Page 160 - 2020_01-Haematologica-web

C. Li et al.
HHT treatment (5 or 10 ng/mL for 48 hours) by The Genomics, Epigenomics and Sequencing Core of the University of Cincinnati. The Gene Set Enrichment Analysis (GSEA)28 was used to analyze the enriched signaling pathway in PBS or HHT treated cell sam- ples. The RNA sequencing data have been deposited in the GEO repository with the accession number GSE103143.
Statistical analysis
Data were analyzed with GraphPad Prism 6 and were present- ed as mean±Standard Deviation as indicated. Two-tailed Student t-test was used to compare means between groups as indicated. P<0.05 was considered significant. The Kaplan-Meier survival curves were produced with GraphPad Prism 6 and P-values were calculated using the log rank test. The densitometric analysis of the bands from Western blot or dots from dot blot were per- formed with Gel-Pro analyzer and normalized to the loading con- trols.
A detailed description of all materials and methods is available in the Online Supplementary Appendix.
Results
Homoharringtonine potently inhibits cell growth and viability and promotes cell cycle arrest, apoptosis and differentiation in human acute myeloid leukemia
To systematically investigate the therapeutic potential of HHT in AML, we first analyzed the responses of human AML cells to HHT in vitro. Three AML cell lines with various backgrounds, including MONOMAC 6, MA9.3ITD and MA9.3RAS, were included for the analy- ses (Figure 1A). Remarkably, we found that all three AML cell lines were highly sensitive to HHT treatment, with very low IC50 values (5~20 ng/mL; or 9.2~36.7 nM) (Figure 1A), and HHT significantly inhibited their growth and via- bility in a dose- and time-dependent manner (Figure 1B and Online Supplementary Figure S1A). HHT dramatically induced apoptosis (Figure 1C and D, and Online Supplementary Figure S1B and C) and cell cycle arrest in G0/G1 phase (Figure 1E and F, Online Supplementary Figure S1D and E) in AML cells. Furthermore, we also assessed the potential effect of HHT on myeloid differentiation of MONOMAC 6 and NB4 (carrying t(15;17)/PML-RARA) AML cells. Notably, HHT also significantly promoted myeloid differentiation of AML cell as detected by both flow cytometry and qualitative polymerase chain reaction (qPCR) (Figure 1G and H, Online Supplementary Figure S1F and G), including PMA-induced monocytic differentiation and ATRA-induced granulocytic differentiation. Thus, HHT exhibits a broad-spectrum antileukemic activity involving the inhibition of AML cell growth/viability and the promotion of apoptosis, cell cycle arrest, and myeloid differentiation.
Homoharringtonine substantially inhibits murine and human acute myeloid leukemia progression in vivo
We next examined the effect of HHT on survival and proliferation of primary mouse AML cells via colony form- ing assays. Leukemic BM blast cells collected from pri- mary BMT recipient mice carrying MLL-AF9- or NRAS+AE9a (AML1-ETO9a fusion gene29 plus NRASG12D)- induced full-blown AML were seeded into semi-solid medium containing PBS or HHT (5 ng/mL or 10 ng/mL) for serial plating. After three rounds of plating, HHT sig- nificantly suppressed colony-forming activity and
decreased cell proliferation of primary AML cells in a dose-dependent manner (Figure 2A). HHT treatment also markedly reduced the colony size (Figure 2B).
We then utilized leukemic mouse BMT model to assess the effect of HHT on AML progression in vivo. Briefly, pri- mary mouse MLL-AF9 AML cells (CD45.2) were injected via tail vein (i.v.) into semi-lethally irradiated recipient mice (CD45.1). Ten days post transplantation, the recipi- ents were treated with either HHT (1 mg/kg body weight) or PBS once daily for ten consecutive days (Figure 2C). As expected, HHT treatment significantly inhibited AML progression and substantially prolonged survival in the AML mice (102 days vs. 63 days; P=0.0007) (Figure 2D). Compared to the PBS-treated control group, HHT treat- ment dramatically reduced leukemic burden in PB, BM, spleen and liver in mice (Figure 2E-G and Online Supplementary Figure S2A and B).
We also employed "human-in-mouse" xenograft models to further evaluate the effect of HHT on human AML pro- gression in vivo. Human MA9.3ITD AML cells were i.v. injected into NSGS mice and ten days post xenotransplan- tation, the mice were treated with PBS or HHT (1 mg/kg body weight) for ten days (Figure 2H). HHT substantially delayed leukemia progression and prolonged survival of treated mice, associated with significantly inhibited engraftment of human AML cells and remarkably reduced leukemia burden in recipient mice (Figure 2I and Online Supplementary Figure S2C-F). Pathological morphologies also identified a significant decrease of leukemia blasts in PB, BM, liver, and spleen tissues in HHT-treated group compared with the control group (Figure 2J). Similar effects of HHT treatment were also observed in NSGS mice xeno-transplanted with human MONOMAC 6 AML cells (Online Supplementary Figure S2G-I). Collectively, HHT treatment can substantially inhibit leukemia pro- gression and prolong survival of mice carrying human or murine AML, demonstrating the potent therapeutic effica- cy of HHT in treating AML.
Homoharringtonine down-regulates global 5hmC level by targeting SP1/TET1 in acute myeloid leukemia cells
Altered epigenetic modification at DNA levels is a well- known feature of AML and displays critical effects during AML initiation, progression, and prognosis.30,31 Strikingly, we found that HHT treatment significantly reduced global 5hmC level, but not 5mC level, both in MA9.3RAS and MA9.3ITD AML cells (Figure 3A and B). Notably, amongst the genes encoding methylcytosine dioxygenase TET pro- teins (including TET1, TET2, and TET3) that convert 5mC to 5hmC,13 we found that only TET1, but not TET2 or TET3, was significantly down-regulated upon HHT treat- ment (in a dose-dependent manner) in AML cells as detected by qPCR (Online Supplementary Figure S3A). Furthermore, we also confirmed HHT-induced suppres- sion of TET1 expression through our RNA-seq data analy- sis and Western blotting assay (Figure 3C). Notably, our qPCR results indicated that the significant downregulation of TET1 started as early as at 18 hours and continued after- wards in MA9.3ITD upon HHT treatment (Figure 3E). Thus, HHT-induced decrease of 5hmC level is owing to the downregulation of TET1. To further determine whether HHT-mediated TET1 inhibition is due to tran- scriptional inhibition, we employed nuclear run-on assay,32 with biotin-labeled uridine 5'-triphosphate (UTP) (Online Supplementary Figure S3B) and showed that HHT
150
haematologica | 2020; 105(1)