The Role And Mechanisms Of ATP-citrate Lyase In PD-L1 Mediated Tumor Immune Evasion

Background:Malignant melanoma originates from melanocytes in skin,characterized by rapid proliferation,high incidences of metastasis and mortality.For the past decades,the morbidity of melanoma in China has increased progressively year by year.So,it is of great importance to investigate the pathogenesis of melanoma.It has been reported that,upregulation of PD-L1 in tumor cells is among the key mechanisms underlying the exhaustion of tumor infiltrated T cells,which leads to the evasion of tumor cells to anti-tumor immunity.Although anti-PD-1/PD-L1 antibodies have been widely used as first-line therapies in the treatment of melanoma,about 60-80%of patients showed poor clinical response,and responsive patients showed adaptive resistance to anti-PD-1/PD-L1therapies induced by interferon.Up-regulation of immune checkpoint PD-L1 mediated tumor cell evasion of anti-tumor immunity was demonstrated as the major mechanism of adaptive resistance to anti-PD-1 therapies.Therefore,further elucidation of mechanisms underlying PD-L1 regulation to provide the potential targets of melanoma treatment and to explore potential combinatory therapies for melanoma treatment are of great importance to improve patients’outcome.De novo fatty acid synthesis pathways are prominently activated to provide the material for membrane biogenesis,substrates for energy metabolism,as well as the second messenger for lipid signaling transduction in rapidly proliferating tumor cells.ATP-citrate lyase(ACLY)catalyzes citrate to Ac Co A,which is the starting material for lipogenesis.More importantly,Ac Co A is the sole donor of the acetyl groups for histone and nonhistone proteins acetylation in the nucleus.Histone acetylation modification has a great impact on the interactions between histones and DNA,which has critical roles in regulating genes transcription.Our previous work demonstrated that,upregulated ACLY promoted histone acetylation of MITF promoter region,activated MITF-PGC1?and mitochondrial biogenesis,thus led to melanoma cell proliferation and resistance to targeted therapy.However,the function of ACLY besides regulating melanoma cell proliferation and the sensitivity to targeted therapy is still unknown.Through bioinformatics analysis,we found ACLY expression was in negative correlation with TIL and Treg signatures,suggesting ACLY might be involved in tumor immune evasion.Further studies revealed that ACLY expression was positively correlated with PD-L1 expression in melanoma tissues.In addition,acetylation of histones located in CD274 promoter region plays a vital role in the expression of PD-L1.Based on these premises,we proposed that upregulated ACLY might promoted histone acetylation of CD274 promoter to activate PD-L1 transcription and promote PD-L1 mediated tumor cell immune evasion.Objectives:1.To identify the function of ACLY in the regulation of PD-L1 expression and PD-L1mediated T cells exhaustion.2.To elucidate the exact mechanism of ACLY in regulation of PD-L1 expression.3.To explore the potential combinatory therapies to enhance the sensitivity of melanoma patients to immune therapies.Methods:1.Analysis on the correlation of ACLY and TIL,Treg signatures and immune checkpoints.Western blot and q RT-PCR were employed to determine the expression level of lipogenesis enzymes in melanoma cell lines.Bioinformatics analysis was used to analyze the expression of ACLY in different types of cancer.GSVA analysis and immunohistochemical staining assay were performed to identify the correlation between ACLY and TIL signatures,as well as PD-L1 expression.The role of ACLY to predict the prognosis of melanoma patients was also analyzed using TCGA database.PBMCs were isolated from peripheral blood,and then co-cultured with melanoma cells transfected with ACLY si RNA or control si RNA.The number of melanoma cells after co-culture was analyzed to detect T cell mediated tumor cell killing.2.Analysis on the role of ACLY in the regulation of PD-L1.IFN-?was used to stimulate melanoma cells.Western blot,cell fluorescence,flow cytometry and q RT-PCR were used to detect the expression of PD-L1.3.The RNA synthesis inhibitor was used to examine the half-life of PD-L1.Proteasome inhibitor and lysosome inhibitor were used to validate the exact role of ACLY in the degradation of PD-L1.4.Western blot was used to determine whether ACLY regulated PD-L1 expression via the production of palmitate.Western blot and q RT-PCR were used to identify the role of Ac Co A on the expression of PD-L1.Western blot was used to determine whether ACLY regulated PD-L1 through Ac Co A.5.Multiple interventions of Ac Co A metabolism pathways were employed to validate Ac Co A regulate