(PQ4) - Tools for simultaneous disruption of multiple epigenetically silenced genes for studying their roles in tumorigenesis using ex vivo human and mouse colon organoid and in vivo mouse models

我们将解决PQ 4-需要开发工具，同时操纵多个基因在人类 癌症相关的模型-因为该领域的一个关键挑战是了解多个基因如何受到 表观遗传或拷贝数改变共同影响肿瘤发展的过程。为 表观遗传学影响的基因，我们已经表明，多个重要的肿瘤抑制和发展， 调节基因通过启动子CpG岛（CGI）DNA维持在永久沉默状态 在不同癌症类型中的高甲基化。重要的是，来自相同生物途径的许多基因 在同一个病人样本中表观遗传学上沉默了。多重表观遗传学的累积效应 沉默基因（ESG）在肿瘤发展中的作用仍然是癌症生物学的一个未解决的方面，主要 由于缺乏靶向多个基因的工具，易于处理的生物模型和测定方法， 肿瘤发展在这项提案中，我们试图通过利用我们目前在人类癌症方面的工作来填补这些空白 相关模型。我们进行这些研究的动机源于我们过去3-4年的工作， 我们已经通过致癌BRAFV 600 E模拟了人类结直肠癌（CRC）发展的早期阶段， 和KRASG 12 D突变。我们发现，在小鼠近端结肠源性类器官中，BrafV 600 E的诱导，但 而不是突变体KrasG 12 D，导致Wnt途径激活、干细胞表型和转化。肿瘤 转化的类器官的外植体表型模仿人近端CRC，包括粘液组织病理学 和CGI高甲基化表型（CIMP）。我们发现，衰老样的CGI超甲基化的获得 在类器官中，与衰老的人结肠中的情况不同，通过诱导BrafV 600 E介导的肿瘤发生， 早期Wnt通路激活和干细胞表型，这是肿瘤发生过程中的早期特征， 模型我们的研究与其他实验室的工作一致，表明类器官是一个高度相关的模型， 它模仿了人类肿瘤发展的特征。此外，我们已经建立了基于Cas9和Cpf 1的 在类器官中进行筛选，以鉴定促进肿瘤发生的ESG的缺失。该项目的主要目标是 开发同时灭活多种ESG的工具，以研究它们在癌症中的联合作用。在这 在这方面，我们已经开发了基于诱导CRISPR的人结肠源性类器官，用于多基因表达。 基因敲除研究ESG和癌症驱动突变之间的相互作用。我们将继续这些， 开发具有可诱导Cas9/Cpf 1的人和小鼠结肠类器官系统用于系统筛选 多个ESG的随机组合失活，赋予干细胞维持和阻断分化。 接下来，我们将集中在重要的候选基因，生物信息学挖掘癌症数据库，研究 它们在癌症引发中的同时损失的协同作用。最后，我们将应用并验证我们的研究在体内 小鼠模型。总之，这项建议将开发通用工具，用于同时操纵多个 人类癌症相关模型中的基因，可以很容易地转化为其他癌症模型。

We will address PQ4 – the need to develop tools for simultaneous manipulation of multiple genes in human cancer-relevant models – because a key challenge in the field is to understand how multiple genes affected by epigenetic or copy number alterations act in concert to influence the process of tumor development. For the epigenetically affected genes, we have shown that multiple important tumor suppressor and developmental regulator genes are maintained in a permanently silenced state by promoter CpG-island (CGI) DNA hypermethylation across various cancer types. Importantly many genes from the same biological pathways are epigenetically silenced within the same patient sample. The cumulative effect of multiple epigenetically silenced genes (ESGs) on tumor development remains an unaddressed aspect of cancer biology, mainly because of a lack of tools for targeting multiple genes, tractable biological models and assays to measure tumor development. In this proposal we seek to fill these gaps by leveraging our current work in human cancer relevant models. Our motivation to pursue these studies originate from our work in the past 3-4 years where we have modeled the early stages of human colorectal cancer (CRC) development by oncogenic BRAFV600E and KRASG12D mutations. We show that in mouse proximal colon-derived organoids, induction of BrafV600E, but not mutant KrasG12D, results in Wnt pathway activation, stem cell phenotype and transformation. Tumor explants of the transformed organoids phenocopy human proximal CRC, inclusive of mucinous histopathology and CGI hypermethylator phenotype (CIMP). We show that an aging-like acquisition of CGI hypermethylation in organoids, not unlike that in aging human colon, promotes BrafV600E-mediated tumorigenesis by inducing early Wnt pathway activation and stem cell phenotype, which are early features during tumorigenesis in this model. Our studies, in concert with work from other labs, show that the organoids are a highly relevant model that phenocopies features of human tumor progression. Further, we have established Cas9 and Cpf1-based screening in organoids to identify loss of ESGs that promote tumorigenesis. The major goal of this project is to develop tools for simultaneous inactivation of multiple ESGs to study their combined roles in cancers. In this regard, we have developed inducible-CRISPR based human colon-derived organoids for multiple gene knockout to study the collusion between ESGs and cancer driver mutations. We will continue on these by developing human and mouse colon organoid system with inducible Cas9/Cpf1 for systematically screening random combinatorial inactivation of multiple ESGs that confer stem-cell maintenance and block differentiation. Next we will focus on important candidate genes, bioinformatically mined form cancer databases, to study the synergy of their simultaneous loss in cancer initiation. Finally, we will apply and validate our studies in in-vivo mouse models. In summary, this proposal will develop generic tools for simultaneous manipulation of multiple genes in human cancer-relevant models that can be easily translated to other cancer models.