Defining the function of SETD8 in colorectal cancer

项目摘要 结直肠癌(CRC)预计将成为2018年与癌症相关的死亡的第二大原因。 超过90%的结直肠癌肿瘤在规范的Wnt信号通路中包含突变，该突变解除了对 β-连环蛋白-Tcf转录活性和驱动致癌基因表达。事实上，经典的WNT-Target LGR5被建议标记CRC干细胞，这是原发和转移性肿瘤生长所必需的。此外 对于基因突变，结直肠癌具有表观遗传学特征的变化，包括全局DNA低甲基化， CpG岛高甲基化和组蛋白翻译后修饰的各种变化。尽管这些 染色质的改变被认为是结直肠癌发生的重要因素，即结直肠癌的致癌染色质因子。 在很大程度上仍未确定，阻碍了表观遗传过程在临床上的靶向。 为了解决这一知识差距，我进行了一项体外CRISPR-Cas9辍学筛查，使用的是 原代小鼠结直肠癌模型中染色质集中的慢病毒引导RNA(GRNA)文库。GRNAs靶向 Kmt5a(编码SETD8)，唯一的组蛋白4赖氨酸20(H4K20)单甲基转移酶，缺失 在此筛选过程中显著，表明该基因在结直肠癌增殖中的重要性。SETD8和 H4K20me1在转录调控、DNA损伤修复和细胞周期进程中发挥重要作用。 与结直肠癌特别相关的是，β-连环蛋白-Tcf转录需要SETD8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刺激下，SETD8直接相互作用 用β-连环蛋白-三氯甲烷络合物和沉积的H4K20单甲基(H4K20me1)修饰在规范的Wnt 靶基因。基于这些发现，我推测SETD8促进结直肠癌的发生部分是通过 与β-连环蛋白-Tcf协同增强典型的Wnt信号通路。为此，在目标1中，我将 通过调控SETD8在小鼠体内的表达探讨SETD8在体内结直肠癌发生中的作用 CRC有机化合物，并将这些细胞系原位植入同基因宿主。此外，我将在体外定义一种 SETD8抑制剂UNC-0379在原发正常和结直肠癌中的治疗指数 人类病人。在目标2中，我将阐明SETD8在β-连环蛋白-Tcf转录中的机制作用 通过操纵SETD8水平和通过转录组图谱测量规范的WNT靶标激活来实现活性 和功能报告分析，以及通过量化Lgr5+CRC干细胞的丰度和活性 活着。我还将定义β-连环蛋白、TCFLEF1和SETD8之间的蛋白质-蛋白质相互作用为 以及这些蛋白和H4K20me1在规范的Wnt靶基因座上的富集性。 这项研究将首次确定SETD8在结直肠癌发生中的作用。建议数 实验可能会找到一种有希望的治疗途径来对抗这种致命的疾病。

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.