Site-specific epigenetic activation of TP53 to improve cancer therapy

摘要 表观遗传途径的破坏是导致无数人类疾病的关键驱动机制。近期 染色质生物学和高通量基因测序的进展已经发现，这种干扰是 相当数量的癌症。虽然有助于癌症病理生理的受影响的表观遗传途径 不同的，其中一个共同的主题是，表观遗传酶的异常调节可以导致表达失调 关键的疾病驱动基因。例如，DNA甲基化异常、组蛋白H3甲基化和/或组蛋白H3 去乙酰化可以抑制基因，并导致更有害的疾病表型。事实上，广泛作用的泛表观遗传学 抑制剂在临床上已经显示出初步的前景，它通过改变表达来严重扰乱疾病信号通路。 关键基因图谱。然而，与这些传统表观遗传抑制中心的机制有关的一个基本问题 关于它们同时影响数千个基因的潜力(包括靶上和靶外)。因此，在用组蛋白治疗的同时 脱乙酰酶(HDAC)抑制剂会引起特定靶基因的激活，这种激活也是 潜在的成百上千个额外基因的非靶标激活。最近，技术进步 在CRISPR/Cas9系统上，已经开发出允许诱导位置特异的染色质修饰，该修饰可以 调节基因表达。我们的技术进步建立在这些方法的基础上，并允许 开发具有临床翻译潜力的有针对性的、基于表观遗传学的基因疗法。 在这里，我们概述了一种方法，它利用特定部位的dCas9-FKBP融合蛋白，与合成的 双功能化学表观遗传修饰剂(CEM)CEM由三个模块化组件组成：(1)FK506(绑定 FKBP)；(2)短惰性化学聚乙二醇链联体；以及(3)与宿主相互作用的化学实体 表观遗传机制。最终，dCas9-FKBP CEM有能力靶向基因组中的任何一个位点，并“激活” 调节基因表达的表观遗传活性。我们建议将这一策略应用于临床相关的靶点：TP53。 我们提出的技术旨在将内源性组蛋白乙酰化酶靶向TP53基因座，以逆转其表观遗传学 抑制，从而增加肿瘤细胞对化疗药物的敏感性。作为第一步，我们建议评估 我们在结直肠癌临床前模型方面的技术。结直肠癌是第三种最常见的癌症。 第三，癌症是导致男性和女性癌症相关死亡的最常见原因。此外，结直肠癌 已知由突变和表观遗传沉默的TP53驱动，并且有可用的临床前模型系统 这一总结是临床观察。从长远来看，我们的新平台可以代表一个创新和治愈的 治疗许多表观遗传驱动的人类癌症。

ABSTRACT The disruption of epigenetic pathways are key driving mechanisms that contribute to myriad human diseases. Recent advances in chromatin biology and high-throughput genetic sequencing have discovered that such disruptions underlie a substantial number of cancers. While the affected epigenetic pathways that contribute to cancer pathophysiology are quite diverse, a common theme among them is that aberrant regulation of epigenetic enzymes can lead to dysregulated expression of key disease driver genes. For example, aberrant DNA methylation, histone H3 methylation and/or histone H3 deacetylation can repress genes, and lead to a more deleterious disease phenotype. Indeed, broad-acting pan-epigenetic inhibitors have shown initial promise clinically by grossly disrupting disease signaling pathways by altering the expression profile of key genes. However, a fundamental issue related to the mechanism of these traditional epigenetic inhibitors centers on their potential to affect thousands of genes simultaneously (both on- and off-target). Thus, while treatment with histone deacetylase (HDAC) inhibitors causes the desired effect of activation of specific target genes, this activation also comes with off-target activation of potentially hundreds to thousands of additional genes. Recently, technological advancements on the CRISPR/Cas9 system have been developed to allow for induction of site-specific chromatin modifications that can modulate gene expression. Our technological advancements build on these approaches and allow for the possibility of developing targeted, epigenetically based gene therapeutics with real potential for clinical translation. Here, we outline an approach that leverages a site-specific dCas9-FKBP fusion protein, coupled with a synthetic bifunctional chemical epigenetic modifier (CEM). The CEM consists of three modular components: (1) FK506 (which binds FKBP); (2) a short inert chemical polyethylene glycol (PEG) linker; and (3) a chemical entity that interacts with host epigenetic machinery. Ultimately, the dCas9-FKBP CEM has the ability to target any locus in the genome, and “activate” epigenetic activity to modulate gene expression. We propose to implement this strategy to a clinically relevant target: TP53. Our proposed technology seeks to target endogenous histone acetylation enzymes to the TP53 locus to reverse its epigenetic repression, and thus increase the sensitivity of tumor cells to chemotherapeutic agents. As a first step, we propose to evaluate our technology in preclinical models of colorectal cancer. Colorectal cancer is the third most commonly diagnosed cancer and third cancer most common cause of cancer-related death among both men and women. Additionally, colorectal cancer is known to be driven by both mutated and epigenetically silenced TP53, and there are available preclinical model systems that recapitulate was is observed clinically. Long-term our novel platform could represent an innovative and curative treatment for many epigenetically driven human cancers.