1.The Impacts Of Protein Interaction And Transcriptional Regulatory Relationship Between RUNX1 And KLF4 On The Biological Functions Of T(8;21) Leukemia Cells 2.the Biological Effects And Mechanism Studies Of A Novel LSD1 Inhibitor JL1037 On Leukemia Cells

Objective:Our previous study has reported that sodium phenylbutyrate(PB),one of the HD AC inhibitors,could induce Kasumi-1 cells to undergo differentiation and apoptosis.By analyzing the microarray data of Kasumi-1 cells treated with PB,we found that the expression levels of RUNX1,KLF4 and P57 were significantly upregulated.Bioinformatics analysis further demonstrated that there might be some relationship among these three genes.In this study,we investigated the transcriptional regulatory mechanism and the protein interaction relationship among RUNX1,KLF4 and P57 and performed both in-vitro and in-vivo experiments to explore their biological roles on t(8;21)leukemia cells.Methods:Real-time quantitative PCR(qRT-PCR)and Western blot assay were performed to evaluate the expression changes of RUNX1,KLF4 and P57 in Kasumi-1 cells after PB treatment.A KLF4 promoter/enhancer reporter plasmid containing RUNX1 binding sites and a P57 promoter/enhancer reporter plasmid containing KLF4 binding sites were constructed.Then luciferase assay was performed to investigate the roles of RUNX1 and RUNX1-ETO on transactivation of KLF4 and the role of KLF4 on transactivation of P57.Western blot assay was used to verify the results of luciferase assay on protein level.Co-immunoprecipitation(CO-IP)and immunofluorescence confocal imaging were used to investigate the physical intereaction and the colocalization between RUNX1/RUNX1-ETO with KLF4 and to further determine their specific binding domains.The roles of PB on the intereaction between RUNX1/RUNX1-ETO with KLF4 was evaluated by CO-IP assay.Luciferase assay was then performed to investigate the effects of RUNX1 and RUNX1-ETO on KLF4-dependent transactivation of target genes.RUNX1,KLF4 and P57 were overexpressed in Kasumi-1 cells by a pCDH lentivirus system,and their biological functions were investigated by MTS assay and flow cytometry.Finally,KLF4 and P57 were overexpressed in bone marrow leukemic cells from t(8;21)leukemia mice and their effects on the incidence of leukemia development and the survival time of recipient mice were evaluated.Results:RUNX1,KLF4 and P57 expression at both mRNA and protein level were significantly up-regulated in Kasumi-1 cells after PB treatment.RUNX1 exhibited strong transcriptional activation effect on KLF4 promoter/enhancer reporter plasmid in a dose-dependent manner,while RUNX1-ETO showed slight effect.KLF4 exhibited dose-dependent transcriptional activation effect on P57 promoter/enhancer reporter plasmid.The above transcriptional regulatory relationships were further confirmed by Western blot assay.Transient expression of exogenous RUNX1 up-regulated the expression of KLF4 and P57 in 293T cells and Kasumi-1 cells,while RUNX1-ETO had no effect.RUNX1 interacted with KLF4 and co-localized with it in nucleus,Runt homology domain(RHD)was responsible for this interaction.RUNX1-ETO also interacted with KLF4 and co-localized with it in nucleus.Domain analysis demonstrated that KLF4 not only bind with RHD but also the ETO part of RUNX1-ETO.Further Co-IP assay showed RUNX1-ETO competed with RUNX1 for binding to KLF4 in a dose-dependent manner and PB has no effect on the interactions between RUNX1/RUNX1-ETO with KLF4.RUNX1 could coactivate KLF4 target genes while RUNX1-ETO had little effect.The overexpression experiments showed all of RUNX1,KLF4 and P57 could inhibit proliferation,block cell cycle and induce apoptosis of t(8;21)leukemia cells.Moreover,in vivo experiment demonstrated that overexpression of KLF4 and P57 in mouse bone marrow leukemic cells could reduce the incidence of leukemia development in recipient mice.Conclusions:In this study,we reported "RUNX1→KLF4→P57" as a novel signaling pathway in t(8;21)leukemia cells.RUNX1 transactived KLF4 and then P57 in leukemia cell to inhibit cell proliferation,block cell cycle and induce cell apoptosis,while oncoprotein RUNX1-ETO had little effect.Moreover,RUNX1 interacted with KLF4 and coactivated its target genes,which could be blocked by the competed binding of RUNX1-ETO with KLF4.Our study revealed the relationship between RUNX1 and KLF4 and the impacts of RUNX1-ETO on this relationship.Objective:Lysine-specific demethylase 1(LSD1)is highly expressed in leukemia cells and correlated with poor prognosis.LSD1 knockdown by shRNA or pharmacological inhibition reduces the colony-forming cell(CFC)frequencies of leukemia stem cell and prolongs the survival of leukemia mice.So developing small-molecule LSD1 inhibitors of high efficiency and low toxicity is expected to be a new strategy to treatment leukemia.In this study,a novel specific and reversible LSD1 inhibitor