The Roles And Mechanisms Of Sirt6 And Its Inhibitor OSS?128167 In Diffuse Large B-cell Lymphoma

Diffuse Large B-cell Lymphoma(DLBCL)accounts for the highest proportion of newly-treated adult non-Hodgkin lymphoma(NHL).DLBCL is a highly aggressive lymphoma with heterogeneity.In recent years,with the application of rituximab and novel targeted drugs,the therapeutic effect of DLBCL has been significantly improved.However,there are still about 30%-40%of DLBCL patients with initial treatment resistance or early relapse after complete remission,and the vast majority of patients still have difficulty in achieving long-term survival.Therefore,it is an urgent need to explore the abnormal expression of biomolecules and aberrant activation of signaling pathways related to the development of DLBCL,so as to effectively improve the prognosis of patients.Sirtuin 6(Sirt6)is derived from a large molecule group known as the mammalian Sirtuins(Sirts,Sirt1-7)family(1).It is a highly conserved ADP-ribosylase and NAD+dependent deacylase,involved in a myriad of biological processes including DNA repairment,glucose/lipid metabolism,transcriptional regulation as well as telomere maintenance.Sirt6 functions as a cellular pathway checkpoint controller and is responsible for the regulation of cellular proliferation and survival.Its aberrant expression has been implicated in various cancers.There is controversy regarding the role of Sirt6 in tumor development.In mouse embryonic fibroblasts(MEFs),Sirt6 knockout led to tumorigenesis independent of the activation of oncogenes,suggesting that this molecule may function as a tumor suppressor.Clinical sample analysis revealed that Sirt6 is down-regulated across several malignancies such as rectal and pancreatic tumors,with its level of expression strongly associated to patient prognosis.Sirt6 suppresses tumor growth mainly through suppression of anaerobic glycolysis and co-suppression of MYC,thereby inhibiting cancer occurrence and development.In contrast,Sirt6 has been reported to be oncogenic in breast cancer,prostate cancer,hepatocellular carcinoma and skin cancer.Knock out of Sirt6 gene leads to an increased level of DNA damage and enhanced sensitivity to chemotherapy.Michele Cea,et al.reported that Sirt6 was highly expressed in MM cells and closely linked to resistance against DNA damaging agents,an effect that has been attributed to MAPK/ERK2/p90RSK signaling inhibition.Sirt6 is a double-edged sword in carcinogenesis,as it possesses both oncogenic and tumor suppressive abilities due to the complexity of its upstream and downstream signaling pathways.Research on the function of Sirt6 might provide new insight into the development of chemotherapeutic regimens.OSS?128167 is a novel specific inhibitor targeting Sirt6 with IC50 values of 89,1578 and 751?M for Sirt6,Sirt1 and Sirt2,respectively.It is able to permeate cell membrane and is biologically active in cultured cells.OSS 128167 increases H3K9 acetylation and GLUT-1 expression.Research on it is still in the early stages given its relatively new introduction to the market.Michele Cea,et al.suggested that OSS 128167 was able to sensitize primary MM cells,as well as doxorubicin-and melphalan-resistant MM cell lines,towards chemotherapy,and have certain clinical application prospects.However,the role and molecular mechanism of Sirt6 in DLBCL is still unclear,and whether OSS?128167 has regulatory effect and therapeutic potential in DLBCL remains to be explored.The purpose of this study is to investigate the expression pattern,functional mechanism and potential clinical relevance of Sirt6 in DLBCL,and to further study the regulatory role of its targeted small molecule inhibitor OSS?128167,in order to provide theoretical and experimental basis for the diagnosis,prognostic risk assessment and novel targeted therapy of DLBCL.Part ?.Expression of Sirt6 and Its Biological Role,ClinicalPerspectives in Diffuse Large B-cell LymphomaObjective:DLBCL is a clinically,pathologically and molecularly heterogeneous disease.Accordingly,various clinical,pathological and molecular markers have been developed to characterize this disease.Patients most often present with rapidly growing tumors