPCR in MONOMAC 6 cells and showed that TET1 was especially enriched at the CpG area (Site 1) rather than the distal upstream area (Site 2) of FLT3 (in Figure 5E MLL and H3K79me2 are included as positive controls). Moreover, we showed that knock- down of Tet1 resulted in decreased expression of Flt3 in MLL-AF9 transformed colony cells (Figure 5F) and in BM cells of MLL-AF9 leukemia mice (Figure 5G). Conversely, forced expression of wild-type Tet1 (but not catalytically inactive mutant Tet1) in non-MLL rearranged human AML cells, such as Kasumi-1 cells (carrying t(8;21)/AML1-ETO), results in a significantly elevated expression of FLT3 (Online Supplementary Figure S5C). Similarly, in human CD34+ hematopoietic stem/progenitor cells (HSPC), we
observed a strong positive correlation between FLT3 and TET1 in expression during both granulocytic and mono- cytic differentiation models (Online Supplementary Figure S5D). Furthermore, both HHT treatment and TET1 knockdown suppressed FLT3 expression in human AML cells (Online Supplementary Figure S5E). Taken together, our results suggest that FLT3 is a direct target of TET1 and HHT treatment-induced TET1 inhibition or knockdown of TET1 suppresses FLT3 expression through a 5hmC- related mechanism.
We previously reported that HOXA9 and MEIS1 were directly targeted by TET1 in AML cells.17 Consistently, here we showed that HHT treatment could also decrease expression of HOXA9 and MEIS1 in human AML cells (Online Supplementary Figure S5F). Interestingly, we and others have also reported previously that HOXA9/MEIS1 and FLT3 may each positively regulate the expression of the other.23,40,41 Indeed, here we showed that forced expres- sion of either wild-type FLT3 or FLT3-ITD could signifi- cantly up-regulate expression of HOXA9 and MEIS1 in human 293T cells (Online Supplementary Figure S5G) in a manner similar to FLT3-ITD-mediated upregulation of HOXA9 and MEIS1 in human MONOMAC 6 AML cells, as we had reported previously.23 Conversely, we also showed that forced expression of HOXA9 and MEIS1 could substantially increase FLT3 level in mouse bone marrow progenitor cells (Online Supplementary Figure S6A). Thus, there may be a reciprocal positive regulatory loop between TET1 targets, such as FLT3 and HOXA9/MEIS1.
Figure 7. Acute myeloid leukemia (AML) with FLT3 mutations are highly sensitive to homoharringtonine (HHT) treatment. (A) The sensitivity of AML cells with and without FLT3 mutations to HHT treatment. The AML cells were treated with a series of concentrations of HHT for 48 hours. (B) The HHT-based treatment regimen used for seven relapsed/refractory FLT3-ITD AML patients in clinic. HA: HHT plus cytarabine; HAA: HHT plus cytarabine and aclarubicin. (C) The IC50 values of HHT and sorafenib in primary FLT3-ITD AML patients' samples.(C) The HHT-based treatment regimen used for seven relapsed/refractory FLT3-ITD AML patients in clinic. HA: HHT plus cytarabine; HAA: HHT plus cytarabine and aclarubicin. (D) Schematic illustration of the molecular mechanism underlying the anti-tumor effects of HHT main- ly through suppression of the SP1/TET1/5hmC/FLT3-HOXA9-MEIS1/MYC axis.
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