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S. Liu et al.
A large number of studies on RUNX1 and RUNX1-ETO have been performed to investigate their roles in normal hematopoiesis and leukemogenesis. However, the mecha- nisms by which RUNX1 and RUNX1-ETO regulate their target genes and interact with binding partners are far from clear. Identification of RUNX1 and RUNX1-ETO novel target genes and interacting proteins are still of great importance in order to develop new therapeutic strategies for t(8;21) leukemia.
Our previous study reported that sodium phenylbu- tyrate (PB), one of the HDAC inhibitors, could induce t(8;21) leukemia cells to undergo differentiation and apop- tosis.11 In another study, we identified PIG7 as a direct tar- get gene of RUNX1 that was responsible for differentia- tion and apoptosis induction of t(8;21) leukemia cells.12 In addition to PIG7, a series of genes were also up-regulated in the process, including RUNX1, KLF4 and P57. Among them, KLF4, a transcription factor frequently deregulated in a variety of malignant tumors, including several exam- ples in hematologic malignancies, was considered a tumor suppressor. In AML, KLF4 was negatively regulated by HDAC1 and overexpression of KLF4 could markedly repress proliferation of AML cells by blocking cell cycles and inducing the expression of P21 and P27.13 In T-cell acute lymphoblastic leukemia, KLF4 delayed disease pro- gression through directly inhibiting T-cell associated genes, such as NOTCH1, BCL11B, GATA3 and TCF7, and activating the BCL2/BCLXL pathway.14 Another study by Morris et al. reported that KLF4-mediated anti-leukemic effects through regulation of microRNA networks, includ- ing MIR-150 and the MYC/MAX/MXD network, provid- ing novel mechanistic support for AML treatment via increasing KLF4 expression.15
In this report, we identified KLF4 as both a novel target gene and a binding partner of RUNX1, which induced the expression of cell cycle inhibitor P57. We further con- firmed that overexpression of either of RUNX1, KLF4 or P57 could inhibit proliferation and induce apoptosis of t(8;21) leukemia cell. Our results support the hypothesis that RUNX1, KLF4 and P57 might compose a transcrip- tional activation cascade in t(8;21) leukemia cells. RUNX1 inhibited proliferation and induced apoptosis of t(8;21) leukemia cells via KLF4-mediated upregulation of P57. We believe that reactivating the “RUNX1-KLF4-P57” signaling pathway might be a potentially potent therapeutic strate- gy for t(8;21) AML.
Methods
Cell culture
All cell lines used in this study were purchased from ATCC (Manassas, VA, USA). Kasumi-1 and SKNO-1 cells were cultured in RPMI-1640 medium (Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco). SKNO-1 was also supplemented with 10 ng/mL recombi- nant human granulocyte-macrophage colony-stimulating factor (Peprotech, USA). CV-1 and HEK-293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS.
Plasmids construction
Construction of the plasmids for luciferase assays, co-immuno- precipitation assays and overexpression experiments are described in the Online Supplementary Appendix.
Lentiviral preparation and transduction of Kasumi-1 cells
Details of lentiviral preparation and transduction of Kasumi-1 cells are available in the Online Supplementary Appendix.
Real-time polymerase chain reaction and western blot analyses
Real-time quantitative polymerase chain reaction (PCR) and western blot analyses for gene expression are described in the Online Supplementary Appendix.
Co-immunoprecipitation assay
Cells were collected and lysed in Cell lysis buffer for western blotting and immunoprecipitation (IP) (Beyotime, China) on ice for 30 minutes (min). Then the lysates were pre-cleared with protein A/G-Sepharose beads (Santa Cruz, CA, USA) at 4°C for 1h before IP with indicated primary antibodies or anti-IgG anti- body (Beyotime) overnight. The protein-antibody complexes were incubated with protein A/G-Sepharose beads for another 4 hours (h). The beads were subsequently washed three times with cold lysis buffer and the bound proteins were separated by SDS-PAGE, followed by western blot assay.
Immunofluorescence analysis
Cell preparation and immunofluorescence staining proce- dures have been described previously.16 Fluorescence images were taken on a spinning disk confocal microscope (PerkinElmer, USA) by a 100X oil-immersion objective.
Luciferase assay
5×104 CV-1 cells were transfected with 100 ng indicated pGL3 luciferase reporter plasmid, 50 ng control Renilla luciferase plas- mid together with different combinations of transcription factor expression plasmid. At 48 h after transfection, cells were har- vested and the luciferase activities were analyzed according to the Dual-Luciferase Reporter 1000 Assay System Technical Manual (Promega, USA) on a luminometer (Lumat LB 9507, Berthold Technologies GmbH & Co. KG, Baden-Württemberg, Germany).
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were per- formed with a Pierce Agarose ChIP Kit according to the manu- facturer’s instructions (Thermo Scientific, USA). Each ChIP reac- tion contained chromatin from 1×107 cells and 2 μg indicated antibody. Following IP, the binding sites of transcription factor to target gene promoter were analyzed by PCR. Anti-RUNX1 anti- body (ChIP Grade, Abcam, ab23980) and Rabbit IgG antibody (Beyotime) were used in this study. Primers used in ChIP assays were listed in the Online Supplementary Table S1.
Cell proliferation, cell cycle, cell apoptosis and differentiation analyses
Details are available in the Online Supplementary Appendix.
Statistical analysis
All results were presented as mean with error bars indicating standard deviation of triplicate experiments. Statistical differ- ences were determined by Student t-test using GraphPad Prism (v.5.0). P<0.05 was considered statistically significant; *P<0.05; **P<0.01; ***P<0.001.
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haematologica | 2019; 104(8)