in single or multiple lymph nodes or extranodal sites.The treatment of DLBCL patients is an active area of research,and the 5-year overall survival rate with standard first-line treatment is about 60-70%.As many as 1/3of patients with DLBCL eventually develop resistance to the R-CHOP regimen,while the remaining treatment options are limited.A precision medicine approach for the design of new therapies based on molecular findings,the invention of targeted drugs and immunotherapy have opened new doors for improving the treatment of DLBCL.The biological functions of Sirt6(deacetylation,deacylation and single ADP-ribosylation,etc.)play a key role in regulating lifespan and controlling several basic processes of aging,such as DNA repair,gene expression and telomere maintenance.In the past few decades,abnormal expression of Sirt6 has been widely observed in various life-threatening human diseases.Sirt6 acts as a cellular pathway checkpoint controller,responsible for the regulation of cell proliferation and survival.Its abnormal expression is related to a variety of cancers.There is controversy about the role of Sirt6 in tumor development.In MEFs,Sirt6 knockout led to tumorigenesis independent of the activation of oncogenes,indicating that the molecule may have a tumor suppressor effect.Analysis of clinical samples showed that Sirt6 was down-regulated across several malignancies(such as rectal and pancreatic tumor).Sirt6 mainly inhibits tumor growth by inhibiting anaerobic glycolysis and co-inhibition of MYC,thereby inhibiting the occurrence and development of cancer.In contrast,Sirt6 is reported to be carcinogenic in breast,prostate,hepatocellular,and skin cancers.Knockout of Sirt6 lead to increased levels of DNA damage and increased sensitivity to chemotherapy.Sirt6 was highly expressed in MM cells and was closely related to resistance to DNA disruptors.However,there is no literature refers to the expression level and biological function of Sirt6 in DLBCL.Therefore,this part mainly discusses the expression level,biological function and clinical significance of Sirt6 in DLBCL.Materials and Methods:1.Use the Gene Expression Omnibus datasets to analyze the expression of Sirt6 in DLBCL.2.DLBCL/Reactive hyperplasia lymphoid(RHL)patients' selection and specimen collection;3.Immunohistochemistry(IHC);4.DLBCL cell culture;5.Isolation of peripheral blood mononuclear cells(PBMCs);6.Extract RNA,reverse transcription and real-time fluorescent quantitative polymerase chain reaction(qRT-PCR);7.Protein extraction and Western blot analysis(WB);8.Cell transfection by lentivirus vectors encoding shSirt6,lvSirt6 or an empty lentiviral vector;9.RNA-sequencing(RNA-seq)detects the differentially expressed genes of shSirt6 and shControl groups;10.Cell Counting Kit-8(CCK-8)experiment to detect cell proliferation and drug sensitization;11.Annexin V-PE/7AAD double staining method to detect cell apoptosis;12.PI staining assay to detect cell cycle;13.DLBCL xenotransplantation mouse model to test the influence of Sirt6 on the growth of DLBCL in vivo;14.Statistical analysis.Results:1.Firstly,we examined Sirt6 expression in GEO database.Sirt6 was markedly upregulated in DLBCL in contrast to normal samples(p<0.001).Both the mRNA and protein levels of Sirt6 were aberrantly higher in DLBCL cell lines than those in the control cells from healthy volunteers.Compared with RHL,expression level of Sirt6 was significantly higher in DLBCL tissues upon IHC staining,with a Sirt6 positive rate of 80%in DLBCL tissues compared to 8.6%in tissues with RHL.To clarify the clinical significance of Sirt6 expression in DLBCL,the clinicopathological characteristics of DLBCL patients were analyzed.Sirt6 expression levels were positively related to age(p=0.009),Ann Arbor stage(p=0.036),and international prognostic index(IPI,p=0.045)score of DLBCL patients(Table 1).There was no significant correlation n with patients' gender,B symptoms,disease subtype,serum lactate dehydrogenase(LDH)level,and extranodal invasion(p>0.05).2.Kaplan-Meier survival curve analysis of the GSE32918 indicated higher Sirt6 expression in DLBCL(n=249)was correlated with shorter overall survival times(p=0.0023).Consistent with