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C. Li et al.
Notably, MA9.3ITD cell line was established from human cord blood CD34+ cells virally transduced with MLL-AF9 and FLT3-ITD.21 Thus, one may expect that ectopic expression of FLT3-ITD in this cell line would not be suppressed by HHT/TET1 and thereby should show resistance to HHT, which is somewhat opposite to what we observed (e.g. Figure 1A and B). We presumed that such a discrepancy might be due to the possibility that virally transduced FLT3-ITD in MA9.3ITD cells was by chance integrated to a locus that is also under control of TET1. Actually, there are a total of 11,632 genes that are associated with Tet1 enrichment in their promoter regions [-2kb to +2kb relative to annotated transcription start sites (TSS)] as detected by at least 2 out of 3 genome-wide ChIP-on-chip or ChIP-seq analyses in mouse embryonic stem cells (mESC).37-39 Therefore, although many of such putative targets identified from mESC might not be gen- uine targets of TET1 in human AML cells, there is still a good chance that the virally transduced FLT3-ITD in MA9.3ITD cell line was integrated, by chance, to a locus that is also under control of TET1. Indeed, HHT treatment could dramatically decrease the overall FLT3 (including FLT3-ITD) protein level, suggesting that is highly likely that FLT3-ITD expression in MA9.3ITD cell line is also under control of HHT/TET1 (Figure 6E). To determine whether non-TET1-controlled ectopic expression of FLT3 or FLT3-ITD can cause resistance to HHT in transduced AML cells, we virally transduced human Kasumi-1 AML cells with a high titer of FLT3 or FLT3-ITD viruses. In this way, each transduced cell had multiple copies of FLT3 or FLT3-ITD, and thus there would be a good chance that at least one copy was integrated into a locus not controlled by TET1. We sorted transduction-positive cells (i.e. RFP+ cells) 48 hours post transduction and then treated the cells with HHT or PBS for 24 hours. Forced expression of FLT3 or FLT3-ITD conferred at least partial resistance in trans- duced Kasumi-1 cells to HHT, while TET1 expression was still suppressed by HHT (Online Supplementary Figure 5H- J). Taken together, our data suggest that FLT3 is a down- stream target of HHT/TET1 and mediates the sensitivity of AML cells to HHT.
MYC signaling is a major downstream pathway affected by the homoharringtonine⊣SP1/TET1/5hmC axis
To further identify downstream pathways affected by the HHT⊣TET1/5hmC axis, we conducted an integrative analysis of our RNA-seq data of HHT-induced TET1 inhi- bition in AML cells and RNA-seq data of Tet1 knockout in mouse HSPCs [Lin–/c-Kit+/Sca1+ (LSK) and multipotent progenitor (MPP) cells] reported by Cimmino et al.42 Through gene set enrichment analysis (GSEA), we identi- fied six gene sets strongly enriched in both HHT-induced TET1 inhibition and Tet1 knockout, including MYC targets V1, MYC targets V2, E2F targets, G2M checkpoints, MTORC1 signaling, and DNA repair (Figure 6A). The nor- malized enrichment score (NES) of the six co-enriched sig- naling pathways in all four pairs of samples are shown in Figure 6B. Among the six gene sets, MYC targets V1, MYC targets V2, E2F targets, and G2M checkpoints were significantly suppressed upon HHT treatment and Tet1 knockout (Figure 6C). The violin plots showed the down- regulated expression of the clustering genes in MYC tar- gets V1 and MYC targets V2 after HHT treatment in AML cell lines (Figure 6D). Among these suppressed signal path-
ways, MYC functions as universal transcriptional amplifi- er and directly and indirectly regulates expression of mul- tiple core enriched genes.43 More interestingly, MYC was reported as a downstream target of FLT3, and was signifi- cantly enriched in FLT3 constitutively activated cells and largely suppressed with the treatment of FLT3 inhibitor.10,12 Indeed, we showed that forced expression of either FLT3 or FLT3-ITD can substantially increase expres- sion of MYC (Online Supplementary Figure S6B). In addi- tion, consistent with previous studies showing that HOXA9/MEIS1 can up-regulate expression of MYC,44 we found that forced expression of HOXA9 and MEIS1 could also substantially increase MYC level (Online Supplementary Figure S6A). Our Western blot results also confirmed the decreased expression of TET1, FLT3 and MYC in HHT-treated or TET1-knockdown human MLL-rearranged or non-MLL-rearranged AML cells (Figure 6E and Online Supplementary Figure S6C). These findings suggest that MYC is an essential downstream target of the HHT⊣SP1/TET1/5hmC/FLT3-HOXA9-MEIS1 axis and MYC signaling is a major pathway inhibited by HHT treatment in AML.
Homoharringtonine treatment represents a promising therapeutic strategy for the treatment of acute myeloid leukemia with FLT3 mutations
In line with the above discoveries, we found that human AML cell lines with FLT3 mutations are indeed much more sensitive to HHT than those without (Figure 7A). Next, we collected four primary AML samples from de novo or relapse/refractory patients with FLT3-ITD muta- tion (Online Supplementary Table S4). Notably, all the pri- mary AML samples are highly sensitive to HHT treat- ment, with IC50 values < 30 nM; in contrast, these AML samples are relatively resistant to sorafenib, a tyrosine kinase inhibitor that was usually recommended to patients with FLT3-ITD mutation in clinic,45 with IC50 val- ues >2.7 mM (Figure 7C). The superior effect of HHT, rel- ative to sorafenib, might be owing to the fact that HHT can suppress expression of not only FLT3 but also other critical oncogenic targets of TET1 (e.g. HOXA9 and MEIS1), as mentioned above. Furthermore, we have suc- cessfully applied the HHT-based salvage chemotherapy in treating relapse/refractory patients in Zhejiang, China, and some of them were successfully bridged to BMT (Figure 7B and Jie et al., unpublished data). Together with our mechanistic studies described above, our data suggest that the high sensitivity of HHT in AML with FLT3 muta- tions is largely attributed to the HHT-induced inhibition of FLT3/HOXA9/MEIS1 expression/function through the HHT⊣SP1/TET1/5hmC axis (Figure 7D).
Discussion
Previous studies have reported that HHT-based chemotherapy exhibited a high efficiency in treating de novo AML patients,4,5 but the underlying mechanism has not been well elucidated. In the present study, we showed that HHT treatment alone caused potent inhibition of AML cell growth/survival in vitro and substantial suppres- sion of AML progression in vivo, and such inhibitory effects are likely attributed to HHT-induced cell cycle blockage and apoptosis, as well as enhanced myeloid dif- ferentiation. Mechanistically, we showed that, by target-
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