Project Summary/Abstract The field of cancer research and treatment has greatly advanced due to breakthroughs in DNA sequencing techniques. These developments have accelerated the identification of dependency genes and paved the way for precision medicine. Many encoded dependency proteins, however, are not known to bind endogenous small molecules and are consequently more difficult to gauge in terms of their potential for targeting by chemical probes. "Binding-first" platforms including Activity-Based Protein Profiling (ABPP) technology have revealed numerous cysteine sites amenable to small-molecule binding. However, it is unclear whether these ligandable sites are important for protein functions and could serve as starting points for small molecule drug development. To address this challenge, I have recently innovated a base editing - ABPP platform that assesses the essentiality of ligandable cysteines in cancer dependency proteins. Notably, I have uncovered several highly ligandable and functionally important sites in dependency proteins, including TOE1. In this proposal, I will utilize biochemistry, sequencing and chemical proteomic methods to 1) dissect the mechanisms of how covalent probes affect TOE1 activity and remodel RNA interactions and 2) explore the consequences of TOE1 inhibition and determinants of sensitivity in cancer. I will also describe plans for continued technology innovation to 3) identify novel functional cysteines in cancer dependency proteins using prime editing. In the long-term, I aspire to create first-in-class therapeutics targeting ligandable cysteines in Strongly Selective cancer dependencies. During the award period, I will improve my communication, writing and scientific skills and learn new lab techniques including eCLIP sequencing and prime editing. The proposed studies will be performed at the Scripps Research Institute (TSRI), a top-ranking institution renowned for its integration of state-of-the-art chemistry and biomedicine research. My mentor, Dr. Benjamin Cravatt, a leading figure in chemical biology and chemical proteomics, and co-mentors Dr. Gene Yeo, a pioneer in RNA-binding protein research, and Dr. David Liu, a leader in the field of genome editing, will provide me invaluable guidance. I will also collaborate with molecular biologist Dr. Lykke-Andersen and medical oncologist Dr. Park at UCSD. Collectively, this research and professional development plan will offer me crucial training during this transitional phase and support me in launching my own laboratory in a research university.

Project Narrative This research program aims to use innovative technologies (chemical proteomic and prime editing) to discover and functionally characterize new sites for drug action in oncogenic proteins. We have already identified novel druggable proteins involved in RNA processing in cancer cells and our platform will reveal more druggable cancer dependencies for therapeutic development.