Project Summary/Abstract: A role for SWI/SNF complexes in cancer was first suggested when SMARCB1 was identified as inactivated in virtually all malignant rhabdoid tumor (RT) cases, a highly aggressive pediatric cancer. It is now clear that mutations in genes encoding subunits of SWI/SNF (BAF) chromatin-remodeling complexes are frequent, collectively occurring in over 20% of all cancers. Our group established SMARCB1 as a bona fide and potent tumor suppressor. We later made high-impact discoveries that helped define mechanisms by which SWI/SNF mutations lead to dysregulated cell proliferation. Our findings suggest a model whereby SWI/SNF- facilitated control of transcription underlies cellular fate specification, with disruption of this control being the basis for cancer formation. Our long-term goals are to elucidate the function of SWI/SNF complexes, to determine how their loss leads to oncogenesis, and to translate this knowledge into novel therapies. Based on our findings, we hypothesized that loss of SMARCB1, while driving cancer growth, also creates unique vulnerabilities. To identify such vulnerabilities, we collaborated with the Pediatric Dependencies Project to perform a rigorous genome-wide CRISPR deletion screen involving 21 RT cell lines compared to over 800 other cancer cell lines. Notably, rhabdoid tumors are typically diploid and contain few other mutations, making them a powerful model with which to study the effects of gene deletions. We have now identified and validated a novel gene that is specifically essential for rhabdoid cell survival from this screen. Our preliminary data reveal that this gene's product, previously only linked to gene repression, has unanticipated connections with SWI/SNF and transcriptional activity. We localize this protein to enhancers and promoters of genes bound by SWI/SNF and hypothesize that this protein performs a key regulatory role in determining whether activating SWI/SNF complexes or repressive complexes are recruited, thus serving a pivotal role in dictating cell fate. We previously demonstrated that SWI/SNF and another repressive protein, EZH2, serve antagonistic roles in chromatin regulation, a discovery that ultimately led to FDA approval of an EZH2 inhibitor (EZH2i) for SMARCB1-mutant cancers. Despite this success, the mechanisms that control the interplay between these complexes are poorly understood, as are the mechanisms that underlie resistance to EZH2i in SWI/SNF-mutant cancers. To establish mechanisms driving drug resistance, we have performed a near genome-wide CRISPR screen in EZH2 inhibitor-treated RTs. We previously identified NSD1 as a gene that, when inactivated, causes resistance to EZH2i. Here, we plan to investigate the mechanism of resistance conferred by mutations in a second chromatin regulatory gene not previously associated with SWI/SNF functions. Together, these experiments have the potential for broad impact including understanding the roles of these critical chrom...