Effect And Molecular Mechanism Of R-2-HG On Myelodysplastic Syndromes

Research purposes:Myelodysplastic syndromes(MDS)are a group of heterogeneous hematopoietic stem cell disorders and is manifested as ineffective hematopoiesis,refractory cytopenia and hematopoietic failure.MDS frequently progress to acute myeloid leukemia(AML)and are the most common cause of acquired bone marrow failure in adults over 65.The pathogenesis of MDS is so complicated that so far scientists have not been able to fully explain it.It is now recognized that karyotype abnormalities(including+8,-7/7q-,-5/5q-,20q-,-Y,etc.)and reproducible gene mutations(including TET2,DNMT3A,IDH1/2,SF3B1,U2AF1,ASXL1,TP53,etc)have priming effects.IDH2 mutation is a recurrent gene mutation in adult myelodysplastic syndrome,and its role in MDS is receiving more and more attention.The tumor metabolite(2R)-2-Hydroxyglutarate(R-2-HG)derived from isocitrate dehydrogenase 1/2(IDH1/2)mutation is a competitive inhibitor of a-KG-dependent dioxygenases.In the past,R-2-HG is considered to blocks differentiation and promote the proliferation in hematopoietic cells,which ultimately promote leukemogenesisis.However,recent studies have demonstrated that R-2-HG can promote cell cycle arrest and apoptosis to exert extensive anti-leukemia activity.By now,the specific mechanism by which R-2-HG leads to anti-tumor effects is still unknown.Research methods:(1)R-2-HG inhibits the proliferative activity of high-risk MDS cells.Different concentrations of(2R)-Octyl-2-HG(100,200,300μmol/L)were used to treat SKM-1,THP-1,Molm-13,HL-60 cell lines and bone marrow mononuclear cells derived from MDS and AML patients for 24,48 and 72h.The inhibition rate of cell proliferation was detected by MTS assay.We stably infected cell lines with lentiviral vectors encoding flag-tagged versions of IDH2 R140Q variant which was induced by doxycycline and the proliferative rate were examined via MTS assay.(2)Flow cytometry was used to detect the apoptosis rate and cell cycle of SKM-1 and THP-1 cells treated with different concentrations of(2R)-Octyl-2-HG.Western blot was used to detect protein expression in SKM-1 and THP-1 cells.(3)Agilent SurePrint G3 Human Gene Expression v3 chip was used to detect transcriptome data of SKM-1 cells after the treatment of(2R)-Octyl-2-HG.In combination with gene expression profiles from the GEO database,Gene set enrichment analysis(GSEA)was performed.RT-qPCR and Western blot were used to detect the change of mRNA and protein expressions in SKM-1 and THP-1 cells.(4)The cell death pattern after(2R)-Octyl-2-HG treatment was detected by FAN method.Co-immunoprecipitation(CO-IP)was used to detect the physical intereaction between RIPK1 and caspase8.The RIPK1 shRNA cell line was constructed by lentiviral transfection,and the knockdown was confirmed by westernblot and RT-qPCR.MTS,FAN and co-immunoprecipitation were used to detect the cytotoxicity of R-2-HG to RIPK1 shRNA cell line.(5)We also established AML xenograft mouse models through intravenously injecting RIPK1 shRNA or scramble shRNA SKM-1 cells into NOD/SCID-1L2R7null-SGM3(NSGS)mice.The survival of the mice was recorded.The occupancy of SKM-1 cells in mouse bone marrow was examined by flow cytometry.(6)The expression level of RIPK1 in 86 patients with MDS from the First Affiliated Hospital of Zhejiang University was detected by RT-qPCR.The relationship between RIPK1 expression and clinical features,risk stratification and survival of patients with MDS were analyzed.The mRNA expression data of RIPK1 in 152 patients with MDS and 17 normal controls were obtained from public databases.The relationship between RIPK1 expression level and clinical features of MDS patients was analyzed.The gene expression data of bone marrow from AML patients were obtained from public databases to explore the expression distribution of RIPK1 and its influence on the prognosis of patients.Research results:(1)(2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1,THP-1,Molm-13,HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose-and time-dependent manner.The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines,and the inhibition rate was related to the dose of doxycycline.(2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest.(2)R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells.The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in(2R)-Octyl-2-HG groups,and the expression of RIPK1 gene was increased in all three(SKM-1,NOMO-1 and MA9.3ITD)(2R)-Octyl-2-HG groups.After treatment with(2R)-Octyl-2-HG,the mRNA and protein expression levels of RIPK1 gene were increased.(3)R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis.Within 10 hours after treatment with(2R)-Octyl-2-HG,cell membrane permeability was disrupted,interactions between RIPK1 and caspase8 increased,and phosphorylated MLKL increased.At this time,caspase activity did not increase significantly,suggesting that necroptosis occurs earlier than apoptosis.With RIPK1 inhibitor Necrostatin-1 and(2R)-Octyl-2-HG co-treating cells,proliferation inhibition was reduced,cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form.The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus,suggesting that RIPK1-dependent necroptosis is involved in cell death caused by(2R)-Octyl-2-HG.(4)In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK.In RIPK1 shRNA MDS mice,the tumor burden showed a decreasing trend,but did not show a significant change in the R-2-HG treatment group.Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+cells in bone marrow.(5)Low RIPK 1 expression predicts poor prognosis in MDS patients.Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls.Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival(OS)than patients with higher RIPK1 expression levels.In MDS patients,patients with lower RIPK1 expression levels progress to leukemia more frequently.Conclusion:This study confirmed that R-2-HG inhibited the activity of MDS and AML cells.R-2-HG increased the expression of RIPK1 in MDS cells,inducing necroptosis.Necroptosis occurred earlier than apoptosis.Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells.Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML.MDS patients with low expression of RIPK1 was more likely to progress to leukemia.