EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor Natural Killer (NK) and T cell immunity. Here we found the pancreas tumor microenvironment (TME) suppresses NK and T cell surveillance following therapy-induced senescence through EZH2-mediated epigenetic repression of pro-inflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in PDAC patients. These results demonstrate that EZH2 represses of the pro-inflammatory SASP, and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC. Overall design: For RNA-seq analysis of PIP, PIL, LIL, and LIP tumor samples, GFP+ tumor cells were FACS sorted on a FACSAria (BD Biosciences) from the lungs or pancreas of tumor-bearing mice following 2-week treatment with vehicle or combined trametinib (1 mg/kg body weight) and palbociclib (100 mg/kg). Total RNA was extracted from tumor cells using the RNeasy Mini Kit (Qiagen). Purified polyA mRNA was subsequently fragmented, and first and second strand cDNA synthesis performed using standard Illumina mRNA TruSeq library preparation protocols. Double stranded cDNA was subsequently processed for TruSeq dual-index Illumina library generation. For sequencing, pooled multiplexed libraries were run on a HiSeq 2500 machine on RAPID mode. Approximately 10 million 76bp single-end reads were retrieved per replicate condition. RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic, version 0.36, aligning sequencing data to GRCm38 (Ensembl, version 101) with STAR, version 2.5.3a, and genome wide transcript counting using featureCounts, version 1.6.3 to generate a RPKM matrix of transcript counts. Genes were identified as differentially expressed using R package DESeq2, version 1.28.1 with a cutoff of absolute log2FoldChange = 1 and adjusted p-value <0.05 between experimental conditions. Heatmaps were generated using pheatmap, version 1.0.12. Over-representation analysis of differentially expressed genes (DEGs) against KEGG Pathways was performed using clusterProfiler, version 4.0.5.

在部分恶性肿瘤中可产生持久应答的免疫治疗，因严重的免疫抑制及较差的肿瘤免疫原性，在胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）中未能取得理想疗效。本团队及其他研究者均已证实，诱导衰老相关分泌表型（senescence-associated secretory phenotype, SASP）可有效激活抗肿瘤自然杀伤（Natural Killer, NK）细胞与T细胞免疫。本研究发现，胰腺肿瘤微环境（tumor microenvironment, TME）可在治疗诱导细胞衰老后，通过EZH2介导的促炎SASP基因表观遗传抑制，削弱NK细胞与T细胞的免疫监视功能。EZH2阻断可促进SASP趋化因子CCL2与CXCL9/10的产生，进而增强小鼠模型中NK细胞与T细胞的浸润，并实现PDAC的清除。在PDAC患者中，EZH2的活性亦与趋化因子信号通路及细胞毒性淋巴细胞的抑制状态相关，且与患者不良生存期呈显著负相关。上述研究结果表明，EZH2可抑制促炎型SASP的表达；将EZH2抑制与衰老诱导治疗联合，有望成为实现PDAC免疫介导肿瘤管控的有效策略。

整体实验设计：针对PIP、PIL、LIL及LIP肿瘤样本的RNA测序分析：将荷瘤小鼠经载体对照或曲美替尼（1 mg/kg 体重）联合帕博西尼（100 mg/kg）处理2周后，通过FACSAria流式细胞分选仪（BD Biosciences）从其肺部或胰腺组织中分选GFP阳性肿瘤细胞。采用RNeasy Mini试剂盒（Qiagen）从肿瘤细胞中提取总RNA。随后对纯化的polyA mRNA进行片段化，并按照Illumina mRNA TruSeq文库制备标准流程完成第一、第二链cDNA合成。将双链cDNA进一步处理以构建TruSeq双索引Illumina文库。测序时，将混合的多重文库置于HiSeq 2500测序仪上以RAPID模式进行测序，每个重复样本组可获得约1000万条76 bp单端读取序列。

RNA测序数据的分析流程如下：使用Trimmomatic v0.36去除接头序列，采用STAR v2.5.3a将测序数据比对至GRCm38参考基因组（Ensembl v101），并使用featureCounts v1.6.3进行全基因组转录本计数，以生成转录本计数的RPKM矩阵。使用R包DESeq2 v1.28.1筛选差异表达基因，筛选阈值为绝对log2倍数变化=1且校正后P值<0.05。使用pheatmap v1.0.12绘制热图。采用clusterProfiler v4.0.5对差异表达基因（DEGs）进行KEGG通路富集分析。