PROJECT ABSTRACT. Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) the predominant histologic subtype of lung cancer and lung adenocarcinoma the major subset of NSCLC. ALK gene rearrangements (e.g., EML4-ALK fusions) are validated targets in NSCLC and current ALK kinase inhibitors yield impressive responses. Despite this clinical progress drug resistance remains a problem that limits patient survival. Improved therapeutic strategies are critical to identify to improve clinical outcomes. We propose an innovative, multidisciplinary, and collaborative project to hopefully improve the survival of NSCLC patients by defining a new mechanism of oncogenic signaling that we uncovered by studying ALK fusion oncoproteins. We aim to capitalize on our discovery of membraneless cytoplasmic protein granules (condensates) as a distinct mechanism of oncogenic kinase signaling in cancer. Our data suggest an emerging paradigm in which certain ALK fusion oncoproteins, as well as other clinically-relevant oncoprotein kinase fusions such as RET fusions, form de novo their own phase separated protein-based subcellular compartment devoid of lipid membranes and utilize higher-order protein assembly as distinguishing principles underlying oncogenic output. These membraneless cytoplasmic protein granules comprise a mode of oncogenic signaling that is different from that of native receptor tyrosine kinase (RTK) signaling and oncogenic, mutant forms of other RTKs such as EGFR, which use classical lipid membrane-based signaling. The pathogenic biomolecular condensates formed by ALK (and other RTK) fusion oncoproteins locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in cells. We identified a set of protein granule signaling components and established structural rules that define ALK protein granule formation. For instance, protein granule formation requires the adaptor proteins GRB2 and SHC, in addition to the ALK fusion oncoprotein. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling in cancer. We propose 2 complementary Specific Aims using innovative methodologies to probe condensate biology to understand the role of phase separation in ALK fusion oncogenic signaling. We further define the protein architecture of ALK fusion protein granules and identify the key interacting proteins required for ALK fusion protein granule formation, oncogenic signaling and tumor growth. The proposed studies will establish a mechanistic understanding of RTK fusion condensate biology to lay a firm foundation for the future design of mechanism-based therapeutic strategies to interfere with ALK protein granule assembly per se and that complement conventional ALK-targeted clinical agents, which ...