探討含溴結構域抑制劑對肺癌反應T細胞的代謝重整作用

Abstract

肺癌為全球癌症死亡率之冠，即使現已開發免疫檢查點抑制劑，為肺癌治療帶來新曙光，仍有一定比例的患者因體內耗竭T細胞(Exhausted T cell, TEX)受到既有表觀遺傳基因體 (epigenetic landscapes)的限制，無法完全恢復成為功能T細胞，而導致其治療效果不彰。惡性胸水 (Malignant pleural effusion, MPE) 為晚期肺腺癌患者常見的併發症，胸水內含有轉移的癌細胞以及大量耗竭T細胞，為免疫失能之腫瘤微環境。本實驗室先前的研究中，透過145個表觀遺傳藥物進行藥物篩選，發現含溴結構域抑制劑 (bromodomain inhibitors, BETi) 中的JQ1在體外能夠顯著改善惡性胸水內耗竭性T細胞的功能。然而JQ1改善耗竭性T細胞功能的機制仍未清楚，因此本研究的目標為進一步探討JQ1調節耗竭性T細胞免疫功能的機制。首先，我們進行 RNA定序以鑑定在耗竭性T細胞中受JQ1影響的基因譜，從中發現JQ1治療後顯著上調耗竭性T細胞中代謝相關途徑的基因群，其中包括氨基酸代謝及五碳糖磷酸途徑等。我們進一步藉由不同的代謝抑制劑阻斷相關的代謝途徑，發現氨基酸代謝抑制劑Difluoromethylornithine (DFMO)可顯著降低JQ1提升T細胞的多功能性，表示JQ1可能是透過氨基酸代謝調控T細胞的功能。接著，我們透過即時聚合?連鎖反應驗證JQ1治療顯著上調編碼鳥胺酸脫羧? (ornithine decarboxylase, ODC)的基因ODC1，並利用小分子干擾RNA (small interfering RNA, siRNA)的方式抑制ODC1表現後，釐清JQ1確實透過提升ODC1基因表現而增加分泌細胞因子。最後，我們使用定序轉座?可接觸的染色質分析法(Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) 發現經JQ1處理後，惡性胸水中耗竭性T細胞的ODC1基因，其位於啟動子附近的染色質可及性增加，並由單細胞定序 (single-cell RNA sequencing, scRNA-seq) 發現JQ1可能透過組蛋白修飾影響衰竭性T細胞的基因表現。綜合上述結果，本研究探討JQ1透過表觀遺傳調控代謝重整的方式於晚期肺癌患者體內的耗竭性T細胞進行免疫調節作用，而可為發展肺癌惡性胸水的表觀遺傳治療提供新方向。

Lung cancer causes the leading mortality rate worldwide. Even if immune checkpoint inhibitors (ICIs) have been developed to bring light to lung cancer treatment, there are still patients experiencing poor therapeutic efficacy of ICIs, possibly due to static epigenetic landscapes in exhausted T cells (TEX). Malignant pleural effusion (MPE) is a common complication in patients with advanced lung adenocarcinoma. MPE is an immune dysfunctional tumor microenvironment containing metastatic cancer cells and TEX. In our previous study, we performed a drug screening of 145 epigenetic drugs and found that JQ1 and other bromodomain inhibitors (BETi) significantly improved the function of TEX in MPE ex vivo. However, the mechanism by which JQ1 improved the function of exhausted T cells remained unclear, and we aimed to further explore the mechanism in this study. First, we performed genome-wide RNA-seq analysis to investigate transcriptomic profiles affected by JQ1 in the TEX of MPE. Gene set enrichment analysis (GSEA) analysis revealed that several metabolic pathway-related gene sets were significantly upregulated by JQ1 treatment, including amino acid metabolism and pentose phosphate pathway, etc. Next, we blocked individual metabolic pathways with respective inhibitors and found that difluoromethylornithine (DFMO), an inhibitor for amino acid metabolism, significantly abolished the JQ1-mediated enhancement of T cell polyfunctionality. The data indicated that JQ1 may regulate the function of T cells through amino acid metabolism. We also verified that JQ1 significantly upregulated ornithine decarboxylase (ODC1), an enzyme involved in polyamine synthesis, in primary malignant pleural effusion T cells by real-time polymerase chain reaction (RT-PCR). Through siRNA-mediated knock-down experiments, we discovered that ODC1 enhanced effector functions in T cells. Finally, we dissected the epigenetic landscape of exhausted T cells in malignant pleural effusions after JQ1 treatment by performing an Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We found that JQ1 increased chromatin accessibility at the promoter region in ODC1. Moreover, analysis of single-cell RNA sequencing (scRNA-seq) indicated that JQ1 may affect histone modifications in exhausted CD8 T cells. In summary, JQ1 exerted immunomodulatory effects on TEX in patients with advanced lung cancer through epigenetic regulation of metabolic reprogramming. This study may provide a new direction for the development of epigenetic immunotherapy for malignant pleural effusion in lung cancer.