Suppressing oncogenic RNA regulons using engineered zinc finger ribonucleases

基因表达在癌症中广泛重编程。这些失调的基因包括 RNA调节子是由转录后机制协调调节的， RNA水平和控制肿瘤侵袭性的关键特征。为了理解 癌症中的RNA调节子失调，这个探索性的，高风险/高回报的R21提案的目标是 开发一种锌指导向的RNA切割剂，以抑制RNA调节子，这些调节子在许多 肿瘤的我们的原型将来自tristetraprolin（TTP）的串联锌指（TZF）结构域连接到 核糖核酸内切酶4（R4）。在细胞中，预期嵌合TZF-R4蛋白结合并快速降解 TTP的mRNA底物，但我们的设计将允许底物特异性进行系统性修改。的TTP TZF结构域被选择用于我们的原型，因为它已被进化选择为靶向RNA 含有富含AU的元件（战神）的调节子，其包括许多编码细胞调节因子的mRNA 周期、血管生成和转移。此外，TTP的表达或活性经常被抑制， 人类癌症;特别是乳腺肿瘤中的低TTP水平与患者预后不良相关。 该提案旨在提供“概念证明”，即TZF-R4嵌合体可以作为指导性的免疫调节剂发挥作用。 癌细胞中的RNA降解系统抑制促肿瘤发生RNA调节子并减弱相关的 肿瘤细胞表型纯化的TZF-R4在体外显示选择性RNA识别和切割， 当瞬时转染到细胞中时，抑制两种已知的TTP底物mRNA。此外，TZF-R4 当在克隆的、稳定转染的乳腺癌细胞系中表达时，显著减缓细胞增殖。 在这些主要初步成果的基础上，将努力实现两个具体目标。首先，我们将使用转录组- 广泛的方法来定义RNA调节子，其在稳定时被TZF-R4靶向和去稳定化 在侵袭性乳腺癌细胞模型中表达，并证明TZF-R4抑制多种mRNA 比目前的技术更有效。其次，我们将评估TZF-R4对乳腺癌的影响。 癌细胞增殖、干性、侵袭、迁移和体内肿瘤发展。 设想了该技术的若干未来应用，包括：（i）用于以下的发现工具： 表征RNA介导的生物途径，（ii）开发用于促进纯化的 TZF-R4进入细胞，绕过转染并打开直接体内施用这种药物的可能性。 试剂，（iii）恢复或增强TTP功能以抑制肿瘤侵袭性或炎性 通过改变TZF-R4平台的RNA靶向特异性来扩展TZF-R4平台的特异性。 拓宽范围的策略包括TZF部分的迭代或组合修饰， 取代其他RNA结合结构域以“引导”嵌合蛋白，产生可调的靶向蛋白家族。 具有长期影响的核糖核酸酶。

Gene expression is extensively reprogrammed in cancer. These dysregulated genes include subpopulations called RNA regulons that are coordinately regulated by posttranscriptional mechanisms at the RNA level and control key features of tumor aggressiveness. To understand the molecular consequences of dysregulated RNA regulons in cancer, the goal of this exploratory, high-risk/high-reward R21 proposal is to develop a zinc finger-directed RNA-cleaving agent to suppress RNA regulons that are upregulated in many tumors. Our prototype links the tandem zinc finger (TZF) domain from tristetraprolin (TTP) to the endoribonuclease RNase4 (R4). In cells, the chimeric TZF-R4 protein is expected to bind and rapidly degrade mRNA substrates of TTP, but our design will allow substrate specificity to be systematically modified. The TTP TZF domain was chosen for our prototype because it has been evolutionarily selected to target an RNA regulon containing AU-rich elements (AREs), which includes many mRNAs that encode regulators of the cell cycle, angiogenesis, and metastasis. Furthermore, TTP expression or activity is frequently suppressed in human cancers; in particular, low TTP levels in breast tumors are associated with poor patient prognosis. This proposal is aimed at providing the “proof of concept” that a TZF-R4 chimera can function as a guided RNA degradation system in cancer cells to suppress a pro-tumorigenic RNA regulon and attenuate associated tumor cell phenotypes. Purified TZF-R4 shows selective RNA recognition and cleavage in vitro, and suppressed two known TTP substrate mRNAs when transiently transfected into cells. Furthermore, TZF-R4 dramatically slows cell proliferation when expressed in a clonal, stably-transfected breast cancer cell line. Building upon these key preliminary results, two specific aims will be pursued. First, we will use transcriptome- wide approaches to define the RNA regulon that is targeted and destabilized by TZF-R4 when stably expressed in aggressive breast cancer cell models and demonstrate that TZF-R4 suppresses multiple mRNA targets more efficiently than current technologies. Second, we will assess the impact of TZF-R4 on breast cancer cell proliferation, stemness, invasion, migration, and in vivo tumor development. Several future applications of this technology are envisioned, including: (i) discovery tools for characterizing RNA-mediated biological pathways, (ii) developing methods for promoting uptake of purified TZF-R4 into cells, bypassing transfection and opening possibilities for direct in vivo administration of this reagent, (iii) restoration or augmentation of TTP function to suppress tumor aggressiveness or inflammatory signaling, and (iv) expanding the specificity of the TZF-R4 platform by altering its RNA-targeting specificity. Strategies to broaden the scope include the iterative or combinatorial modification of the TZF moiety and substitution of other RNA-binding domains to `guide' the chimeric protein, creating a tunable family of targeted ribonucleases with long-term impact.