The Researches On DHODH Inhibitor Promote Differentiation Of Acute Myeloid Leukemia Cells By Inhibiting Ribosome Biogenesis

Objective: To explore the intrinsic mechanism of how DHODH inhibitor promotes the differentiation of AML cells.DHODH inhibitor,Brq,was applied to AML cell lines THP-1 and U937 in order to observe the degree of differentiation;To explore the targeted pathways and physiological processes after the exposure of Brq;To explore the change of transcription level of r RNA and ribosome proteins;To explore the change of the protein level of MYC after the exposure of inhibitors relevant to ribosome;To explore the relationships between the ribosome biogenesis and differentiation.Methods: THP-1 and U937 cell lines,which are generated from human AML monocytes and macrophages,was cultured and subcultured;RNA-seq was used to detect the changes of genes at transcription level of THP-1 cell line induced by Brq,and differential gene analysis,functional enrichment analysis and Gene Set Enrichment Analysis were performed to detect the changed pathways;The m RNA-purification reagents were used to detect the impact of Brq on different types of RNA;Western blotting verificates that the protein level of MYC was changed after the exposure of different inhibitors such as Brq,CX-5461,rbin-1,CHX and puromycin;Flow cytometry was used to detect the effects of Brq,CX-5461,rbin-1,CHX and puromycin on differentiation of THP-1 and U937 cell line;Results: 1.The results of RNA-seq and enrichment analysis indicates that after the exposure of Brq,the differential genes are enriched in pathways related to ribosome.And also,these pathways are almost down-regulated.2.After exposure of Brq,r RNA,ribosomal large subunit protein RPL5 and ribosomal small subunit protein RPS6 were all down-regulated at the transcription level;ribosomal biogenesis was down-regulated.3.After the exposure of Brq,non-m RNA,which is consituted mainly by r RNA,is down-regulated more than m RNA.4.After the inhibition of ribosome biogenesis,MYC is down-regulated at the protein level.However,if we only inhibit the translation,MYC will not be down-regulated,it will be stable or up-regulated.5.Inhibition of ribosome biogenesis can promotes the differentiation of AML cells,but the inhibition of translation cannot.Conclusion: Brq,which can inhibit the de novo synthesis of pyrimidine,leads to the depletion of nucleotides,the inhibition of synthesis of r RNA,and ribosome biogenesis.After the exposure of Brq,r RNA and ribosome protein were down-regulated at the transcription level.Inhibition of ribosome biogenesis can promote the differentiation of AML cells and down-regulate MYC at the protein level,but blocking ribosome assembly and translation cannot.