The prevailing approach to precision cancer medicine relies on genetic profiling of patients, followed by identification of the malignant gene product, and delineation of the mechanisms of that protein product in causing disease. As a result, much of the future of precision oncology is built on the hope of tailoring therapeutic interventions based on diagnostic technologies that acquire complex genomic and transcriptomic data. Despite the focus on cancer genetics, the unique functional capabilities acquired by normal cells during tumor development are driven by the aberrantly activated tumor cell proteome that arises not only from gene mutations but also from epigenetic reprogramming, post-translational alterations, or rewiring of signaling pathways. Unfortunately, integrating traditional measurements of protein biochemistry that reflect tumor cell biology and the therapeutics to which a tumor would respond into clinical decision-making for cancer patients is challenging due to the uniqueness of each protein and limitations in existing technologies. Thus, our proposal focuses on mechanism-based cancer research at the interface of chemistry and cancer biology to develop quantitative approaches that evaluate dynamic changes in the proteome in order to characterize unique features of tumor biology with the long-term goal of motivating novel targeted therapies. Specifically, we aim to establish an innovative new development and discovery platform termed Probe Enabled Activity Reporting (PEAR) for tumor proteome profiling by leveraging chemical biology approaches to understand the molecular complexity of proteomic changes necessary for tumor cell function, as well as cellular adaptations to cancer therapy. The foundation of our bedside-to-bench and back again approach is rooted in the hypothesis that novel chemical probe reactomes exist in cancer cells themselves and changes in the reactome profile in response to cancer therapeutics will reflect alterations in protein function that drive cancer cell adaptations and thus, would be ideal for new treatment modalities in the future. In interconnected and interdisciplinary discovery and elucidation modules, we will utilize state-of-the-art patient derived cancer models to both visualize and identify the protein targets of chemical biology probes in pre- and post-treatment with the hypothesis that the differential reactomes will be indicative of proteomic liabilities, therapeutic response, and unique aspects of tumor cell biology. The major outcomes from investing in PEAR for tumor proteome profiling to enable therapeutic development will be development of methodology to visualize reactive targets, identification of treatment induced reactive targets and establishing their functional relevance, and unraveling unique tumor cell biology based on a novel compartmentalized reactive target method. Taken together, our proposal will establish and validate novel concepts and methodologies that can be applied across...