IDO1 Promotes The Survival Of Diffuse Large B Cell Lymphoma Via MDM2-p53-CDC20 Signaling Pathway

Background:Diffuse large B cell lymphoma(DLBCL)is the most frequently diagnosed subtype of hematological malignancy.Although with the development of diagnosis and treatment,up to 30% of DLBCL patients succumb to this cancer.The pathology and mechanism of DLBCL remains unclear and developments are urged to understand the molecular mechanisms of the occurrence and progression of DLBCL.Indoleamine 2,3-dioxygenase 1(IDO1)catabolizes tryptophan through kynurenine pathway.IDO1 plays the traditional immunosuppressive effect via the reduced level of tryptophan and increased level of kynurenine,which lead to immunosuppressive functions by activating dendritic cells and T regulatory cells,suppressing the functions of effector T and natural killer cells.IDO1 is frequently overexpressed in cancers including DLBCL and high expression of IDO1 is associated with a poor clinical prognosis in a variety of human tumors,however,the correlation between IDO1 overexpression and clinical outcomes in DLBCL is inconsistent.Furthermore,the molecular mechanisms underlying the IDO1 activity in DLBCL remains unclear.In recent years,more and more researchers began to pay attention to the role and mechanism of IDO1 independent of immune-tolerance in promoting tumor development.Recent studies have reported that IDO1 inhibitor 1-L-MT induces mitotic death of HCT116 and HT-29 colon cancer cells by inhibiting CDC20 transcription,and 1-L-MT also inhibits tumor cell proliferation and leads to cell cycle arrest in G2/M phase.However,there has been few research on the role and mechanism of IDO1 in DLBCL independent of immune-tolerance.We performed RNA-Seq analysis between IDO1 knocked down(KD)and control(Ctrl)OCI-Ly10 cell and found that MDM2 was down-regulated and while TP53 was up-regulated in IDO1 KD cells.Our previous study uncovered that cell division cycle 20(CDC20)was upregulated and linked with poor outcome in DLBCL based on bioinformatic analysis.In addition,previously study observed that CDC20 is negatively regulated by p53.Thus,we hypothesized that CDC20 could be a downstream factor of the MDM2-p53 signaling pathway and a novel MDM2-p53-CDC20 pathway has been demonstrated.We hypothesized that IDO1 plays a tumor-promoting role via MDM2-p53-CDC20 pathway in DLBCL.Objective:The current research mainly studied the expression level and clinical significance of IDO1 in DLBCL patients and explored the role and mechanism of IDO1 in DLBCL biological function.Furthermore,we investigated the mechanistic role of a novel IDO1-MDM2-p53-CDC20 signaling pathway in DLBCL,providing the theoretical basis for the application of IDO1 inhibitor in DLBCL.Methods:1)Bioinformatic analysis was performed to evaluate the IDO1 expression level in DLBCL tissues based on GEO and TCGA datasets.RT-q PCR was used to detect the expression of IDO1 in DLBCL cells;IHC was conducted to detect the IDO1 protein expression in DLBCL tissues;then the relationship between IDO1 expression and overall survival time and clinicopathological characteristics were performed.2)We knocked down(KD)IDO1 in OCI-Ly10 DLBCL cell and performed RNA-seq analyses.MDM2-p53 was selected as the downstream singling pathway of IDO1 and q RT-PCR was performed to validate the RNA-seq results.The expression of IDO1/MDM2 in DLBCL cells was inhibited,and the regulatory relationship between IDO1 and MDM2-p53 pathway was verified by western blot.Then,the effects of IDO1 in cell proliferation,cell cycle progression and apoptosis were determined by CCK8 assay,EDU cell proliferation assay and flow cytometry analysis.3)Xenograft tumorigenesis in NOD/SCID mice was conducted to evaluate the role of IDO1 in suppressing tumorigenicity in vivo.4)Based on bioinformatic analysis and literature reports,we hypothesized that CDC20 could be a downstream factor of the MDM2-p53 signaling pathway.Furthermore,we investigated the role and molecular mechanism of IDO1-MDM2-p53-CDC20 pathway in DLBCL.Then the relationship between CDC20 and OS and clinicopathological characteristics were performed.The effects of CDC20 in cell proliferation,cell cycle progression and apoptosis were determined by CCK8 assay,EDU cell proliferation assay and flow cytometry analysis.Results:1)IDO1 was elevated in DLBCL cells and tissues.The IDO1 expression level was significantly associated with Ann Arbor stage and IPI risk.Survival analysis showed that IDO1 overexpression was linked with an inferior OS.2)Functional assays showed that the downregulation of IDO1 could inhibit cell proliferation,induce cell apoptosis and G2/M arrest in vitro.Targeting IDO1 in OCI-Ly10 cell suppressed tumorigenesis in NOD/SCID mice in vivo.3)RNA-Seq and bioinformatic analysis results demonstrated that MDM2-p53-CDC20 was the the downstream pathway of IDO1.The above RNA-seq results were validated by RT-q PCR.The study showed CDC20 and MDM2 expression were upregulated in DLBCL tissues and cells and high CDC20 expression was correlated with adverse clinical features and poor outcome.And functional assays showed that the CDC20 could promote cell proliferation and inhibit apoptosis in vitro.4)IDO1 was inhibited by 1-L-MT and western blot showed that downregulation of IDO1 could restore p53 expression and inhibit MDM2 and CDC20.Next,we upregulated p53 using RG7388 and found that protein expression of CDC20 was decreased in OCI-Ly3 and OCI-Ly10 cells.In addition,while down regulation of CDC20 did no effects on IDO1,MDM2 and p53 expression.Conclusion:1)IDO1 and CDC20 were overexpressed in DLBCL and correlated with poor outcome and adverse clinical features in DLBCL.2)The functional assays showed that downregulation of IDO1 in DLBCL cells could suppress the proliferation,induce apoptosis and G2/M cell cycle arrest in vitro.3)IDO1 affected the survival of DLBCL cells through the MDM2-P53-CDC20 signaling pathway.