Project summary: Essential regulation of the cellular machinery is achieved by a network of highly dynamic signaling molecules, which, when dysregulated, allow cancer cells to misinterpret or ignore signals that normally tell cells to stop dividing or begin apoptosis, leading to uncontrolled tumor growth. For the last 18 years, my laboratory has been at the forefront of applying a native biochemistry approach to cell signaling and cancer research. We have developed enabling technologies, and established spatiotemporal regulation as a fundamental paradigm in cell signaling, and discovered that its alteration leads to uncontrolled cell growth. This NCI R35-supported research program seeks to establish a new conceptual framework to specifically understand the cellular organization of molecular activities. We hypothesize that cellular biochemical activities are spatially organized into an “activity architecture” and dysregulated driver molecules can re-organize and re-structure this activity architecture, leading to loss of control over cell growth, division and death. In the past 6 years, we published 51 peer-reviewed articles, and made significant advances in establishing this framework. We developed first-in-class technologies for imaging protein-protein interactions and enzymatic activities in living cells at molecular length-scales and first kinase biosensor that achieved high- resolution imaging in awake mice, which have provided evidence for the biochemical activity architecture across different scales. We also made a breakthrough discovery that a regulatory subunit of Protein Kinase A (PKA), RIα, undergoes liquid-liquid phase-separation (LLPS) to enable the dynamic buffering and spatial compartmentalization of a ubiquitous second messenger, cAMP, providing an answer to a long standing question. We further showed that the oncogenic fusion in fibrolamellar carcinoma (FLC) potently inhibits RIα LLPS and induces aberrant cAMP signaling, which leads to increased cell proliferation and cell transformation. We have also discovered novel regulation in the Ras/ERK pathway and developed a novel Ras biosensor. In the proposed research, we will have three focuses. First, we will develop innovative technologies including super-resolution activity imaging to illuminate the biochemical activity architecture across different scales. Secondly, we will elucidate how the disorganized cAMP-PKA activity architecture leads to tumorigenesis in FLC, and further discover novel, cancer-relevant biomolecular condensates. Thirdly, we will investigate the spatiotemporal regulation of ERK that is critical for its physiological functions and identify the vulnerable connections in the re-organized cancer-driving architecture in pancreatic cancer, which is a deadly cancer that is addicted to the Ras-ERK pathway. We will also facilitate the development of new therapeutics by developing novel assays for evaluating Ras inhibitors and measuring target engagement.