Page 169 - 2020_01-Haematologica-web

Therapeutic effect and mechanism of homoharringtonine in AML
ing SP1/TET1, HHT treatment causes a substantial decrease in global 5hmC abundance and thereby marked- ly changes DNA epigenome and reprograms the down- stream pathways. We demonstrated that SP1 is a direct drug target of HHT and a positive transcriptional regulator of TET1, and HHT competitively inhibits the binding of SP1 to the promoter region of TET1 and thereby suppress- es SP1-mediated TET1 transcription; knockdown of either SP1 or TET1 can largely recapitulate the effects of HHT in AML. In addition, we have previously showed that deple- tion or suppression of TET1 expression dramatically inhib- ited AML progression and substantially prolonged survival in AML mice,17-19 recapitulating the potent in vivo anti-AML effect of HHT. Moreover, depletion of TET1 expression could make AML cells much less sensitive to HHT, further suggesting that the anti-AML activity of HHT relies on the suppression of the SP1/TET1/5hmC axis. However, fur- ther systematical studies are warranted to determine which particular sites/domains of SP1 are bound by HHT; such information would help us better understand how HHT disrupts the transcription-factor activity of SP1. Interestingly, SP1 has also been reported to positively reg- ulate expression of BCR-ABL in chronic myeloid leukemia (CML) cells.46 Thus, the antileukemic effect of HHT in CML might not be solely due to its binding to ribosome,6 but likely also through targeting SP1 directly and thereby suppressing SP1-mediated activation of the BCR-ABL and TET1 signaling pathways.
Furthermore, our 5hmC-seq and RNA-seq analyses identified FLT3 as a critical target of the HHT⊣SP1/TET1/5hmC axis; HHT treatment or TET1 knockdown markedly reduced 5hmC abundance on FLT3 locus and decreased FLT3 expression in AML cells, and our ChIP-qPCR assay confirmed that FLT3 is a direct target of TET1. Interestingly, consistent with previous reports,23,40,41 here we showed that FLT3 exhibits a positive reciprocal regulation relationship with HOXA9/MEIS1, two known targets of TET1.17 Thus, our data suggest that, by suppres- sion of TET1 expression, HHT simultaneously inhibits expression of multiple target genes of TET1 (which may form a reciprocal positive regulatory loop) in AML cells and thereby displays a potent antileukemic effect.
FLT3 encodes a class III receptor tyrosine kinase that regulates hematopoiesis and the mutation of FLT3 is the most common driven mutation found in more than 30% of AML patients.47 Both ITD and tyrosine kinase domain mutation of FLT3 result in its constitutive activation and thus lead to leukemogenesis by promoting expression of a number of critical oncogenic downstream targets such as MYC.10-12,48 Despite the extensive efforts in developing and testing FLT3 inhibitors in the clinic, AML patients with high allelic ratio FLT3-ITD are still classified as adverse risk category in 2017 European LeukemiaNet recommen- dation due to the high relapse rate and poor overall sur- vival.7,11,12 Thus, the development of improved therapeu- tics for treating FLT3-ITD AML is still an unmet need.
Here we also showed that primary AML patients with FLT3 mutations, including both newly diagnosed and relapsed patients, exhibit a high sensitivity to HHT treat- ment (with IC50 <30 nM). Consistent with our findings, another group also reported recently that HHT exhibited preferential antileukemic effect against AML carrying FLT3-ITD as detected by an in vitro drug screening on patients' samples.49 In addition, they conducted a phase II
clinical trial in relapsed/refractory FLT3-ITD AML patients, in which 20 out of 24 patients achieved complete remission with sorafenib and HHT combination treat- ment (median leukemia-free survival and overall survival: 12 and 33 weeks, respectively).49 While they showed sorafenib alone reduced the amount of pFLT3 protein, and HHT alone reduced the amount of both FLT3 and pFLT3 protein in FLT3-ITD AML cell lines, no further mechanis- tic studies were carried out.49 Our studies elucidated the molecular mechanism underlying the high sensitivity of FLT3-mutated AML to HHT treatment. This mechanism involves HHT-induced reprogramming of DNA epigenome by targeting the SP1/TET1/5hmC axis and thereby inhibition of transcription of a set of critical onco- genic targets, especially FLT3, which in turn leads to the suppression of FLT3 downstream pathways, such as MYC signaling. Notably, it is well known that FLT3-ITD muta- tion patients under therapy often develop secondary FLT3 mutations which result in drug resistance. Interestingly, we found that MONOMAC 6, which carries the FLT3 V592A mutation,50 was also sensitive to HHT. Moreover, the relapsed/refractory FLT3-ITD AML patient also showed sensitivity to HHT treatment and could be bridged to transplantation in subsequent treatment49 (Figure 7B). Therefore, HHT-based therapeutics (i.e. HHT plus other therapeutic agents such as FLT3 inhibitors and/or standard chemotherapy) represent effective novel treatments for de novo or relapsed/refractory FLT3-mutated AML patients.
In summary, here we show that HHT, approved by the FDA for CML treatment, also exhibits potent therapeutic efficacy in AML and decreases global 5hmC abundance by targeting the SP1/TET1/5hmC axis. Although other mech- anisms, such as inhibition of protein synthesis,6 may also contribute to the overall antileukemic effects of HHT in AML, which warrant further systematical studies in the future, our work reveals a novel mechanism involving suppression of the SP1/TET1/5hmC/FLT3-HOXA9- MEIS1/MYC signaling through which HHT exhibits potent therapeutic activity in treating AML. Since AML is characterized by cytogenetic and molecular heterogeneity, targeted therapy is a growing trend for selected subtypes of AML, especially for those with adverse prognosis. Here we provide compelling functional and mechanistic data suggesting that an HHT-based therapeutic approach repre- sents effective target therapeutics to treat AML carrying FLT3 mutations and/or with overexpression of endoge- nous TET1/FLT3/HOXA9/MEIS1, which accounts for more than 40% of total human AML cases.7
Acknowledgments
The authors would also like to thank Dr. James Mulloy for the generous gift of MA9.3ITD and MA9.3RAS cell lines, and Dr. Ravi Bahtia for the kind gift of MV4-11 and MOLM-13 cell lines.
Funding
The work was supported in part by the National Institutes of Health (NIH) R01 Grants CA214965 (JC), CA211614 (JC), CA178454 (JC), CA182528 (JC), and CA236399 (JC) and a R56 grant DK120282 (JC), as well as grants from National Natural Science Foundation of China 81820108004 (JJ) and 81900154 (CL). JC is a Leukemia & Lymphoma Society (LLS) Scholar.
haematologica | 2020; 105(1)
159