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Therapeutic effect and mechanism of homoharringtonine in AML
tyrosine kinase inhibitors.2 However, it is still awaiting approval for use in the treatment of acute myeloid leukemia (AML). In China, HHT has been used in the treatment of leukemia for more than 30 years due to its low price and high efficiency.3 In a pilot clinical trial we launched in 2006 in Zhejiang Province, China, we used an HHT-based induction regimen, namely HAA (HHT 4 mg/m2/day, days 1-3; cytarabine 150 mg/m2/day, days 1-7; aclarubicin 12 mg/day, days 1-7) to treat de novo AML and achieved a complete remission rate of 83%.4 Afterwards, a multi-center, open-label, randomized, controlled phase III trial was carried out in China and confirmed modified HAA regimen (HHT 2 mg/m2/day, days 1-7; cytarabine 100 mg/m2/day, days 1-7; aclarubicin 20 mg/day, days 1-7) as an alternative therapeutic strategy in treating de novo AML, especially for those patients with favorable and intermediate prognosis.5 A potential mechanism by which HHT exerts its biological function is through its binding to the A site of the ribosome, resulting in the inhibition of protein synthesis.6 However, it is unclear whether there is any other mechanism(s) underlying antileukemic effect of HHT, in particular in AML.
Acute myeloid leukemia is one of the most common and fatal forms of hematopoietic malignancies, character- ized by blockage of myeloid differentiation and malignant proliferation of immature myeloid blasts.7 With contem- porary therapies, the vast majority (over 70%) of patients with AML cannot survive over five years. Despite the common myeloid background, molecular and cytogenetic alterations contribute to the heterogeneity of the disease and the variable responses to treatment. For instance, mutations in FLT3, including internal-tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations, occur in over 30% of AML cases and are often associated with poor prognosis.7-9 Meanwhile, overexpression of FLT3 has also been reported in more than 60% of AML with a variety of AML subtypes, such as AML carrying FLT3-ITD or t(11q23) [i.e. chromosome rearrangements involving the mixed lineage leukemia (MLL) gene].9 FLT3-ITD activates multiple signaling pathways, leading to the aberrant overexpression of a set of oncogenes including MYC.10 Despite extensive efforts in developing and testing various FLT3 tyrosine kinase inhibitors (TKI) in clinical trials, the long-term therapeutic effects are still not promising,11,12 indicating that the development of more effective therapeutics to treat FLT3-mutated AML remains an unmet need.
The Ten-eleven translocation (TET) proteins (including TET1/2/3) are a family of methylcytosine dioxygenases that convert 5-methylcytosine (5mC) to 5-hydrox- ymethylcytosine (5hmC), leading to active or passive DNA demethylation.13 TET1, the founding member of the TET family, was first identified as a fusion partner of the MLL gene associated with t(10;11)(q22;q23) in AML.14,15 In contrast to the frequent loss-of-function mutations and tumor-suppressor role of TET2 observed in hematopoietic malignancies,16 we reported recently that TET1 plays a critical oncogenic role in the pathogenesis of various sub- types of AML and represents a promising therapeutic tar- get for AML treatment.17-19 The oncogenic role of Tet1 in the development of myeloid malignancies was also observed by others.20
In the present study, we show that HHT exhibits potent anti-AML effects both in vitro and in vivo, and affects DNA epigenome by directly targeting SP1 and inhibiting SP1-
mediated transcriptional regulation of TET1 expression, thereby reducing global 5hmC levels. Moreover, we demonstrate that FLT3 is a direct target of the HHT┤SP1/TET1/5hmC axis, and therefore HHT treat- ment substantially inhibits the FLT3/MYC pathways. Consistently, human primary FLT3-ITD AML cell samples display particularly high sensitivity to HHT treatment. Taken together, our studies reveal a previously unrecog- nized mechanism involving HHT-induced 5hmC reduc- tion in treating AML, and suggest that HHT-based regi- mens hold great therapeutic potential for the treatment of AML, especially that carrying FLT3 mutations.
Methods
Cell lines and cell culture
MA9.3ITD (MLL-AF9 fusion gene plus FLT3-ITD mutation; MLL, also known as KMT2A) and MA9.3RAS (MLL-AF9 fusion gene plus NRASG12D mutation) were established by Dr. James Mulloy and cultured in IMDM supplemented with 20% FBS.21 MONOMAC 6, MV4-11, MOLM13, Kasumi-1, THP-1, SKNO-1 and ML-2 are obtained and maintained as previously described.22 Homoharringtonine purchased from Sigma-Aldrich was used in this study.
Mouse model
B6.SJL-Ptprc (CD45.1) mice and NOD/LtSz-scid IL2RG-SGM3 (NSGS) mice were obtained from the Jackson Lab (Bar Harbor, ME, USA), and bone marrow transplantation (BMT) or xeno- transplantation assays were carried out as previously described.17,19,22,23 All mice were maintained in the animal core facility of the University of Cincinnati. All experiments were con- ducted according to our research protocol which was approved by the Institutional Animal Care and Use Committee. In all mouse models, HHT (1 mg/kg body weight) or phosphate-buffered saline (PBS) was intraperitoneally injected daily for ten consecutive days from day 11. The mice were euthanized by CO2 inhalation once typical leukemic symptoms, i.e. paralysis, hunched posture, labored breathing and decreased activity, had been observed. The peripheral blood (PB), bone marrow (BM), spleen and liver sam- ples were harvested for further analysis.
Patients’ samples
Bone marrow aspirates and PB samples were obtained from AML patients with their written informed consent. Mononuclear cells (MNC) were isolated and used for subsequent experiments. The genetic mutations were tested by the First Affiliated Hospital, Zhejiang University College of Medicine. The study was approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University College of Medicine.
DNA 5hmC sequencing
DNA samples were collected and sent for 5hmC sequencing. 5hmC library construction and sequencing were performed by Dr. Chunxiao Song’s lab as previously reported.24 The identification of 5hmC peaks in each sample was performed using MACS2,25 gene expression was analyzed by RSEM,26 5hmC and expression target analysis was performed by BETA.27 5hmC sequencing data have been deposited in the Gene Expression Omnibus (GEO) reposito- ry with the accession number GSE103144.
RNA sequencing and RNA-sequencing analysis
RNA sequencing was performed with total RNA samples isolat- ed from MA9.3RAS and MA9.3ITD AML cells with or without
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