GSE201495
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.