Defining the function of SETD8 in colorectal cancer

定义 SETD8 在结直肠癌中的功能

• 批准号: 10359781
• 负责人: ZVI Cramer
• 金额: $ 3.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359781
• 关键词: APC mutation; Address; Binding; Biological Assay; CRISPR/Cas technology; Cancer Etiology; Cancer Model; Candidate Disease Gene; Cell Cycle Progression; Cells; Cessation of life; Chromatin; Clinic; Co-Immunoprecipitations; Collaborations; Colonoscopy; Colorectal; Colorectal Cancer; Complex; CpG Islands; DNA; DNA Methylation; DNA Repair; DNA Sequence Alteration; Deposition; Disease; Dropout; Drops; Drosophila genus; Embryo; Engineering; Enzymes; Epigenetic Process; Epithelial; Event; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Guide RNA; Histones; Human; Human Cell Line; Hypermethylation; Implant; In Vitro; Injections; Internal Ribosome Entry Site; Intestines; KRAS2 gene; Kidney; Knowledge; LGR5 gene; Lysine; MADH4 gene; Maintenance; Malignant Neoplasms; Measures; Metastatic Carcinoma; Metastatic Neoplasm to the Liver; Metastatic to; Methyltransferase; Modeling; Modification; Mucous Membrane; Mus; Mutation; Neoplasm Metastasis; Normal Cell; Oncogenes; Oncogenic; Organoids; Patients; Play; Post-Translational Protein Processing; Primary Neoplasm; Process; Proteins; RNA library; Reporter; Research; Retrotransposon; Role; Shapes; TCF Transcription Factor; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic Index; Time; Transcriptional Activation; Ursidae Family; WNT Signaling Pathway; Zebrafish; base; beta catenin; cancer stem cell; cancer type; cell transformation; chromatin immunoprecipitation; colon cancer cell line; colorectal cancer progression; combat; experimental study; genetic regulatory protein; histone methyltransferase; histone modification; implantation; in vivo; inhibitor; knock-down; loss of function; mortality; mutant; novel; novel therapeutics; overexpression; pre-clinical; prognostic; promoter; protein function; protein protein interaction; recruit; small hairpin RNA; stemness; therapeutic development; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; tumor; tumor growth; tumorigenesis

Project Summary Colorectal cancer (CRC) is predicted to be the second leading cause of cancer-related deaths in 2018. Greater than 90% of CRC tumors contain a mutation in the canonical Wnt-signaling pathway, which deregulates β-CATENIN-TCF transcriptional activity and drives oncogenic gene expression. Indeed, the canonical Wnt-target LGR5 is proposed mark CRC stem cells, which are required for primary and metastatic tumor growth. In addition to genetic mutations, CRC tumors bear alterations in epigenetic features, including global DNA hypomethylation, CpG island hypermethylation and a variety of changes in histone post-translational modifications. Although these chromatin changes are thought to be important for CRC tumorigenesis, the oncogenic chromatin factors in CRC remain largely undefined, impeding targeting of epigenetic processes in the clinic. To address this gap in knowledge, I conducted an in vitro CRISPR-Cas9 dropout screen using a chromatin-focused pooled lentiviral guide RNA (gRNA) library in a primary murine CRC model. gRNAs targeting Kmt5a (encoding SETD8), the sole histone 4 lysine 20 (H4K20) monomethyltransferase, dropped out significantly during this screen, indicating the importance of this gene in CRC proliferation. SETD8 and H4K20me1 play important roles in regulating transcription, DNA damage repair and cell cycle progression. Particularly relevant for CRC, SETD8 methyltransferase activity is required for β-CATENIN-TCF transcriptional function in human cell lines, zebrafish and Drosophila. Indeed, upon Wnt stimulation, SETD8 directly interacts with β-CATENIN-TCF complexes and deposits H4K20 monomethyl (H4K20me1) modifications at canonical Wnt target genes. Based on these findings, I hypothesize that SETD8 promotes CRC tumorigenesis in part by cooperating with β-CATENIN-TCF to potentiate the canonical Wnt-signaling pathway. To this end, in Aim 1 I will investigate the importance of SETD8 in CRC tumorigenesis in vivo by manipulating SETD8 expression in murine CRC organoids and orthotopically implanting these lines into a syngeneic host. Moreover, I will define an in vitro therapeutic index for the SETD8 inhibitor UNC-0379 in pairs of primary normal and CRC organoids derived from human patients. In Aim 2 I will elucidate the mechanistic role of SETD8 in β-CATENIN-TCF transcriptional activity by manipulating SETD8 levels and measuring canonical Wnt-target activation by transcriptome profiling and functional reporter assays, as well as by quantifying the abundance and activity of Lgr5+ CRC stem cells in vivo. I will also define the protein-protein interactions between β-CATENIN, the TCF factor LEF1 and SETD8 as well as the enrichment of these proteins and H4K20me1 at canonical Wnt-target loci. This study will determine, for the first time, the role of SETD8 in CRC tumorigenesis. The proposed experiments will potentially identify a promising therapeutic avenue to combat this deadly disease.

