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。最近 染色质生物学和高通量基因测序的进展已经发现，这种破坏是 大量的癌症。虽然影响癌症病理生理学的表观遗传途径相当复杂， 尽管这些研究多种多样，但其中一个共同的主题是表观遗传酶的异常调节可导致表达失调 关键的疾病驱动基因。例如，异常的DNA甲基化、组蛋白H3甲基化和/或组蛋白H3甲基化可导致异常的DNA甲基化。 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 抑制剂在临床上已经显示出初步的前景，通过改变表达来严重破坏疾病信号传导途径， 关键基因图谱然而，与这些传统的表观遗传抑制剂中心的机制有关的一个根本问题是， 它们同时影响数千个基因的潜力（包括靶基因和脱靶基因）。因此，虽然用组蛋白治疗 脱乙酰酶（HDAC）抑制剂引起特定靶基因的活化的期望效果，这种活化也伴随着 可能有数百到数千个额外基因的脱靶激活。最近，技术进步 已经开发了CRISPR/Cas9系统上的基因修饰，以允许诱导位点特异性染色质修饰， 调节基因表达。我们的技术进步建立在这些方法的基础上， 开发具有真实的临床转化潜力的靶向、基于表观遗传学的基因疗法。 在这里，我们概述了一种利用位点特异性dCas 9-FKBP融合蛋白，结合合成的 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）FK 506（其结合 FKBP）;（2）短的惰性化学聚乙二醇（PEG）接头;和（3）与宿主相互作用的化学实体 表观遗传机制最终，dCas 9-FKBP CEM能够靶向基因组中的任何基因座，并“激活” 表观遗传活性调节基因表达。我们建议将该策略应用于临床相关目标：TP 53。 我们提出的技术旨在将内源性组蛋白乙酰化酶靶向TP 53基因座，以逆转其表观遗传 抑制，从而增加肿瘤细胞对化疗剂的敏感性。作为第一步，我们建议评估 我们的技术应用于结直肠癌的临床前模型。结肠直肠癌是第三个最常见的诊断癌症 第三大癌症是男性和女性癌症相关死亡的最常见原因。此外，结肠直肠癌 已知由突变的和表观遗传学沉默的TP 53驱动，并且存在可用的临床前模型系统 临床上观察到了这一重演。从长远来看，我们的新平台可以代表一种创新和治疗 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.