The Researches On DHODH Inhibitor In Combination With Decitabine Targeting On Acute Myeloid Leukemia

Objective: To explore the synergistic effects of DHODH inhibitor BRQ and demethylation drug DAC,on the proliferation,apoptosis,differentiation and cycle of AML cells in AML cell line THP-1,and to detect the changes of transcriptional level induced by the two drugs by RNA-seq and verify them,so as to explore the possible mechanism of the two drugs.Methods: THP1 cell line was cultured and subcultured;CCK8 assay was used to determine the drug concentration;CCK8 was used to detect the effects of single drug and combined drug on the cell viability of THP1 cell line;flow cytometry was used to detect the effects of single drug and combined drug on apoptosis,differentiation and cell cycle of THP1 cell line;RNA-seq was used to detect the changes of gene transcriptome level of THP1 cell line induced by single drug and combined drug,and differential gene analysis and functional enrichment were performed.The m RNA level of enrichment pathway was changed after RT-q PCR verification;the protein level of enrichment pathway was changed after Western blotting verification;bone marrow blood was collected from newly diagnosed AML(non-APL)patients to further verify the anti-AML effect of BRQ and/ or DAC.Results: 1.Cell viability: 0.25 μM Brequinar could inhibit the cell proliferation of THP1 cell line;0.1 μ M decitabine could slightly promote the cell proliferation;0.25 μM Brequinar combined with 0.1 μM decitabine significantly inhibited the cell proliferation at48 hours.two。.Apoptosis: 0.25 μM Brequinar and 0.1 μM decitabine could promote the apoptosis of THP1 cell line,and the combination of the two drugs could promote apoptosis.3.Cell cycle and cell differentiation: 0.25μM buquina and 0.1μM dicitabine both promoted cell cycle arrest and cell differentiation of Th P1 cell line;After the combination of the two drugs,DAC could increase the effect of BRQ on promoting THP-1 cell differentiation.4.RNA-seq: 0.25 μM BRQ and 0.1 μM DAC combined with RNA-seq detection,differential genes were enriched in ribosomal biosynthesis and other pathways.GSEA enrichment analysis showed that the level of c-myc in BRQ+DAC group was significantly lower than that in the control group.5.RT-qPCR and Western Blotting confirmed that BRQ and DAC can exert their antileukemic effect through synergistic inhibition of CMMC and then inhibition of ribosome biosynthesis pathway.6.BRQ and DAC can synergistically inhibit the cell survival of mononuclear cells in newly diagnosed AML patients.Conclusion: BRQ combined with DAC can synergistically inhibit the survival of AML cells and promote apoptosis,which may be due to the synergistic inhibition of C-myc,and then the inhibition of ribosome biosynthesis pathway to exert their anti-leukemia effect.Both BRQ and DAC could induce cell cycle arrest and promote cell differentiation,thus exerting their anti-leukemia effects.DAC could enhance the differentiation of leukemia cells induced by BRQ after the combination of these two drugs.