Mutant-p53 stimulates Cxcl1 Expression from Distal Enhancers to Suppress Immune Response to Pancreatic Cancer [EXOME-seq]. Mutant-p53 stimulates Cxcl1 Expression from Distal Enhancers to Suppress Immune Response to Pancreatic Cancer [EXOME-seq]

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective treatments. It is characterized by activating KRAS mutations and p53 alterations. However, how these mutations alter cell-intrinsic gene programs to influence the immune landscape of the tumor microenvironment (TME) remains poorly understood. Here, we demonstrate that p53R172H enhances tumor growth, establishes a suppressive TME by inducing immune evasion, and blunts the effectiveness of immune checkpoint inhibitors (ICIs). We discovered that the oncogenic function of p53R172H is mediated by upregulation of the immunosuppressive chemokine Cxcl1. Mechanistically, we show that p53R172H binds to the distal enhancers of the Cxcl1 gene and increases enhancer activity and Cxcl1 expression. NF-kB also occupies Cxcl1 enhancers, and p53R172H binds these enhancers in an NF-kB-dependent manner, suggesting a role of NF-kB in commuting p53R172H to the Cxcl1 enhancers. Our findings elucidate how a common mutation in a critical tumor-suppressor gene exploits enhancers to modulate chemokine gene expression and foster an immunosuppressive TME in PDAC that undermines the efficacy of ICI. Overall design: To investigate the role of p53R172H in pancreatic ductal adenocarcinoma (PDAC), we generated isogenic Trp53-/- PDAC cells (KrasG12D/+; Trp53-/-) using CRISPR-Cas9. We compared transcriptional programs via RNA-seq and PRO-seq, exonic mutations using Exome-seq, and genome occupancy with CUT&RUN between Trp53R172H/- and isogenic and syngenic Trp53-/- cells, as well as Trp53-/- cells with ectopically expressed p53R172H. We orthotopically implanted the PDAC cells into the pancreas of mice and evaluated tumor growth, size, and the immune landscape. We further treated the mice with immune checkpoint inhibitors and monitored their survival. To explore the role of Cxcl1 and its enhancers, we generated Cxcl1-/- cells and Cxcl1 enhancer knockout cells. We then assessed gene expression, immune profiling, and the response to immune checkpoint inhibitors in these models.

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）是一种侵袭性极强且缺乏有效治疗手段的恶性肿瘤，其标志性特征为携带激活型KRAS突变及p53基因改变。然而，上述突变如何通过调控细胞内在基因程序，进而重塑肿瘤微环境（Tumor microenvironment, TME）的免疫特征，目前仍不甚明确。本研究证实，p53R172H突变可促进肿瘤生长，通过诱导免疫逃逸构建免疫抑制性肿瘤微环境，并削弱免疫检查点抑制剂（Immune checkpoint inhibitors, ICIs）的治疗疗效。我们发现，p53R172H的致癌功能可通过上调免疫抑制性趋化因子Cxcl1得以介导。机制层面，我们证实p53R172H可结合Cxcl1基因的远端增强子，增强该增强子的转录活性并上调Cxcl1的表达水平。核因子κB（NF-κB）同样可结合Cxcl1增强子区域，而p53R172H以依赖于NF-κB的方式靶向上述增强子，这提示NF-κB可介导p53R172H募集至Cxcl1增强子区域。本研究结果阐明了关键抑癌基因中的常见突变如何通过靶向增强子调控趋化因子基因表达，并在胰腺导管腺癌中构建免疫抑制性肿瘤微环境，进而削弱免疫检查点抑制剂的治疗疗效。实验设计概述：为探究p53R172H在胰腺导管腺癌中的作用，我们利用CRISPR-Cas9技术构建了同基因背景的Trp53敲除（Trp53-/-）胰腺导管腺癌细胞（KrasG12D/+; Trp53-/-）。我们通过RNA测序（RNA-seq）与精准核转录测序（PRO-seq）比较两组细胞的转录组特征，通过外显子组测序（Exome-seq）分析外显子突变，并利用CUT&RUN技术检测Trp53R172H/-细胞、同基因及同系的Trp53-/-细胞，以及异位表达p53R172H的Trp53-/-细胞的基因组结合谱。我们将上述胰腺导管腺癌细胞原位移植至小鼠胰腺内，评估肿瘤生长情况、体积及免疫微环境特征。此外，我们利用免疫检查点抑制剂处理小鼠并监测其生存状态。为探究Cxcl1及其增强子的功能，我们构建了Cxcl1敲除（Cxcl1-/-）细胞及Cxcl1增强子敲除细胞。随后我们在上述模型中评估基因表达水平、免疫特征以及对免疫检查点抑制剂的响应情况。