项目摘要 结直肠癌（CRC）预计将成为2018年癌症相关死亡的第二大原因。 超过90%的CRC肿瘤在经典的Wnt信号通路中含有突变， β-catenin-TCF转录活性并驱动致癌基因表达。事实上，典型的Wnt-靶标 LGR 5被提议标记CRC干细胞，这是原发性和转移性肿瘤生长所需的。此外 对于基因突变，CRC肿瘤具有表观遗传特征的改变，包括整体DNA低甲基化， CpG岛超甲基化和组蛋白翻译后修饰的多种变化。虽然这些 染色质变化被认为是重要的CRC肿瘤发生，致癌染色质因子在CRC 仍然在很大程度上是不确定的，阻碍了在临床上靶向表观遗传过程。 为了解决这一知识差距，我使用一种新的CRISPR-Cas9缺失筛选方法进行了体外CRISPR-Cas9缺失筛选。 图10显示了在原代鼠CRC模型中的聚焦染色质的合并的慢病毒引导RNA（gRNA）文库。gRNA靶向 Kmt 5a（编码SETD 8），唯一的组蛋白4赖氨酸20（H4 K20）单甲基转移酶， 在该筛选过程中，该基因的表达显著增加，表明该基因在CRC增殖中的重要性。SET 8和 H4 K20 me 1在转录调控、DNA损伤修复和细胞周期进程中发挥重要作用。 尤其与CRC相关，β-连环蛋白-TCF转录需要SETD 8甲基转移酶活性 在人类细胞系、斑马鱼和果蝇中发挥作用。事实上，在Wnt刺激后，SETD 8直接与 与β-连环蛋白-TCF复合物结合，并在典型Wnt处沉积H4 K20单甲基（H4 K20 me 1）修饰 靶基因基于这些发现，我假设SETD 8促进CRC肿瘤发生的部分原因是 与β-catenin-TCF协同作用以增强经典Wnt信号通路。为此，在目标1中， 通过操纵小鼠中的SETD 8表达来研究SETD 8在体内CRC肿瘤发生中的重要性 CRC类器官并将这些细胞系原位植入同基因宿主中。此外，我将定义一个体外 SETD 8抑制剂SETD-0379在来源于大肠癌的原发性正常和CRC类器官对中的治疗指数 人类病人在目的2中，我将阐明SETD 8在β-catenin-TCF转录中的机制作用。 通过操纵SETD 8水平和通过转录组谱分析测量典型Wnt-靶标激活来测定活性 和功能性报告基因测定，以及通过定量Lgr 5 + CRC干细胞在大肠癌中的丰度和活性， vivo.我还将β-连环蛋白、TCF因子LEF 1和SETD 8之间的蛋白质-蛋白质相互作用定义为： 以及这些蛋白和H4 K20 me 1在典型Wnt靶基因座处的富集。 这项研究将首次确定SETD 8在CRC肿瘤发生中的作用。拟议 实验将有可能确定一种有希望的治疗方法来对抗这种致命的疾病。