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.