SNF5 (SMARCB1/INI1/BAF47) was the first subunit of the SWI/SNF chromatin remodeling complex linked to cancer when it was found to be specifically mutated in virtually all cases of the highly aggressive pediatric cancer malignant rhabdoid tumor and in a familial cancer predisposition syndrome. Mouse models subsequently established that conditional inactivation of SNF5 results in the rapid development of cancer in 100% of mice. Broad relevance to human malignancy has recently emerged as genome sequencing studies have revealed that 20% of all human cancers carry mutations in genes encoding SWI/SNF subunits. During the current funding cycle, we discovered that the genomes of SNF5-deficient human tumors are remarkably simple, suggesting that the effects of SNF5 loss are epigenetic in nature. Along these lines, we discovered a role for SNF5 in establishing nucleosome occupancy at promoters; discovered epigenetic antagonism between SNF5 and the Polycomb complexes; identified the Cyclin D1/CDK4, Hedgehog, and Wnt/ß-catenin pathways as targets of its tumor suppressor activity; and translated our CDK4 findings into a clinical trial However, major questions remain. Why is there little correlation between SNF5-mediated nucleosome positioning at promoters and changes in gene expression? What is the biochemical mechanism by which SNF5 contributes to the function of the SWI/SNF complex? Also, given that we have demonstrated that SNF5 loss leads to specific genetic dependencies, can we systematically identify vulnerabilities created by SNF5 mutation? We now have substantial preliminary data that begin to address these questions. We have generated evidence for scaffolding and enhancer-targeting roles for SNF5 as well as for a key role of SNF5 in controlling acetylation of H3K27. Based upon our preliminary findings, we hypothesize that a central function of SNF5 is to target the SWI/SNF complex to lineage-specific enhancers and super-enhancers, where it modulates nucleosome position and facilitates H3K27 acetylation to activate transcription. We further hypothesize that SNF5 loss drives cancer due to an impaired ability of SNF5-deficient cells to execute lineage-specific differentiation programs. Using our genetically engineered loss-of-function murine model systems and our gain-of-function systems in which we reintroduce SNF5 into SNF5-deficient cancer cell lines, we will define the contributions of SNF5 to control of nucleosome occupancy and H3K27 acetylation at enhancers and super-enhancers. We will also use these models to characterize roles for SNF5 in the control of lineage-specific transcriptional regulation. Lastly, building upon our recent successes in identifying vulnerabilities in cancer cell lines mutant for other SWI/SNF subunits, we will systematically identify genetic vulnerabilities created by SNF5 loss. Relevance: Mutations of the SWI/SNF complex occur in 20% of all human cancers. Our proposed studies are designed to define the mechanism by which mutation of the core SWI/SNF subunit SNF5 drives cancer and to identify genetic vulnerabilities conferred by SNF5 loss, which represent potential therapeutic targets.