Project Summary/Abstract: SMARCB1 (SNF5/INI1/BAF47) is a conserved subunit of SWI/SNF chromatin remodeling complexes, which utilize the energy of ATP hydrolysis to mobilize nucleosomes. A role for SWI/SNF complexes in cancer was first suggested when SMARCB1 was identified as inactivated in virtually all cases of malignant rhabdoid tumor (RT), a highly aggressive pediatric cancer. It has recently been discovered that nearly 25% of all human malignancies carry mutations in SWI/SNF subunits. We established SMARCB1 to be a bona fide and potent tumor suppressor and later demonstrated broad epigenetic antagonism between SWI/SNF and Polycomb repressor complexes during oncogenic transformation. In subsequent work, including substantial progress in the current funding period, we uncovered novel biological mechanisms and therapeutic vulnerabilities that form the foundation for the aims described in this proposal. For example, we found that SMARCB1 is essential for enhancer regulation, thus identifying a new mechanism underlying the tumor-suppressive activity of SMARCB1. We and others also discovered the existence of a SWI/SNF complex that lacks SMARCB1, previously considered a core subunit. Instead, this non-canonical complex contains the bromodomain protein BRD9, which we also identified as a specific vulnerability in SMARCB1-deficient pediatric RTs. While our findings demonstrate critical interactions of SWI/SNF and Polycomb complexes in transcriptional regulation and cell fate control, the roles and contributions of SWI/SNF sub-families are unknown. Our first aim is therefore to determine how SMARCB1-containing SWI/SNF complexes differ from mutually-exclusive BRD9- containing SWI/SNF complexes in the antagonism of Polycomb interactions and transcriptional regulation. Recent data from our group have shown that SWI/SNF complexes control histone acetylation and enhancer function; however, the mechanism by which this leads to a mitotically heritable cancer phenotype is unknown. Polycomb complexes remain bound to DNA during mitosis, but histone acetylation is erased and the SWI/SNF ATPase, SMARCA4/BRG1 is phosphorylated and degraded. Thus, for our second aim, we propose to determine how SWI/SNF complexes contribute to epigenetic memory during mitosis, including interrogating a potentially paradigm-shifting model that is supported by preliminary data described in this proposal. Finally, we identified the Polycomb subunit EZH2 as a vulnerability in RTs, inspiring the development of several clinical trials and leading to recent FDA approval of EZH2 inhibitors for SMARCB1-deficient sarcomas. While impactful, resistance to these inhibitors has emerged. To establish mechanisms driving drug resistance, we have performed a near genome-wide CRISPR screen in EZH2 inhibitor-treated RTs, and for our third aim plan to investigate the mechanism of resistance conferred by mutations in a gene not previously associated with SWI/SNF functions. Taken together, these questions ...