The Role Of CMTM6 In The Superior Efficacy Of Combination Of HDAC Inhibitors And PD-1 Inhibitors And Its Immunomodulation In Lung Adenocarcinoma

Part Ⅰ:Study on the efficacies and mechanisms of the combination of HDAC inhibitors and PD-1 inhibitors in non-small cell lung cancerObjective:The PD-1/PD-L1 signaling pathway plays a key role in tumor immune escape by inhibiting tumor antigen specific T cells via multiple mechanisms.PD-1/PD-L1 inhibitors have now evolved into one of the most important therapy in non-small cell lung cancer（NSCLC）.However,PD-1/PD-L1 inhibitors monotherapy are observed just a low response rate.Combination therapy is an effective way to improve the response rate and benefit more patients.Clinical trials indicate that the higher PD-L1 expression is associated with higher efficiency of PD-1/PD-L1 inhibitors.At the same time,up-regulating PD-L1 expression is also a resistance mechanism to many anti-cancer drugs.This study aims to identify candidate drugs that can up-regulate PD-L1 expression in NSCLC from traditional anti-tumor drugs and verify that the candidate drugs can mediate synergistic anti-tumor activity with PD-1/PD-L1 inhibitors.The mechanism of up-regulating PD-L1 expression were explored.In general,the fundamental purpose of this study is to provide new ideas and rationale for combination therapy of PD-1/PD-L1 in NSCLC.Materials and Methods:After determined the optimal concentration of drugs by MTT cytotoxicity assay,selecting candidate drugs based on upregulation m RNA,total protein and surface protein of PD-L1 expression in human NSCLC cell line A549 from a variety of antitumor drugs including Cisplatin,Paclitaxel,Vinorelbine,Irinotecan,Doxorubicin,Gemcitabine,Capecitabine,Afatinib,Sorafenib,Idelalisib,Trametinib,Decitabine and Vorinostat using q RT-PCR,Western Blot,and flow cytometry.Next,we explored the molecular mechanism of selected drugs up-regulating PD-L1 expression by constructing knock-out cell lines,q RT-PCR,Western Blot and si RNA libraries at both the transcriptional and post-transcriptional levels.At the same time,the PD-L1 KO cell line was constructed to verify that the selected drugs can enhance the anti-tumor efficiency of PD-1/PD-L1 inhibitors by up-regulating PD-L1 expression on LLC or LLC^PD-L1-tumor-bearing models.Results:The MTT cytotoxicity assay indicated that all drugs tested were not cytotoxic up to 10μM in A549 cell line.In this concentration range,several drugs would up-regulate PD-L1m RNA or total protein expression,but only HDAC inhibitor,Vorinostat could significantly increase PD-L1 surface protein expression in the results of flow cytometry.Further more,our research also confirmed that Vorinostat significantly increased of CKLF like MARVEL transmembrane domain containing 6（CMTM6）expression at the transcriptional level by inhibiting the HDAC3 subtype via HDAC si RNA libraries,sh RNA and other technologies.CMTM6 could stabilize the PD-L1 on the cell membrane surface,prevent PD-L1 from the endocytosis,lysosomal digestion and ubiquitination degradation,thus up-regulating the expression of PD-L1 on the cell membrane surface.After CMTM6 was knocked out,the induction of Vorinostat up-regulating PD-L1 expression was decreased.MTT assay confirmed that PD-L1 knockout did not affect LLC proliferation.In the tumor-bearing mouse model,Vorinostat monotherapy could slow down the tumor growth of wild type（WT）mice to a certain extent but cannot change the trend of tumor growth.PD-1 antibody could control tumor growth very well in the early stage,but tumors began to recur 25 days after subcutaneous inoculation,PD-L1 KO group had similar results with PD-1 antibody treatment group.In the combined treatment group,the tumor volume was well controlled throughout the observation period,and no signs of tumor recurrence were found,PD-L1 KO with Vorinostat treatment group had similar results.Conclusion:HDAC inhibitor Vorinostat can regulate CMTM6 expression via HDAC3subtype,and then up-regulate PD-L1 expression on the cell membrane surface.Up-regulating PD-L1 expression is a resistance mechanism to HDAC inhibitors,and it is also a mechanism mediating synergistic anti-tumor activity for combinating HDAC inhibitors with PD-1/PD-L1inhibitors.HDAC inhibitors combined with PD-1/PD-L1 inhibitors were expected to become a new option for NSCLC.Part Ⅱ: Exploration of The Role of CMTM6 in Lung Adenocarcinoma based on TCGA,GEPIA,TIMER databasesObjective: Immunotherapy is currently one of the most important approaches for NSCLC treatment,and PD-1/PD-L1 inhibitors are hot spots.Clinical trials indicated that the higher PDL1 expression is related to the higher efficacy of PD-1/PD-L1 inhibitors,and PD-L1 expression is currently one