PD-L1 expression in melanoma cells.si RNAs were employed to inhibit the expression of key enzyme,ACSS2,which catalyze acetate to Ac Co A.Whereas,small molecular inhibitors were used to inhibit the function of mitochondrial pyruvate carrier(MPC),which imports pyruvate into mitochondrial or pyruvate dehydrogenase complex(PDC),catalyzing mitochondrial pyruvate to form Ac Co A.Western blot and q RT-PCR were used to identify ACSS2 regulated PD-L1 expression via promoting the generation of Ac Co A.6.P300 inhibitor and acetate were used to pretreat melanoma cells to inhibit P300 or supplement Ac Co A,respectively.In some conditions,IFN-?was added to stimulate melanoma cells.Western blot and q RT-PCR were performed to detect the regulation of PD-L1 and histone acetylation.Ch IP assay was performed to validate Ac Co A and its producer,ACLY,directly regulated histone acetylation of CD274 promoter region,leading to upregulation of PD-L1.7.For the melanoma mouse model,mouse melanoma cells were subcutaneously injected to the flank of C57BL/6,and then CTLA-4 antibody or Ig G,ACLY inhibitor,SB-204990 or control were intraperitoneally administrated on indicated times.The tumor volume was measured to analyze the anti-tumor efficacy of the treatment.Tissue immunofluorescence staining was used to compare PD-L1 and CD8~+T cells in tumors of each group.8.Immunohistochemical staining of melanoma tissue microarray was employed to analyze the correlation of ACLY and H3K27ac,P300,as well as the correlation of H3K27ac,P300 and PD-L1.Results:1.The m RNA and protein levels of multiple lipogenesis enzymes including ACLY were upregulated in melanoma cell lines compared with normal melanocytes.The expressions of multiple lipogenesis enzymes were upregulated in different types of cancer.The expression of ACLY was negatively correlated with TIL and Treg signatures.ACLY expression was positively correlated with PD-L1 expression in melanoma tissues.In addition,high expression of ACLY predicted poor prognosis of melanoma patients.The down-regulation of ACLY in melanoma cells sensitized to human lymphocytes in the T cell-mediated tumor cell killing assay.2.The knockdown of ACLY led to down-regulated PD-L1 m RNA and protein levels,as well as membrane PD-L1.The knockdown of ACLY led to down-regulation of IFN-?induced PD-L1 m RNA and protein levels,as well as membrane PD-L1.3.The half-life assay showed that inhibition of ACLY had no effect on the half-life of PD-L1.The treatment of proteasome inhibitor and lysosome inhibitor showed that ACLY did not regulate the degradation of PD-L1.4.Western blot results revealed that supplementation of palmitate had no effect on ACLY regulated PD-L1 expression.Western blot and q RT-PCR results showed that the supplement of Ac Co A upregulated PD-L1 expression in a dose-dependent manner.In addition,Ac Co A restored PD-L1 expression that was down-regulated by ACLY deficiency.5.The knockdown of ACSS2 inhibited PD-L1 expression,and Ac Co A restored ACSS2 regulated PD-L1 expression.PDC inhibitor treatment caused upregulated PD-L1m RNA and protein levels in melanoma cells,while MPC inhibitor treatment had no effect on PD-L1 m RNA and protein levels.6.Acetate up-regulated the expression of PD-L1 and acetylation of histone H3K27and H3K18,while P300 inhibitor down-regulated endogenous and acetate induced PD-L1expression,H3K27ac and H3K18ac level.P300 inhibitor impaired IFN-?or IFN-?and acetate induced PD-L1 expression and acetylation of H3K27 and H3K18.Moreover,Ch IP assay revealed that Ac Co A and Ac Co A regulator,ACLY,promoted histone acetylation on CD274 promoter.7.We observed that ACLY inhibitor or anti-CTLA-4 antibody treatment had moderate inhibitory effect on tumor growth,whereas ACLY inhibitor combined with anti-CTLA-4 antibody significantly limited tumor progression.Consistently,the combination group displayed remarkably down-regulation of PD-L1 and increased infiltrated CD8~+T cells.8.Immunohistochemical staining of melanoma tissues micro array showed a positive correlation of PD-L1 and P300,H3K27ac level,as well as that of ACLY and P300,H3K27ac level.Conclusions:In this study,we revealed that upregulated ACLY activated Ac Co A biogenesis,which promoted histone acetylation of CD274 promoter,thus leading to PD-L1 expression and PD-L1 mediated tumor cell immune evasion.In addition,we demonstrated the pivotal role of Ac Co A metabolism in the regulation of PD-L1 expression.More importantly,the combination of ACLY inhibitor and CTLA-4 synergistically impaired tumor growth.Our study sheds light on the understanding of ACLY function in the pathogenesis of melanoma,reveals the new mechanisms of PD-L1 regulation,and provides potential combinatorial therapies for the treatment of melanoma patients in the future.