JL1037 was investigated.The anti-tumor effect of JL1037 on leukemia cells and its possible related mechanisms were explored with both in-vitro and in-vivo experiments.Methods:The expression levels of LSD 1 in a variety of AML cell lines and normal bone marrow mononuclear cells(BMMNCs)were measured by real-time quantitative PCR(qRT-PCR)and Western blot.AML cell lines with relatively high expression of LSD1 were chosen for subsequent cell biology studies.Western blot assay was performed to determine the effect of JL1037 on the methylation levels of LSD1 substrates H3K4 and H3K9 and the acetylation level of total H3.Cell viability of leukemia cells treated with JL1037 was determined by MTS assay and the half maximal inhibitory concentrations(IC50)of JL1037 were calculated by SPSS statistical software.The cell cycle distribution and cell apoptosis rate were evaluated by flow cytometry after JL1037 treatment.Morphology analysis with Wright-Giemsa staining was also performed to observe the morphological changes of leukemia cells.For mechanism study,cell cycle-related proteins(P21,P27 and P57),apoptosis markers(cleaved caspase-3 and cleaved PARP)and Bcl-2 family members(BCL-2,BCL-XL and BAX)were examined by Western blot after JL1037 treatment.LSD1 knock-down experiment was further performed to confirm that the anti-leukemia effects of JL1037 on the AML cells were due to JL1037-mediated inhibition of LSD1.Western blot assay and Immunofluorescent staining were used to evaluate the expression level and cellular distribution of autophagy-related protein LC3-Ⅱand transmission electron microscopy(TEM)was utilized to analyze the ultrastructure of leukemia cells after treated with JL1037.The cell apoptosis rate of leukemia cells was further evaluated after JL1037 treatment in the presence or absence of a specific autophagy inhibitor chloroquine(CQ).Bone marrow mononuclear cells from newly diagnosed AML patients and healthy donors and CD34+ hematopoietic stem/progenitor cells from fresh umbilical cord blood samples were isolated by Ficoll gradient solution and immunomagnetic beads,respectively.Cell apoptosis assay was performed to evaluate the effect of JL1037 on those cells in combine with or without CQ.For in vivo experiment,transplantable leukemia mice co-expressing AML1-ETO and C-KITD816V were randomly divided into three groups and received treatment of 2.5 or 5 mg/kg of JL1037 and PBS each day by tail vein injection for 10 days.The body weight and the percent of peripheral blood leukemic cells were dynamic monitored during the administration period.The survival time of each mouse was recorded and the differences between groups were compared.JL1037 toxicity effect was evaluated by comparing organ weight and organ histological structure between mice from PBS and JL1037 treatment group(5 mg/kg)after 10-days of drug delivery.Results:LSD1 expression at both mRNA and protein level was significantly higher in majority of leukemia cell lines,especially in Kasumi-1,K562 and THP-1 cells,compared with that of normal BMMNCs.In THP-1 and Kasumi-1 cells,JL1037 treatment resulted in detectable increases of H3K4mel and H3K4me2,whereas the levels of H3K4me3,H3K9mel,H3K9me2,H3K4me3 and acetylated H3 remained unchanged.JL1037 strongly inhibited the growth of THP-1 and Kasumi-1 cells.The IC50 values of 48 hours were(30.3 ± 2.22)μM and(19.42 ± 3.02)μM,respectively.JL1037 induced caspase-3 dependent apoptosis in a dose-dependent manner,accompanied by the activation of mitochondrial apoptosis signaling pathways,showing that pro-apoptotic protein Bax was up-regulated and anti-apoptosis proteins Bcl-2 and Bcl-XL were down-regulated.The results of cell cycle assay demonstrated that JL1037 treatment arrested cell cycle at S phase when used at lower doses(10～30)μM,however,when the doses increased to(40～50)μM,the cell cycle would be blocked at G0/G1 phase.This is probably because of the up-regulation of cyclin-dependent kinase inhibitors P21 and P57.LSD1 knock-down experiment demonstrated that anti-tumor effect of JL1037 was LSD1 dependent.JL1037 appeared to activate autophage response in leukemia cells by increasing LC3-Ⅱ expression and the formation of autophagosomes and autolysosomes in cytoplasm.Co-treatment with autophagy inhibitor chloroquine(CQ)enhanced JL1037-induced cell apoptosis.The proportion of apoptotic cells increased with the concentration of JL1037 in AML samples,while BMMNCs from healthy donors were slightly affected.Daily intravenous administration of JL1037 tended to reduce tumor burden and prolong the survival of t(8;21)leukemia mice.However,JL1037 might also have some toxic effects along with anti-leukemia effects in vivo.Conclusions:As a novel LSD1 inhibitor,JL1037 could inhibit proliferation,block cell cycle,induce apoptosis and activate autophagy reaction of leukemia cells,which provide a new strategy for the treatment of leukemia.