The goal of this proposal is to apply cutting edge protein engineering technology for helping us understand how to directly inhibit oncogenic RAS mutants and the consequences of such inhibition in RAS-driven cancer cells. RAS is a small membrane bound GTPase that acts as a binary switch for regulating cell proliferation and survival. Mutation, particularly in residue 12 and 13, of the RAS family gene disrupts the de-activation of RAS, and leads to RAS oncogenesis. There is no drug known to date that can selectively target the active state of RAS mutant, thus RAS has earned the reputation of being “undruggable”. Due to the lack of mutant-specific RAS inhibitors, how effective mutant-selective inhibition will be, and how tumor cells will adapt to mutant- selective inhibition are major unanswered questions in RAS cancer biology. To address the dire need for mutant-selective RAS inhibitors, I will engineer mutant-selective binders using the “monobody” technology. Monobody is a synthetic binding protein platform that has been consistently shown to be capable of generating selective binders against difficult targets. I propose the following Aims to accomplish this overall goal: In Aim 1 I will generate potent and highly specific monobodies to KRAS mutants (G12C, G12D, G12V, G13D) frequently found in pancreatic, lung, and colorectal cancer, and validate their specificity. I will be trained in the use of molecular display technology (phage and yeast surface display) for binder development, and advanced protein engineering methods. I have recently generated a monobody that is highly selective to the active state of the G12C mutant, demonstrating the feasibility of this approach. In Aim 2 I will elucidate the underlying molecular mechanism for mutant-specific recognition. Structural based understanding of mutant-selectivity can potentially accelerate the development of direct inhibitor against RAS function, which will significantly contribute to a major goal of NCI’s RAS Initiative ("help discover small molecules that bind to RAS directly"). I will define the epitopes of monobodies using known RAS effectors. To elucidate the structural basis for mutant-selective recognition in atomic resolution, I will perform x-ray crystallography on monobody-RAS complexes. I have obtained preliminary crystals for such a complex. I will be trained in advanced structural biology technologies. In Aim 3, I will examine the effect of mutant-selective inhibition on RAS-driven cancer cells by examining the short and long-term responses. I will introduce mutant-selective monobodies as genetically encoded inhibitors in RAS-driven tumor cell lines, and quantify alterations in various pathways of RAS signaling, cell proliferation, apoptosis, and tumor adaptation. I will be trained in RAS signaling and cancer cell biology. Together results from the proposed project will shed light on the effectiveness of mutant-selective inhibition against RAS-driven cancers, and reveal potential adaptation that tumor cells can undergo in order to offset the therapeutic effect of mutant-selective inhibition. The proposed training will prepare me as a well-rounded biomedical scientist with strong emphasis in protein engineering and cancer biology.

Project Narrative: Mutations in RAS family genes are found in more than 25% of all human cancers, and currently there is no clinically approved drug that can directly inhibit RAS function. In this study, I will utilize cutting edge protein engineering technology to design inhibitors that will serve as tools for uncovering how to selectively target RAS mutants that are commonly found in RAS-driven cancers. I will also use the mutant- selective inhibitors to evaluate the consequences of mutant-selective inhibition in RAS-driven cancer cells, and this will tell us whether mutant-selective inhibition is truly the “silver bullet” at stopping RAS-driven cancers. !