The role of DPF2 stability in SMARCB1-deficient cancers - PROJECT SUMMARY Rhabdoid tumors are one of the most aggressive and lethal cancers in pediatric oncology with overall 5-year survival rates of ~20-25%. Loss of SMARCB1 is the primary recurrent genetic alteration found in over 90% of cases. Recent advances have implicated SMARCB1 loss in a number of other cancers broadly referred to as SMARCB1-deficient cancers. SMARCB1 is a critical component of the BAF chromatin remodeling complex, a complex which controls gene transcription by positioning nucleosomes at gene regulatory regions. Targeted localization of the BAF complex to enhancers has largely been attributed to SMARCB1, yet no domain has been identified on SMARCB1 that is able to recognize and bind specific histone marks. Instead SMARCB1 is only able to nonspecifically bind to nucleosomes. This raises a key question: how does the BAF complex recognize regulatory regions such as enhancers and bind. Recent work has shown that deletion of SMARCB1 depletes an adjacent BAF subunit, DPF2, which is able to recognize and bind histone marks associated with enhancers. Re- expression of SMARCB1 in a SMARCB1-deficient cell line leads to a robust accumulation of DPF2. The histone reader protein, DPF2, has previously been implicated in the recruitment of the BAF complex to regulatory regions. Through deep mutational scanning of SMARCB1, we have identified four missense mutations in SMARCB1 that seem to mimic an oncogenic loss of function nonsense mutation. These residues are predicted to directly interact with DPF2, which suggests SMARCB1 mediates its tumor suppressor role at least partially through its interaction with DPF2. Moreover, we have identified a novel tumor suppressor role of DPF2 in the context of constitutive overexpression in two SMARCB1-deficient cancers cell lines. For this reason, we hypothesize that depletion of DPF2 is required in SMARCB1-deficient cancers. In Aim 1, I will test the hypothesis that reduced DPF2 association with the BAF complex is driving this observed loss of function phenotype by assessing the phenotypic, structural, and regulatory effects of each proposed SMARCB1 mutant. In Aim 2, I will test the hypothesis that stabilization of DPF2 may be a promising therapeutic strategy to restore transcription normalcy despite the loss of SMARCB1. In this aim I will characterize the phenotypic, structural, and regulatory consequences of DPF2 overexpression in SMARCB1-deficent cell lines and determine if this is occurring through either a BAF-dependent or BAF-independent mechanism. Together these aims will provide fundamental insight into the mechanisms through which SMARCB1 exerts its tumor suppressor function, as well as expand our understanding of DPF2 in SMARCB1-deficient cancers as a potential therapeutic target.
