Molecular Mechanisms Underlying Targeting Of MYC In T Cell Leukemia And Neuroblastoma

ObjectiveT-cell acute lymphoblastic leukemia(T-ALL)is a malignant hematological cancer caused by the differentiation of T cell precursors in the bone marrow and clonal abnormal proliferation.There is no small molecule drug that effectively target T-ALL in clinic currently.Hence,it is of great scientific significance and clinical significance to investigate the pathological mechanisms of T-ALL in order to explore more effective targeted therapies.MYC is aberrantly expressed in T-ALL and plays an important role in development of T-ALL.Inhibition of MYC effectively represss the growth of T-ALL cells.In order to elucidate the expression of MYC in T-ALL besides aberrant transcription or splicing and to find new strategies for indirectly targeting MYC in T-ALL treatment,we found that AURKB,an important cell mitotic kinase,binds to MYC specifically.This study is aimed to investigate the molecular mechanisms by which AURKB maintains the stability and transcriptional activity of MYC protein and to validate the possibility of directly targeting AURKB in the treatment of T-ALL.MethodsLC-MS/MS identifies proteins that interact with MYC in T-ALL;explore the relationship between AURKB and MYC protein stability contact in RNAi or inhibitor-treated cells;RNA-seq(RNA transcriptome sequencing)and other techniques to explore the relationship between AURKB and MYC transcriptional activity;isotope labeling assay to detect whether AURKB can phosphorylate MYC;Screening for drugs synergistically with AURKB inhibitor(AZD1152)from the FDA-approved library of small molecule compounds by the CellToxTM-Greencytotoxicity assay;Xenografg mice model to evaluate the anti-cancer effect of AURKB inhibitors in vivo.ResultsAURKB specifically binds to MYC.Inhibition of AURKB activity or its expression in T-ALL cells significantly reduce MYC protein levels,but not mRNA level.Further RNA expression profiling revealed that AURKB-regulated gene populations are enriched for target genes regulated downstream of MYC.AURKB phosphorylates MYC at S67,blocking its binding with GSK3? and degradation via ubiquitination and proteasomal system,maintaining MYC protein stability and transcriptional activity.MYC/TAL1 co-transcriptionally activates AURKB and thus forming a feedforward loop.The combination of AURKB inhibitor and vincristine reduced tumor burden and significantly prolonged the survival of FBW7 wild-type T-ALL mice.ConclusionAURKB and MYC form a positive feedback loop regulation.Indirect inhibition of MYC by AURKB inhibitors provides a new strategy for T-ALL targeted therapy.The combination of AURKB inhibitors with vincristine has a significant synergy effect in FBW7 wild-type T-ALL cells.Taking together,our results provide biomarkers for the treatment of T-ALL.Objective The oncogene MYC family mainly includes three members of C-MYC,MYCN and MYC L genes,which encode C-MYC,N-MYC and L-MYC proteins respectively.The function complementarity of oncogene MYC exists.High expression of C-MYC is common in non-amplified neuroblastoma of MYCN.MYC oncoprotein promotes the growth and proliferation of cancer cells by regulating cell cycle,cell metabolism,splicing and other important biological processes,as well as directly or indirectly assisting other carcinogenic pathways,leading to malignant development of cancer.In mouse models,systemic knockout of MYC can significantly inhibit the occurrence and development of tumors,suggesting that targeted MYC can be used as a therapeutic option for tumors.Although inhibiting MYC seems particularly promising,due to its structural particularity and lack of protein structure regions bound by small molecules,there is no direct targeting of small molecules of MYC at present,so indirect targeting of MYC has become the most studied project.In this part,we use the principle of synergistic lethality to screen tumor cells that can selectively target MYC overexpression from FDA approved drug banks,and hope to find the mechanism of small molecule compounds promoting MYC overexpression of tumor apoptosis.Method Using MYCN-driven neuroblastoma model cells as models,938 FDA-approved drugs were screened through high-throughput screening in the hope of screening small molecular compounds synergistic with MYC to kill tumors.Apoptosis detection and analysis were carried out by apoptotic kit and flow cytometry;RT-q PCR was used to explore the changes of related target gene levels in cells after drug treatment;Western Blot was used to detect the changes of related target protein levels after drug administration;RNA interference technology was used to silence the related target genes in the article,and RT-q PCR was used to detect the gene expression.Levels and Western Blot were used to validate the protein levels.Result We screened proteasome inhibitor BTZ from 938 FDA-approved libraries of small molecule compounds,which could significantly kill tumor cells in cooperation with oncogene MYC.BTZ was the first small molecule drug used to treat multiple myeloma,but had no effect on SHEP cells with low MYC expression.It can also promote the expression of cleaved-caspase 3,and activate NOXA/TRIB3 through BAX to promote the mechanism of apoptosis.Conclusion Bortezomib(BTZ)can synergistically kill MYC-overexpressed cancer cells and induce apoptosis through NOXA/TRIB3-BAX pathway.