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treatment significantly decreased the transcriptional rate of TET1 (Figure 3D), suggesting transcriptional inhibition largely contributes to HHT-induced downregulation of TET1. To elucidate the molecular mechanism by which HHT inhibits the transcription of TET1, we examined the potential binding of transcription factors (TF) on the pro- moter region of TET1 (-530 to +10 bp) and identified mul-
tiple putative binding sites of SP1 (Figure 3F, top panel, and Online Supplementary Figure S3C). SP1 is an important TF in AML and mediates drug resistance to chemotherapy.33,34 Our ChIP-qPCR confirmed the direct binding of SP1 on TET1 promoter region and such binding was remarkably decreased upon HHT treatment in AML cells (Figure 3F, bottom panel). Furthermore, we conducted drug affinity
Figure 2. Homoharringtonine (HHT) inhibits the progression of acute myeloid leukemia (AML) in vivo. (A) Effects of HHT on colony forming activity of mouse hematopoietic stem/progenitor cells (HSPC) transformed by MLL-AF9 or NRAS plus AML-ETO9a (AE9a). Colony numbers (left panel) and cell counts (right panel) from colony forming assay (CFA) were displayed. (B) Representative images of the 3rd generation of colonies under treatment with different HHT concentrations (0, 5 and 10 ng/mL) (5× microscope). (C) Schematic illustration of secondary MLL-AF9 AML transplantation mouse model coupled with HHT or phosphate-buffered saline (PBS) treatment. (D) Kaplan-Meier curves of PBS- and HHT-treated mice that were transplanted with mouse MLL-AF9 AML cells. (E-G) White blood cell (WBC) count (E), spleen (SP) weight (F), and the engraftment ratio of leukemic cells into SP (G) at the end point of the PBS- or HHT-treated MLL-AF9 AML mice. (H) Schematic illus- tration of the MA9.3ITD AML xenograft NOD/LtSz-scid IL2RG-SGM3 (NSGS) model coupled with HHT or PBS treatment. (I) Kaplan-Meier curves of PBS- and HHT-treat- ed NSGS mice that were xenotransplanted with human MA9.3ITD AML cells. (J) Wright-Giemsa staining of mouse peripheral blood (PB) and bone marrow (BM), and Hematoxylin and Eosin (H&E) staining of liver and spleen (SP) from PBS- or HHT-treated MA9.3ITD leukemic mice. Bars represent 50 mM for PB, SP and liver; 30 mM for BM. *P<0.05; **P<0.01; ***P<0.001; t-test. Error bar, mean±Standard Deviation. For Kaplan-Meier curve, P-values were calculated by log-rank test.
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