肿瘤细胞和MDSCs表达的PD-L1在胰腺癌免疫逃逸中的功能

In the last few years, cancer immunotherapy has made significant breakthroughs in the clinic. Immune checkpoint inhibitor (ICI) immunotherapy, parimarily immunotherapy with antibody that blocks program death ligand 1 (PD-L1) or programmed death protein 1 (PD-1), has produced durable efficacy in many types of human cancers. Anti-PD-1 and anti-PD-L1 antibody-based checkpoint blockade immunotherapy has shown a overall response rate from 13% to 69%. It has been approved for treatment of melanoma, non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, bladder cancer, and Hodgkin’s lymphoma, and the list is still fast growing since hundreds of clinical trials are ongoing. However, despite these amazing success of ICI immunotherapy, pancreatic cancer stands out as one of the few human cancers that do not respond to ICI immunotherapy. What distinguishes pancreatic cancer from other human cancers in response to ICI immunotherapy is curently unknown. In the literature, the relative contributions of tumor cell-expressed or the non-tumor cell-expressed PD-L1 in suppression of T cell-mediated anti-tumor immune response is highly controversial in colon carcinoma. However, this relative contributions are totally unknown in pancreatic cancer. The central hypothesis is that both tumor-expressed and tumor-infiltrating MDSCs-expressed PD-L1 function to inhibit cytotoxic T lymphocyte activation to promote pancreatic tumor immune evasion. The objectives of this project are :1) determine the cellular source(s) of PD-L1 that controls pancreatic cancer immune evasion; and 2) determine the regulatory mechanism of PD-L1 expression in non-tumor cells, especially MDSCs. Successful completion of this research project will not only provide significant insight for understanding pancreatic cancer non-response to ICI immunotherapy but also provide the molecular target for the development of targeted therapy to overcome pancreatic cancer resistance to ICI immunotherapy.

在过去的几年中，肿瘤免疫治疗已在临床上取得了突破性进展。免疫检查点阻断疗法已在多种人类肿瘤中显现出持久的疗效，其适应症也在不断地增加。然而，尽管免疫检查点阻断疗法已取得令人瞩目的巨大成功，胰腺癌是少数几种对该疗法不起反应的人类肿瘤之一，其耐受的机制尚不明确。肿瘤细胞和非肿瘤细胞表达的PD-L1在介导肿瘤的免疫抑制中所起的相对作用在文献中存在着较大争议，而其在胰腺癌的免疫抑制中所起的相对作用则鲜见报道。本研究课题的核心假设是：肿瘤细胞和肿瘤侵润的MDSCs表达的PD-L1在抑制CTL功能，以及促进胰腺癌的免疫逃逸方面均起着十分重要的作用。研究目标为：1.揭示介导胰腺癌免疫逃逸的PD-L1的细胞来源；2.揭示PD-L1在MDSCs中的表达调控机制。该研究课题的成功完成将不仅有助于理解胰腺癌对免疫检查点阻断疗法耐受的原理，还可为逆转胰腺癌对免疫检查点阻断疗法的抗性提供有效的分子靶点。

过去几年中，免疫检查点阻断疗法（Immune Checkpoint Immunotherapy, ICI）已在多种类型的肿瘤治疗中展现出显著的疗效和巨大的应用前景。Science杂志将基于T细胞的肿瘤免疫治疗誉为“2013年的年度突破”。遗憾的是，被称为“癌中之王”的胰腺癌却是少数几种对单药PD-1/PD-L1抗体免疫疗法不反应的肿瘤之一，其耐受的免疫学机制尚不明确。因此，阐明胰腺癌对ICI疗法耐受的分子机制具有重要的理论意义和实践价值，并且是肿瘤免疫学和免疫治疗领域的前沿热点。另一方面，表观基因组的失调已成为T细胞耗竭和对ICI疗法耐受的主要机制之一。因此，本研究项目旨在验证假设：胰腺肿瘤微环境中存在着某种表观遗传学机制可弥补PD-L1的功能以赋予胰腺癌对ICI疗法的耐受。. 本项目利用RNA测序（RNA-Seq）和染色质免疫沉淀测序（ChIP-Seq）技术鉴定了H3K4me3在小鼠胰腺肿瘤组织中的OPN及其受体CD44启动子上高度富集；通过免疫组化、ELISA和数据库分析得出：OPN在人胰腺肿瘤组织中的表达水平显著高于正常胰腺组织，其表达水平与胰腺癌病人的生存时间成反相关；流式细胞术分析小鼠胰腺肿瘤浸润髓细胞谱和免疫细胞谱指出：OPN在胰腺肿瘤微环境中的M-MDSCs中高表达，PD-L1在PMN-MDSCs和肿瘤相关髓细胞中高表达。另一方面，在肿瘤细胞和MDSCs中，WDR5对于H3K4me3特异的甲基化转移酶活性具有重要的调控作用。本项目利用RNA-Seq，MTT和Western Blot方法分析得出：三种WDR5小分子抑制剂均能以剂量依赖的方式抑制MLL1-WDR5的酶活性，均可在胰腺肿瘤细胞和J774M细胞中降低OPN的蛋白表达水平。用具有较低细胞毒的抑制剂WDR5-47处理PANC02-H7荷瘤小鼠，结果表明：WDR5-47可有效降低小鼠外周血中的OPN蛋白水平，并能显著改善抗PD-1抗体的疗效。敲除Spp1（编码OPN的基因）并与抗PD-1抗体合用可得到类似的结果。. 该研究工作不仅在胰腺肿瘤微环境中揭示了OPN是可以弥补PD-L1功能的新型免疫检查点，也为临床上提高ICI疗效开辟了新途径。WDR5小分子抑制剂WDR5-47具有较大潜力在我国开发成为具有自主知识产权的新型胰腺癌免疫治疗药物。