Isoform-Selective Targeting of RAF Kinases with Synthetic Binding Proteins - This project aims to develop first-in-class isoform-selective synthetic binding proteins against RAF kinases (A/B/CRAF) as a strategy for precise disruption of RAF-mediated signaling in cancers. Protein-protein interactions (PPIs) are central to signal transduction, and their dysregulation can drive diseases like cancer. Whereas modulating PPIs is a promising therapeutic concept, traditional small molecule drugs struggle to target PPI interfaces with sufficient affinity and specificity. Hence, new approaches for drugging PPIs are needed. I propose that monobody, a high-performance synthetic binding protein with established workflows for intracellular usage, is an effective tool for targeting PPIs. Using RAS-RAF as an archetype, I critically evaluate our approach to disrupt PPIs. The RAS-RAF PPI of the MAPK pathway is a key cancer driver that remains difficult to target. RAS mutations frequently occur in the deadliest cancers, yet current FDA-approved RAS inhibitors target only ~14% of RAS-driven cancers and quickly lead to resistance. Over a decade ago, genetic ablation identified CRAF as a therapeutic target for KRAS-driven lung cancer, yet there still are no drugs that selectively inhibit CRAF. Clinically approved RAF kinase inhibitors (RAFKi) cannot treat RAS-driven cancers because RAFKi paradoxically activate RAS-mediated RAF signaling. These RAFKi also lack isoform specificity, hampering precise biochemical modulation of A/B/CRAF signaling—especially concerning for studying ARAF, an emerging yet understudied player in RAS-driven oncogenesis. These challenges highlight the unmet need for isoform- selective RAF inhibitors that block RAS signaling. The engagement of the RAS-Binding Domain (RBD) with RAS is required for RAS-mediated activation of RAF. I hypothesize that I can develop monobodies that selectively disrupt the RAS-RBD interaction in an isoform-specific manner and that they can inhibit RAF functions. In preliminary work, I have developed monobodies that differentiate the RBDs of A, B, and CRAF, demonstrating the feasibility of achieving RAF isoform selectivity. Remarkably, these monobodies disrupted the RAS-RAF PPI in cells. Based on these strong data, I propose: Aim 1, in which I build upon these RAF-targeting monobodies by improving their affinities and further characterizing their specificity. I also determine the structural basis of RAF- RBD isoform selectivity, which could inform future efforts to target the RBD. Aim 2, in which I investigate how isoform-selective, biochemical inhibition of the RAS-RAF PPIs impacts proliferation, apoptosis and signaling in cancer cells. I also develop cell-deliverable RAF-targeting monobody “therapeutics”. This project establishes novel tools for precise dissection of RAF-mediated signaling and to advance PPI-targeting drug discovery, paving the way for novel therapies in cancer and beyond. This project also has multidisciplinary training potential in protein engineering, structural biology, cell signaling, and therapeutic development, facilitating my development into an independent academic physician scientist in medical oncology.
