Epigenetic regulation of pancreatic cancer subtype identity and tumorigenesis by PHF2

胰腺导管癌(PDAC)的分子亚型定义了两个主要的转录 亚型，经典和基本样型，这是目前最有效的预测患者预后的指标。 因此，迫切需要确定PDAC细胞身份的潜在机制。 我们的初步实验表明，在低传代过程中，组蛋白去甲基酶PHF2失活 患者来源的经典PDAC细胞导致经典基因程序的显著丢失和虚拟 消除肿瘤生长。此外，Phf2基因的缺失抑制了胰腺肿瘤的发生和延长 基因工程小鼠模型的存活率。此外，我们的切割和运行实验表明，PHF2 与CTCF和SMC3共定位在染色体上，这两种蛋白质通过调节 染色质的三维结构。基于这些令人振奋的初步结果，我们的中央 假说是PHF2通过调节三维结构调控PDAC中的经典基因程序 染色质的结构。该方案中的实验将使用一种独特的低通道集合 人类细胞系，染色质相关蛋白和染色质结构的高分辨率图谱，以及 基因工程小鼠模型的NanoStringGeoMx空间转录分析以定义 PHF2在PDAC中的作用提出了三个具体的目标来检验中心假设：1)定义子集 依赖于PHF2的PDAC；2)确定PHF2调节基因表达的机制 以及3)确定Phf2在基因工程小鼠肿瘤发生中的作用。在第一个 目的：CRISPR/Cas9可暂时灭活PHF2的原位异种移植瘤模型 被用来确定PHF2在维持PDAC肿瘤以及它们的 对化疗的反应。在第二个目标中，将进行Cut&Run、HiChIP和RNA-Seq实验 用来确定PHF2在PDAC细胞染色质上的全球定位并确定其在 招募CTCF和对DNA的粘附力来介导DNA环的形成。为了第三个目的，从基因上讲 缺乏Phf2的PDAC的工程化小鼠模型将被用来阐明其在KRAS驱动的 胰腺腺泡细胞转化为浸润性癌所需的细胞特性改变。空间 将进行转录学研究以确定Phf2缺陷如何改变肿瘤的成分 由PDAC开发的微环境是经典程序所不具备的。这项研究 在本申请中提出的是创新的，因为它将定义PHF2在调节中的新功能 染色质结构，进一步阐明了允许细胞类型特定的染色质的机制 建筑。这项拟议的研究具有重要意义，因为它将提供强有力的证据证明 表观遗传学对PDAC细胞特性的调控，最终为PDAC的发展提供新的机遇 治疗PDAC的新疗法。

Molecular subtyping of pancreatic ductal adenocarcinoma (PDAC) has defined two major transcriptional subtypes, Classical and Basal-like, which are presently the most powerful predictors of patient outcomes. Thus, there is a critical need to determine the underlying mechanisms responsible for PDAC cell identity. Our preliminary experiments demonstrate that inactivation of PHF2, a histone demethylase, in low-passage patient-derived Classical PDAC cells results in a striking loss of the Classical gene program and virtually abolishes tumor growth. In addition, deletion of Phf2 suppresses pancreatic tumor initiation and prolongs survival in genetically engineered mouse models. Moreover, our CUT&RUN experiments reveal that PHF2 co-localizes on chromosomes with CTCF and SMC3, proteins that contribute to cell identity by regulating the three-dimensional architecture of chromatin. Based on these exciting preliminary results, our central hypothesis is that PHF2, controls the Classical gene program in PDAC by regulating three-dimensional architecture of chromatin. Experiments in this proposal will employ a unique collection of low-passage human cell lines, high resolution mapping of chromatin-associated proteins and chromatin architecture, and NanoString GeoMx spatial transcriptomic analysis of genetically engineered mouse models to define the role of PHF2 in PDAC. Three specific aims are proposed to test the central hypothesis: 1) Define the subsets of PDAC that are dependent on PHF2; 2) Identify the mechanisms by which PHF2 regulates gene expression in PDAC; and 3) Define the contribution of Phf2 to tumorigenesis in genetically engineered mice. In the first aim, orthotopic xenograft tumor models allowing for the temporal inactivation of PHF2 by CRISPR/Cas9 will be employed to determine the importance of PHF2 in the maintenance of PDAC tumors as well as their response to chemotherapy. In the second aim, CUT&RUN, HiChIP, and RNA-seq experiments will be employed to define the global localization of PHF2 on chromatin in PDAC cells and ascertain its role in recruiting CTCF and cohesion to DNA to mediate DNA loop formation. For the third aim, genetically engineered mouse models of PDAC lacking Phf2 will be used to elucidate its role in the KRAS-driven changes in cell identity required for pancreatic acinar cells to transform to invasive cancer . Spatial transcriptomic studies will be performed to ascertain how Phf2 deficiency alters the composition of the tumor microenvironment by the development of PDAC that is deficient in the Classical program. The research proposed in this application is innovative because it will define a novel function for PHF2 in regulating chromatin architecture, further elucidating the mechanisms that allow for cell type-specific chromatin architecture. The proposed research is significant because it will provide strong evidence for the role of epigenetic regulation of PDAC cell identity, ultimately providing new opportunities for the development of novel therapies for PDAC.