Co-repression Of BET Proteins And METTL3 As Potential Synergic Therapy For Pancreatic Cancer

BackgroundPancreatic cancer is one of the most lethal cancer types worldwide with a survival rate of only 11%at 5 years.As the most common type of pancreatic cancer(PC),pancreatic ductal adenocarcinoma(PDAC)accounts for 95%of PC.Because of anatomic and biological characteristics,PDAC is often diagnosed at an advanced stage.Only 20%of PD AC patients are suitable for surgical resection when diagnosed,while surgical resection is currently the only proved effective therapeutic strategy for PDAC patients.Besides,PDAC shows resistance to chemotherapy and many targeted therapies.JQ1 is the first studied BET inhibitor and has emerged as an effective therapeutic strategy against different cancer types.However,pancreatic cancer cells show great heterogeneity in the sensitivity to JQ1 and clinical studies showed that pancreatic cancer patients barely benefit from BETi-related therapy.Our preliminary work showed that pancreatic cancer cell lines showed great heterogeneity in the sensitivity to JQ1.And sensitive cell lines showed downregulated protein expression of METTL3 and reduced m~6A methylation and in response to JQ1 treatment.N~6-methyladenosine(m~6A)is the most abundant mRNA modification in mammalians,contributing to RNA localization,splicing,stability and translation,finally influencing the phenotype of cancer cells.Recently,many studies identified the interaction between RNA modification and histone modifications.And the influence of small molecule drugs on m~6A modification has also been widely discussed.Therefore,we will further study the role of m~6A in the function of JQ1 and try to overcome JQ1 resistance in pancreatic cancer by combined therapy.ObjectiveIllustrate the role of METTL3-m~6A in the function of JQ1 and explore whether corepression of BET proteins and METTL3 could serve as potential synergic therapeutic strategy for pancreatic cancer.MethodsFirstly,we used SRB assay to test the sensitivity of pancreatic cancer cell lines to JQ1.We then used colony formation,cell proliferation analysis and subcutaneous transplanted tumor model to identify JQ1 sensitive and resistant cell lines for further study.By using LC-MS/MS,Western blot and qPCR,we analyzed the change of m~6A modification and m~6A regulatory enzymes after JQ1 treatment.Then we downregulated METTL3 by siRNA and overexpressed METTL3 by pkMYC-METTL3 plasmid and tested the change of JQ1 sensitivity afterwards.For long-term experiments such as colony formation analysis and subcutaneous transplanted tumor model,we also constructed Tet-inducible METTL3 silencing stable cell line using PATU-8902 cells.Using Combenefit software,we calculated the synergy distribution of the combination of JQ1 with METTL3 inhibitors STM2457 and UZHla respectively.Then we established JQ1 resistant cell line MIA-J using JQ1 sensitive MIA-PaCa2 cells and verified the effect of METTL3 silencing on JQ1 sensitivity.Finally,by using MeRIP-qPCR,MeRIP-seq,RIP-qPCR,polysome fraction,EU-labeling-qPCR and Western blot analysis,we explored the downstream targets of JQ1m~6A axis and possible mechanisms driving the synergic effect of METTL3 silencing and JQ1 treatment.ResultsFrom cell experiments and mouse models,we found that pancreatic cancer cell lines showed great heterogeneity on JQ1 sensitivity.Among them,MIA-PaCa2,HPAF-Ⅱ and BxPC-3 shows high sensitivity to JQ1,with an IC50 lower than 200nM.After JQ1 treatment,METTL3 expression and level of m~6A modification was downregulated in sensitive cell lines.Knockdown of METTL3 by transfection of siRNAs could attenuate the proliferation of pancreatic cancer cells.Tissue microarray and data analysis of public database showed that high METTL3 expression is related to poor prognosis.GO analysis showed that METTL3 silencing could influence various crucial pathways related to cell proliferation and cell cycle transition.Data from MeRIP-seq showed that the function of JQ1 was partially carried out by m~6A.In MIA-PaCa2,we found that JQ1 treatment could attenuate the translation of SP1 in a m~6A-YTHDF1 dependent pathway.Cell proliferation analysis and data from public database showed that SP1 played an oncogenic role in pancreatic cancer.In vivo and in vitro experiments demonstrated that in JQ1 resistant cell lines,METTL3 silencing or inhibiting the function of METTL3 using METTL3 inhibitors could increase JQ1 sensitivity.Mechanically,we found that JQ1 treatment combined with METTL3 silencing could downregulate the m~6A modification of E2F8 transcript and attenuate its transcription.ConclusionCo-repression of BET proteins and METTL3 servers as potential synergic therapy for pancreatic cancer.