it,the survival analysis indicated higher Sirt6 expression in DLBCL was correlated with shorter OS time(p=0.039).3.Three lentivirus mediated RNA interference(RNAi)vectors carrying GFP against Sirt6 demonstrated effective knockdown of Sirt6 in human LY1,LY8,and Val cells,of which shSirt6#1 exhibited the highest efficacy.Effective knockdown(shSirt6)or overexpression(lvSirt6)was verified using western blotting or qRT-PCR experiments.GCK-8 assay was used to detect cell proliferation activity.Compared with cells with empty vector,Stable shSirt6 transfected DLBCL cells exhibited growth suppression(p<0.01),while lvSirt6 had no impact on the proliferative ability of cells(p>0.05).In mouse xenograft model,compared with the shControl group,SCID mice inoculated subcutaneously with shSirt6 cells had significantly reduced tumor growth(p<0.01).Lower Sirt6 and Ki-67 expression levels were observed in the xenograft tumor tissues derived from shSirt6 cells by IHC staining.It suggests that Sirt6 plays a positive role in regulating the proliferation of DLBCL cells.4.RNA sequencing analysis showed that Sirt6 appeared to be closely related to cell cycle,DNA damage repair(DDR)and cell apoptosis.Annexin V-PE/7AAD apoptosis assays indicated that shSirt6 promoted cell apoptosis in LY1 cells(shControl:8.63±0.44%vs.shSirt6:14.23±0.35%,p=0.0006),LY8 cells(shControl:10.80±1.00%vs.shSirt6:19.03±1.85%,p=0.0014)and Val cells(shControl:10.73±0.76%vs.shSirt6:17.43±0.69%,p=0.0029).In contrast,Sirt6 overexpression inhibited cell apoptosis(LY1:lvControl:8.63±0.44%vs.lvSirt6:4.400±0.15%,p=0.0008;LY8:lvControl:10.80±1.00%vs.IvSirt6:2.77±0.32%,p=0.0016).In addition,Sirt6 knockdown promoted the increase of cleaved-PARP expression.We then monitored cell cycle by flow cytometry.It is suggested that G2/M phase cell arrest occurred after Sirt6 knockdown,and the protein expression levels of p27 and CDK2 also changed accordingly.5.The effect of Sirt6 in the drug response to doxorubicin and bendamustine was studied by cytotoxicity assays.shSirt6 cells and control cells were exposed to a predetermined concentration of doxorubicin or bendamustine for 48 hours to detect cell proliferation.It was found that the proliferation of shSirt6 DLBCL cells was significantly reduced(p<0.05).The ATM-Chk2 and ATR-Chkl signaling cascade regulates DDR and cell cycle checkpoints.Western blot experiments showed that the expression of phosphorylated ATR and phosphorylated Chk1 were reduced in cells transfected with shSirt6,which may reveal Sirt6 knockdown can make DLBCL cells sensitive to chemotherapy drugs.Conclusions:1.Sirt6 expression is significantly increased in DLBCL.Sirt6 expression levels were positively related to lymphoma progression and shorter OS than those with lower expression.Sirt6 is a potential prognostic marker of DLBCL.2.Sirt6 promotes the proliferation of DLBCL;3.Sirt6 inhibits DLBCL cell apoptosis and promotes the progression of cell cycle G2/M phase;4.Sirt6 inhibition can enhance the chemo-sensitivity in DLBCL.Part ?.The Biological Role of Sirt6 Inhibitor OSS?128167 in Diffuse Large B-cell LymphomaObjective:In recent years,small molecule targeted therapy has achieved good clinical benefit in cancer treatment.OSS?128167 is a specific small molecule inhibitor of Sirt6,which can permeate cell membranes and has biological activity in cultured cells.It can increase H3K9 acetylation and GLUT-1 expression.The Harvard University research team found that OSS?128167 can increase the sensitivity of MM cells to chemotherapy and has certain clinical application prospects.However,there is a lack of report on the systematic detection of Sirt6 and its inhibitor OSS?128167 in DLBCL currently.The research on OSS?128167 is still in the early stage.Thus we aim to explore whether OSS?128167 have a regulation effect in DLBCL.Materials and Methods:1.DLBCL primary cells and cell line culture;2.CCK-8 experiment to detect cell proliferation and drug sensitization;3.Annexin V-PE/7AAD double staining method to detect cell apoptosis;4.PI staining assay to detect cell cycle;5.DLBCL xenotransplantation mouse model to test the influence of OSS?128167 on the growth of DLBCL in vivo;6.IHC staining;7.Statistical