of most important biomarkers for predicting the efficacy of PD-1/PD-L1 inhibitors in NSCLC.CMTM6,as a key regulator of PD-L1 expression,can stabilize the the cell surface protein of PD-L1,and may have important research value.Lung adenocarcinoma is the most important pathological type in lung cancer.This article aims to explore the expression and prognostic value of CMTM6 and its possible biological functions in lung adenocarcinoma using the TCGA,GEPIA,and TIMER database.Materials and Methods: Transcriptome data and clinical data of lung adenocarcinoma were downloaded from the TCGA database.The transcriptome data of gene expression were standardized by TPM.Using the GEPIA database to analyze the differences in expression levels of CMTM6 between lung adenocarcinoma samples and normal lung tissue samples.Kaplan-Meier curve and Log-rank test were used to evaluate the effect of CMTM6 expression on the prognosis.The best cut-off value was determined using the "surv_cutpoint" function of the survminer package in R,and the research indicators included OS and PFS.Wilcoxon rank sum test and chi-square test were used to analyze the correlation between CMTM6 expression and clinicopathological characteristics of lung adenocarcinoma.The correlation between CMTM6 expression and immune cell infiltration abundance or immune checkpoints expression were explored to evaluate the CMTM6 immunomodulatory effect.CMTM6 expression value was regarded as the phenotype for gene sequencing,GSEA was used to perform gene set pathway enrichment analysis on genes related to CMTM6 expression,and annotation was performed with the GO \ KEGG \ HALLMARK database.Using three immunotherapy data sets to preliminarily evaluate the ability of CMTM6 expression predicting the efficiency of immunotherapy.Results: A total of 510 lung adenocarcinoma samples from the TCGA database were included.The median age was 66 years,of which 235（46.1%）were male,385（75.5%）were white,422（82.8%）had a history of smoking,and right lung cancer sample accounted for 297（58.2%）.Most of the lung adenocarcinoma samples included in the TCGA database were early stage.The proportion of TNM stage I-II samples was 77%,specifically,443（86.9%）patients were in T1 and T2 stages,N0 and M0 accounted for 64.1% and 67.3%,respectively.The proportion of patients receiving chemotherapy was 34.7%.The median values of CMTM6 and PD-L1 expression levels after TPM standardization were 91.66 and 6.99,and the average values were 97.67 and 13.31.Compared with the normal lung tissue samples,CMTM6 expression in lung adenocarcinoma samples was significantly increased.In lung adenocarcinoma,OS in the high CMTM6 expression group was shorter than the low expression group,but was no statistical difference,and the PFS induced a statistical significant difference.CMTM6 expression had nothing to do with clinicopathological characteristics except T stage.CMTM6 expression were also significantly correlated with infiltration abundance of CD8 + T cells,macrophages,neutrophils,and dendritic cells,but not associated with the infiltration abundance of B cells and CD4 + T cells.CMTM6 expression was also significantly correlated with the expression of dozens of immune checkpoints,most of them were positively correlated.We then performed gene set enrichment analysis（GSEA）and annotated it with three databases: GO,KEGG,and HALLMARK.All significantly enriched gene sets were positively correlated with CMTM6 expression.Identified sets that were significantly enriched in all three databases.CMTM6 expression was significantly associated with various functional activities such as inflammatory responses,cytokine signals,antigen presentation and processing,immune cell proliferation in lung adenocarcinoma tissues.Additionally,high CMTM6 expression may indicate a better clinical response and overall survival outcome of immunotherapy.Conclusion: CMTM6 expression in lung adenocarcinoma tissues was significantly higher than normal lung tissues.the high CMTM6 expression group showed worse prognosis trend than the low CMTM6 expression group.There was no statistically difference in OS,whereas there was a statistically difference in PFS.CMTM6 has a clear immunoregulatory effect,which is CMTM6 expression was significantly associated with the expression of many immune checkpoints and immune cell infiltration abundances,indicating that CMTM6 has an immunoregulatory effect.GSEA results showed that CMTM6 expression was significantly related to the inflammatory and immunomodulatory properties in lung adenocarcinoma.In addition,CMTM6 expression may have a predicting ability on the prognosis of immunotherapy.CMTM6 play an important role and be a potential therapeutic target in lung adenocarcinoma.