(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

(PQ4) - 同时破坏多个表观遗传沉默基因的工具，用于使用离体人类和小鼠结肠类器官以及体内小鼠模型研究它们在肿瘤发生中的作用

• 批准号: 10471240
• 负责人: STEPHEN B. BAYLIN
• 金额: $ 36.71万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471240
• 关键词: Address; Affect; Aging; BRAF gene; Bioinformatics; Biological; Biological Assay; Biological Models; CRISPR/Cas technology; Cancer Biology; Cancer Model; Candidate Disease Gene; Cell Maintenance; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Colon; Colorectal Cancer; Communities; CpG Islands; DNA; Data; Databases; Development; Epigenetic Process; Epithelial; Experimental Models; Future; Gene Combinations; Gene Expression; Gene Silencing; Gene Targeting; Genes; Goals; Histopathology; Homeostasis; Human; Hypermethylation; In Situ; Individual; KRAS2 gene; KRASG12D; Learning; Malignant Neoplasms; Measures; Mediating; Modeling; Molecular; Molecular Abnormality; Motivation; Mucinous; Mus; Mutation; Oncogenic; Organoids; Pathway interactions; Patients; Phenocopy; Phenotype; Play; Pre-Clinical Model; Predisposition; Regulator Genes; Research; Role; Sampling; Screening for cancer; Signal Pathway; Structure; System; Technology; Tissues; Translating; Tumor Suppressor Proteins; Tumor-Associated Process; Work; anticancer research; base; cancer initiation; cancer type; combinatorial; course development; driver mutation; epigenetic silencing; epigenomics; experience; genome editing; in vivo; in vivo Model; innovation; knockout gene; mouse model; novel; preclinical development; promoter; screening; stem cell differentiation; stem cells; synergism; tool; tumor; tumor initiation; tumor progression; tumorigenesis

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.

我们将解决 PQ4——开发同时操作人类多个基因的工具的需求 癌症相关模型——因为该领域的一个关键挑战是了解多个基因如何受到影响 表观遗传或拷贝数改变共同影响肿瘤的发展过程。 表观遗传影响基因，我们已经证明多个重要的肿瘤抑制和发育 调节基因通过启动子 CpG 岛 (CGI) DNA 维持在永久沉默状态 重要的是，来自相同生物途径的许多基因都存在高甲基化。 同一患者样本中的表观遗传沉默 多个表观遗传的累积效应。 沉默基因（ESG）对肿瘤发展的影响仍然是癌症生物学中尚未解决的一个方面，主要是 由于缺乏针对多个基因的工具、易于处理的生物模型和检测方法 在这项提案中，我们寻求利用我们目前在人类癌症方面的工作来填补这些空白。 我们进行这些研究的动机源于我们过去 3-4 年的工作。 我们通过致癌 BRAFV600E 模拟了人类结直肠癌 (CRC) 发展的早期阶段 我们发现，在小鼠近端结肠源性类器官中，可诱导 BrafV600E 和 KRASG12D 突变。 不是突变的 KrasG12D，导致 Wnt 通路激活、干细胞表型和转化。 转化类器官的外植体表型人类近端结直肠癌，包括粘液组织病理学 和 CGI​​ 超甲基化表型（CIMP） 在类器官中，与衰老的人类结肠中的类似，它通过诱导促进 BrafV600E 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 我们的研究与其他实验室的工作相结合，表明类器官是一个高度相关的模型。 此外，我们还建立了基于 Cas9 和 Cpf1 的表型特征。 筛选类器官以确定促进肿瘤发生的 ESG 丢失。 开发同时灭活多种 ESG 的工具，以研究它们在癌症中的综合作用。 就此而言，我们已经开发了基于诱导型 CRISPR 的多基因人类结肠源性类器官 我们将继续淘汰这些 开发具有诱导型 Cas9/Cpf1 的人类和小鼠结肠类器官系统，用于系统筛选 多个 ESG 的随机组合失活可赋予干细胞维持并阻止分化。 接下来我们将重点关注从癌症数据库中生物信息挖掘的重要候选基因，以研究 最后，我们将在体内应用并验证我们的研究。 总之，该提案将开发用于同时操作多个小鼠的通用工具。 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。