analysis.Results:1.Primary DLBCL cells,LY1 and LY8 cells were exposed to different concentrations of OSS?128167 for 24-72 hours,respectively.As a result,OSS?128167 reduced the cell viability of primary DLBCL cells and DLBCL cell lines in a time-and dose-dependent manner.2.After 48 hours incubation with 80 ?M OSS?128167,the number of early apoptotic cells in primary DLBCL cells and DLBCL cell lines increased significantly.3.Increasing concentration of OSS 128167 also affected the cell cycle progression of DLBCL cells.G2/M phase cell counts elevation was consistent with prior studies.4.In the SCID beige mouse xenograft model,the tumor growth of mice treated with OSS?128167 was significantly inhibited compared with the PBS treatment group.Additionally,the expression level of Ki-67 in the OSS?128167 exposed mice tumor tissue was reducedConclusions:OSS?128167 inhibits cell proliferation,promotes cell apoptosis,and arrests the cell cycle in G2/M phase in DLBCL cells.OSS 128167 exerts anti-lymphoma activity in DLBCL.It is expected to become a new targeted therapy for DLBCL.Part ?.Sirt6 Promotes the Growth of Diffuse large B-cell Lymphoma by Activating the PI3K/Akt Signaling PathwayObjective:Our previous study indicated that Sirt6 expression was raised in DLBCL,with its high levels corresponding to poor patient outcomes.Sirt6 was also found to promote tumorigenesis.Targeting Sirt6 exerted promoted cell apoptosis,cell cycle arrest and enhanced chemo-sensitivity.Sirt6 inhibition by OSS?128167 exerted anti-tumor activity in DLBCL cells.The mechanism by which Sirt6 regulates DLBCL cells is unclear.In the study,we aim to explore the mechanism of Sirt6 on its regulation upon DLBCL cells.Our study may lay a theoretical basis for further study on its biological function and targeted therapy.Materials and Methods:1.Cell culture;2.Lentiviral vectors mediated the knockdown of Sirt6 gene;3.Differential gene enrichment analysis(GSEA)of RNA sequencing;4.Protein extraction and Western Blot;5.CCK-8 assay to detect cell proliferation;6.Establishment of DLBCL xenotransplantation mouse model;7.Statistical analysis.Results:1.We explored the potential mechanisms involved in the regulation of Sirt6,using GSEA according to RNA-seq data.Sirt6 appeared to be functionally enriched in the PI3K/Akt and FOXO signaling pathways.The phosphorylation levels of PI3K p110? and its downstream targets,Akt(Ser473)and mTOR proteins,were noted to be dramatically decreased in shSirt6 cells compared to control cells.Moreover,increased expression levels of PTEN,a negative regulator of PI3K,phosphorylated 4EBP1,and FoxO1 protein were found in shSirt6 cells.2.In tumor tissues of xenotransplanted mice with DLBCL cells,the expression of Sirt6 in the shSir6 group was lower than that in the shControl group.Mice bearing shSirt6 cells were noted to have lower levels of phosphorylated PI3K and its downstream targets compared to those in the shControl group.3.Insulin-like growth factor-1(IGF-1),a known activator of PI3K signaling,was used to further investigate the ability of Sirt6 to modify PI3K signaling in DLBCL cells.DLBCL cells transfected with either shSirt6 or shControl were exposed to IGF-1 or vehicle control in 0.5%FBS culture medium for 24-96 h.shSirt6 cells that did not receive IGF-1 treatment demonstrated lower LY8 and LY1 cellular proliferation in contrast to cells transfected with empty vectors.Cells that received IGF-1 treatment were noted to have a partial restoration in their ability to proliferate.This effect was less in shSirt6 cells in contrast to control cells.shSirt6 and shControl LY1 cells,starved of serum for 48 h and subsequently exposed for 30min to either IGF-1 50 ng/ml or vehicle control.Cells that were transfected with shSirt6 were noted to have decreased levels of phosphorylated PI3K and its downstream targets,Akt and mTOR.Conclusions:Sirt6 may promote the growth of DLBCL cells by activating the PI3K/Akt/mTOR signaling pathway,thereby promoting the occurrence and development of DLBCL.These findings provide insights into the mechanism of Sirt6's carcinogenic activity in DLBCL,and provide a theoretical basis for the accurate prognostic evaluation and new targeted therapies for DLBCL.