The Mechanisms Of AGK-Mediated Regulation Of The Sensitivity To Venetoclax In DLBCL

Background:Diffuse large B cell lymphoma(DLBCL)is the most common type of non-Hodgkin lymphoma(NHL)worldwide.Significant progress has been made in the development of small molecule inhibitors in recent years,and many of them have been approved by FDA for the trearment of haemological maglinancies.Venetoclax,a specific BCL-2 inhibitor,has shown promising effects in chronic lymphocytic leukemia(CLL)and acute myeloid leukemia(AML).While the efficacy and overall response of DLBCL patients to Venetoclax are low.We ananlysed the expression profiling of two parental cell lines and venetoclaxresistant cell lines and identified the enriched pathways in this study.The results showed that the signaling pathways related to mitochondrial structure and function and lipid metabolism were enriched in venetoclax-resistant cell lines.Furthermoore,differentially expressed genes were identified,and we found that acylglycerol kinase(AGK)was increased in venetoclax-resistant cell lines,which indicated that AGK is related to the resistant of Venetoclax in DLBCL cells.AGK,a lipid kinase,is not only involved in the regulation of cellular lipid metabolism,but also to the mitochondrial protein transport and glycolysis.Whether AGK regulates the development and Venetoclax sensitivity of DLBCL remain unclear.Objective:In this study,we compared the expression levels of AGK and the sensitivity to Venetoclax in various DLBCL cell lines,and used lentiviruses to knockdown or overexpress AGK in DLBCL cell lines to explore whether AGK expression regulates the sensitivity to Venetoclax of DLBCL and its mechanism through a series of experiments both in vitro and in vivo.Methods:Bioinformatics were used to analyze and identify important factors regulating the sensitivity of DLBCL to Venetoclax by bioinformatics,and Western blot and CCK-8 were used to determine the correlation between AGK expression and DLBCL sensitivity to Venetoclax;AGK knockdown and overexpression plasmids were constructed;Ki67immunofluorescence and transwell assay were used to detect the effects of AGK on the cell proliferation and migration;Western blot was used to detect the effect of AGK expression on PTEN/AKT/FOXO1 pathway and BCL-2 expression;Chromatin immunoprecipitation was used to detect the effect of FOXO1 on the transcription of BCL-2 in DLBCL cells;The correlation of AGK,FOXO1 nuclear localization with BCL-2 expression in tumor sections from DLBCL patients were detected by immunohistochemistry.Results:1.AGK genes were increased in venetoclax-resistant cell lines.AGK protein expression was significantly negatively correlated with their sensitivity to Venetoclax in DLBCL cell lines.2.AGK knockdown or overexpression stable SU-DHL4,TMD8,and OCI-LY1 cell lines were constructed successfully,and the results showed that AGK expression does not affect the proliferation and migration,but negatively regulates the sensitivity to Venetoclax of DLBCL.3.AGK knockdown promotes Venetoclax-induced apoptosis in DLBCL cells,which was dependent on the Caspase3 signaling pathway.4.BCL-2 expression is negatively correlated with AGK expression in DLBCL cells.5.AGK knockdown inhibited the phosphorylation of PTEN/AKT/FOXO1,promoted the nuclear translocation of FOXO1,and then induced the transcription and expression of BCL-2 in DLBCL cells.6.In tumor xenograft models,AGK knockdown induced the tumor volume reduction,weight loss,slower growth curve,and promoted tumor cell apoptosis and inhibited tumor cell proliferation after the treatment of Venetoclax.7.In DLBCL tumor xenograft models,AGK knockdown inhibited the phosphorylation of AKT/FOXO1 and promoted the nuclear localization of FOXO1,and upregulated the transcription of BCL-2,then increased the sensitivity to Venetoclax of tumor cells.8.The expression of AGK was negatively correlated with the expression of BCL-2,and the nuclear localization of FOXO1 was positively correlated with the expression of BCL-2,but negatively correlated with the expression of AGK in clinical DLBCL tumor tissues.Conclusions:AGK knockdown inhibits the phosphorylation of PTEN,AKT and FOXO1,then promotes the nuclear translocalization of FOXO1 and transcription of BCL-2,and enhances the Venetoclax-induced cell apoptosis then promotes the sensitivity to Venetoclax in DLBCL cells.