Probing RAS-mediated Signaling with Monobody Inhibitors

项目摘要 癌症是美国和世界范围内的主要死亡原因。这一创新的多PI项目已进入第六个阶段 去年建立了一种新的方法来应对发现癌症控制策略的挑战。致癌作用 Ras家族GTP酶的激活在约30%的癌症中发生，使其成为最常见的突变 人类癌症中的癌基因。尽管我们对RAS和RAS的生物化学的了解取得了令人印象深刻的进展 它在过去30年中在肿瘤发生中的作用以及第一个被批准的直接 针对特定的致癌RAS突变体，针对RAS的有效治疗方法的开发仍然是一件大事 挑战。我们率先使用一体式技术来定义以前未识别的漏洞 在RAS。单体体是一种小的人工结合蛋白，它能达到亲和力和选择性水平 靶标类似于抗体，可用作生化、结构、细胞和体内研究的工具生物制品。 在目前的项目期内，我们已经开发并使用了两个单体，NS1和R15，以获得新的见解 RAS的功能和漏洞。Ns1揭示了α4-α5接口在RAS中的重要性 二聚化。R15揭示了靶向致癌肿瘤亚集的无核苷酸(Apo)状态的可行性。 RAS突变体，尽管传统观点认为一个人不能有效地与紧密捆绑的人竞争 RAS中的核苷酸。此外，我们已经确定了发展单一机构的可行性，该机构 非共价选择性地抑制致癌RAS突变体和选择性降解RAS突变体 单体-VHL融合。在这些成功和强大的初步数据的基础上，该项目的下一阶段 目标是实现以下目标：1、我们将利用NS1、R15和其他单体作为高度选择性 微扰剂用于解决RAS生物学中的重要机械问题，包括二聚化/自我调节的作用 RAS效应器激活中的关联，野生型KRAS在杂合KRAS突变细胞中的作用，以及 KRAS4A在肿瘤发生中的作用2、我们将使用基因编码建立细胞系和小鼠模型 单体法确定特定的RAS抑制模式如何影响致癌驱动的体内肿瘤形成 RAS突变体。3、我们将开发具有新的特异性轮廓的单体，以扩大我们的项目范围， 特别是对NRAS、KRAS4A和常见的RAS突变具有选择性的那些。这个项目的结果将是 促进我们在生化、细胞和体内水平上对RAS功能的机械性理解并提供信息 直接针对RAS的治疗学的发展。此外，在此基础上开发的独特强大的工具 该项目将增强整个RAS社区的能力。

Project Summary Cancer is a leading cause of death in the United States and worldwide. This innovative multi-PI project in its 6th year has established a novel approach to the challenge of discovering strategies to control cancer. Oncogenic activation of the RAS family of GTPases occurs in ~30% of cancers making it the most frequently mutated oncogene in human cancers. Despite impressive progress in our understanding of the biochemistry of RAS and its role in tumorigenesis over the past 3 decades and the excitement of the first approved drug that directly targets a particular oncogenic RAS mutant, development of effective therapeutics targeting RAS remains a grand challenge. We have pioneered the use of monobody technology to define previously unrecognized vulnerabilities in RAS. Monobodies are small synthetic binding proteins that achieve levels of affinity and selectivity for their target similar to antibodies and can be used as tool biologics in biochemical, structural, cellular and in vivo studies. In the current project period, we have developed and used two monobodies, NS1 and R15, to gain new insights into RAS function and vulnerabilities. NS1 revealed the importance of the α4-α5 interface implicated in RAS dimerization. R15 revealed the feasibility of targeting the nucleotide-free (apo) state of a subset of oncogenic RAS mutants, despite the conventional wisdom that one cannot effectively compete against tightly bound nucleotides in RAS. Furthermore, we have established the feasibility of developing monobodies that noncovalently and selectively inhibit oncogenic RAS mutants and of selectively degrading RAS mutants using monobody-VHL fusions. Building on these successes and strong preliminary data, the next phase of this project aims to accomplish the following: 1, We will utilize NS1, R15 and additional monobodies as highly selective perturbants to address important mechanistic questions in RAS biology, including roles of dimerization/self- association in RAS effector activation, roles of wild-type KRAS in heterozygous KRAS mutant cells, and roles of KRAS4A in tumorigenesis. 2, We will establish cell lines and mouse models using genetically encoded monobodies to determine how specific modes of RAS inhibition affect tumorigenesis in vivo driven by oncogenic RAS mutants. 3, We will develop monobodies with new specificity profiles to expand the scope of our project, specifically those selective to NRAS, KRAS4A, and common RAS mutations. Results from this project will advance our mechanistic understanding of RAS function at the biochemical, cellular and in vivo levels and inform the development of therapeutics directly targeting RAS. Furthermore, uniquely powerful tools developed in this project will empower the entire RAS community.