Mutational activation of RAS occurs in approximately 30% of human tumors with some cancers such as pancreatic cancer having RAS mutations in >90% of tumors. Although mutational activation of RAS shifts RAS to the GTP-bound state, a number of oncogenic RAS proteins are characterized by high intrinsic nucleotide dissociation rates. Thus, these RAS mutants, termed “fast-exchange” mutants, transiently release GTP and transit through the nucleotide-free (apo) state. Following on our previous MERIT award that resulted in development of high affinity tool biologics (Monobodies) that specifically and selectively bind apoRAS, we have made a paradigm shifting discovery that these apo-specific Monobodies function as potent inhibitors of fast- exchange, oncogenic RAS mutants. Preliminary studies suggest that these Monobodies inhibit both the signaling and oncogenic activity of fast exchange but not slow exchange RAS mutants. Using these powerful tool biologics, we will: 1. determine the selectivity of these apoRAS Monobodies toward various RAS mutants and RAS family members; 2. assess the viability and selectivity of targeting apoRAS as a strategy to inhibit RAS-mediated signaling and oncogenic transformation in vitro; 3. determine the efficacy of targeting apoRAS in vivo as a therapeutic strategy for treating RAS-mutant pancreatic cancers; and 4. develop our apoRAS- specific Monobodies into a preclinical anti-RAS therapeutic. Given the prevalence of RAS mutations in human cancers and the importance of RAS in driving cancer development and progression, particularly pancreatic cancers, it is critical to develop new approaches to therapeutically inhibit RAS in patients. Although decades of research in the RAS field has resulted in a dearth of FDA-approved pharmacological inhibitors, recent pioneering work has renewed hope in finally developing an anti-RAS therapeutic. Several companies have launched clinical trials of drugs that specifically target the RAS(G12C) mutant protein based on the thiol reactivity of the G12C mutation. Although initial results are promising, these inhibitors will only be useful in G12C-mutant tumors which constitute roughly15% of all RAS-mutant cancers and less than 2% of pancreatic tumors. Our findings, however, would be applicable to more than >50% of RAS mutant cancers including more than 45% of RAS-mutant pancreatic cancers. Thus, our proposed studies in this MERIT renewal will provide critical insights into a novel approach to inhibit RAS-mediated tumorigenesis and are thus likely to have broad translation significance. More importantly, this work will be beneficial to Veterans as well as the general population, both of which suffer from cancer. However, the incidence for RAS mutant cancers, such as lung and pancreatic cancer, has been reported to be up to 5-7x higher in Veteran populations making these studies particularly relevant to the health of our Veterans.