Organoid modeling to determine and reverse age-related epigenetic changes contributing to risk of colorectal cancer

用于确定和逆转导致结直肠癌风险的年龄相关表观遗传变化的类器官建模

• 批准号: 10206053
• 负责人: STEPHEN B. BAYLIN
• 金额: $ 24.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206053
• 关键词: APC mutation; Address; Affect; Age; Aging; Animal Testing; Automobile Driving; Azacitidine; BRAF gene; Biological Assay; Biological Markers; CRISPR/Cas technology; Cells; Characteristics; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Colon; Colon Carcinoma; Colonic Polyps; Colorectal Cancer; CpG Islands; DNA; DNA Methylation; Data; Dependence; Development; Distal; Engineering; Epigenetic Process; Evolution; Feedback; Frequencies; Gene Expression; Gene Silencing; Genes; Germ Lines; Germ-Line Mutation; Growth; Histologic; Human; Hypermethylation; In Vitro; Individual; Inflammatory; Inflammatory Bowel Diseases; Intercept; Intestines; KRAS oncogenesis; KRAS2 gene; Left; Malignant Neoplasms; Mediating; Methylation; Modeling; Monitor; Mucinous; Mus; Mutation; Oncogenic; Organoids; Pathway interactions; Patients; Pattern; Pharmacology; Phenotype; Population; Predisposition; Prevention; Process; Repression; Risk; Risk Factors; Role; Side; Standardization; Syndrome; System; Testing; Time; WNT Signaling Pathway; Work; age group; age related; aged; cancer initiation; cancer predisposition; cancer risk; cancer subtypes; cancer therapy; cell transformation; colorectal cancer prevention; colorectal cancer progression; colorectal cancer risk; complement pathway; disorder risk; driver mutation; epigenetic therapy; high risk; in vivo; independency; insight; mouse model; mutant; normal aging; novel; novel strategies; polyposis; pre-clinical; prevent; promoter; stem; stem cells; tumor; tumor progression; tumorigenesis

There is great need to develop systems in which the course for initiation and progression of cancer, and therapies directed at these steps can be studied directly in mouse and human cells, and over time courses relevant to real world tumorigenesis. While engineered mouse models are valuable, there is a need to develop complementary systems where meaningful, relatively rapid in-vitro work can yield substantial pre-clinical insights leading to final animal testing. In this regard, we have developed an organoid model which is yielding important data for studying the age-related risks for developing colorectal cancer (CRC). The studies build on extensive data, now just in press, derived from mouse organoids from normal proximal colon wherein we have modeled key steps in the initiation and progression of CRC. The key findings are that despite the organoids being genetically stable over time, they evolve an abnormal, gene promoter CpG island phenotype (CIMP) during long periods of growth. This pattern is very similar to changes seen with aging in normal human colon and which parallels the increasing CRC risk with age. CIMP involves repression of associated gene expression, and/or perhaps even more importantly affects inducibility of genes which otherwise normally function in a feedback fashion to blunt abnormal WNT and other stem pathway activation, prevent abnormal retention of cell renewal, and induce differentiation. In this context, induction of Braf mutations in the older versus younger organoids result in a much more rapid evolution of autonomous Wnt pathway activation, stem cell versus differentiation features and one-step transformation by oncogenic Braf. The resulting cancers have typical histologic features and the epigenetic abnormality of CIMP resembling oncogenic BRAF-driven human proximal colon CRCs. Critically, CRISPR- mediated simultaneous inactivation of multiple CIMP target genes in young organoids rapidly converts these to the old organoid phenotype, importantly resulting in rapid one-step transformation by oncogenic Braf. In the current proposal, we will determine the role of genes affected by age-related CIMP in driving human CRC in the context of KRAS, BRAF and APC mutations, thus addressing the epigenetic dependency of ~75% of CRC evolution. This includes extrapolating our studies to human colon organoids. Importantly, our unique models provide a novel setting to test whether epigenetic therapies can alter the above evolution of CIMP to suppress age-related changes, which may otherwise enhance CRC risk. Results from these studies may allow insights for strategies to prevent and/or intercept CRC evolution. All of the work in our proposal has potential to define management strategies for CRC prevention and interception, which could prove especially valuable for decreasing CRC risk in individuals harboring familial germ line predisposition to colon polyps and for patients with inflammatory bowel diseases.

非常需要开发系统，其中癌症的发生和进展过程以及治疗方法 针对这些步骤可以直接在小鼠和人类细胞中进行研究，并且随着时间的推移，课程与真实的相关 世界肿瘤发生。虽然工程小鼠模型很有价值，但仍需要开发互补的 有意义的、相对快速的体外工作可以产生大量的临床前见解，从而产生最终结果的系统 动物测试。在这方面，我们开发了一种类器官模型，为研究提供了重要数据 与年龄相关的结直肠癌 (CRC) 风险。这些研究建立在广泛的数据基础上，现在刚刚 Press，源自正常近端结肠的小鼠类器官，其中我们模拟了 CRC 的发生和进展。主要发现是，尽管类器官在过去很长时间内都具有遗传稳定性 随着时间的推移，它们在长期的生长过程中进化出异常的基因启动子 CpG 岛表型 (CIMP)。这 模式与正常人结肠随衰老而发生的变化非常相似，并且与增加的 CRC 风险随年龄增长。 CIMP 涉及抑制相关基因表达，和/或可能更多 重要的是影响基因的诱导能力，否则这些基因通常以反馈方式发挥作用，以消除异常 WNT 和其他干通路激活，防止细胞更新的异常保留，并诱导分化。 在这种情况下，与年轻的类器官相比，在较老的类器官中诱导 Braf 突变会导致更快的 自主 Wnt 通路激活的进化、干细胞与分化特征和一步 致癌性 Braf 的转化。由此产生的癌症具有典型的组织学特征和表观遗传特征 CIMP 异常类似于致癌 BRAF 驱动的人类近端结肠 CRC。至关重要的是，CRISPR- 介导年轻类器官中多个 CIMP 靶基因的同时失活，可将这些基因迅速转化为 旧的类器官表型，重要的是导致致癌 Braf 的快速一步转化。在 目前的提案中，我们将确定受年龄相关 CIMP 影响的基因在驱动人类 CRC 中的作用 KRAS、BRAF 和 APC 突变的背景，从而解决约 75% CRC 的表观遗传依赖性 进化。这包括将我们的研究外推到人类结肠类器官。重要的是，我们独特的模型 提供了一种新的环境来测试表观遗传疗法是否可以改变 CIMP 的上述进化以抑制 年龄相关的变化，否则可能会增加结直肠癌的风险。这些研究的结果可能会为以下方面提供见解： 防止和/或拦截 CRC 演变的策略。 我们提案中的所有工作都有可能定义 CRC 预防和治疗的管理策略。 拦截，这对于降低携带家族病菌的个体的结直肠癌风险特别有价值 结肠息肉和炎症性肠病患者的倾向。