(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 - 需要开发工具以简单操纵人类中的多个基因 与癌症相关的模型 - 因为该领域的主要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协同作用以影响肿瘤发育的过程。为了 表观遗传学影响的基因，我们表明多个重要的肿瘤抑制剂和发育 调节基因通过启动子CpG-Island（CGI）DNA保持永久沉默的状态 各种癌症类型的高甲基化。重要的是，来自相同生物学途径的许多基因是 在同一患者样本中表观遗传沉默。多个表观遗传学的累积效应 沉默的基因（ESG）在肿瘤发育中仍然是癌症生物学的未解决方面，主要是 由于缺乏靶向多个基因的工具，可进行易于测量的生物模型和测定法 肿瘤发育。在此提案中，我们试图通过利用我们目前在人类癌症中的工作来填补这些空白 相关模型。我们追求这些研究的动机源于过去3 - 4年的工作 我们已经建模了人类结直肠癌（CRC）发育的早期阶段 和Krasg12d突变。我们表明，在小鼠近端结肠衍生的器官中，诱导Brafv600e，但 不是突变的Krasg12d，导致Wnt途径激活，干细胞表型和转化。瘤 转化的类器官的外植体，近端CRC，包括粘液组织病理学 和CGI高甲基表型（CIMP）。我们表明，CGI高甲基化的衰老样获得 在类器官中，与衰老的人类结肠不同，它促进了诱导的BRAFV600E介导的肿瘤发生 早期Wnt途径激活和干细胞表型，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 人类肿瘤进展的表副膜特征。此外，我们建立了基于CAS9和CPF1的 在器官中筛选以鉴定促进肿瘤发生的ESG丧失。该项目的主要目标是 开发用于简单失活多个ESG的工具，以研究其在取消器中的综合角色。在这个 考虑到，我们为多个基因开发了基于诱导的人类结肠衍生的类器官 敲除研究ESG和癌症驱动器突变之间的勾结。我们将继续通过 用诱导的CAS9/CPF1开发人和小鼠结肠器官系统，用于系统筛选 多种ESG的随机组合失活，这些ESG会遇到干细胞维护并阻止分化。 接下来，我们将重点关注重要的候选基因，生物信息挖掘的癌症数据库，以研究 他们在癌症倡议中同时丧失的协同作用。最后，我们将在体内应用并验证我们的研究 鼠标模型。总而言之，该建议将开发通用工具，以简单地操纵多个 与人类癌症相关的模型中的基因可以轻松地转化为其他癌症模型。