Abstract 986: <i>SMARCB1</i> missense mutants destabilize SWI/SNF complex stability and remodeling activity

SMARCB1-deficient cancers are aggressive and highly lethal pediatric malignancies. Loss of SMARCB1 protein expression, a subunit within the SWI/SNF chromatin remodeling complex, remains the key diagnostic feature of these cancers. This can occur through large deletions, balanced translocations, frameshift mutations, or truncating nonsense mutations. Here, we sought to understand the effect of missense mutations on the tumor suppressor function of SMARCB1 through deep mutational scanning (DMS). Specifically, we developed and introduced a library containing &amp;gt;99% of all possible SMARCB1 amino acid substitutions, including frameshift and nonsense mutants, into three pediatric SMARCB1-deficient cell lines (G401 - malignant rhabdoid tumor of the kidney, BT16 - atypical teratoid/rhabdoid tumor, and CCLF_PEDS9001_T1 - renal medullary carcinoma) and assessed cell fitness after 8-14 days. We observed broad mutational intolerance in three SMARCB1 domains: the winged-helix domain, intrinsically disordered region, and the RPT2 domain. Following our high-throughput study, we then focused on two highly enriched residues predicted to closely interact within the RPT2 domain of SMARCB1. We validated that specific missense mutations in these two residues mimic loss of function while retaining protein expression. Mechanistic studies revealed that these mutations destabilize the SWI/SNF complex, notably resulting in decreased affinity for SWI/SNF subunits known to be associated with cancer pathogenesis. This complex instability leads to diminished nucleosome remodeling and subsequent transcriptional deregulation. These findings challenge our current understanding of what a loss-of-function mutation means in the context of SMARCB1, suggesting that the absence of SMARCB1 protein expression may not be the sole indicator of SMARCB1 deficiency. Furthermore, this dataset provides a valuable resource for researchers to investigate key residues of SMARCB1 that may drive critical intermolecular interactions necessary for proper SWI/SNF complex assembly and function. Citation Format: Garrett W. Cooper, Benjamin Lee, Won Kim, Eliseo Salas, Yongdong Su, Victor Chen, Xiaoping Yang, Robert Lintner, Frederica Piccioni, Andrew Giacomelli, Thomas Howard, Karen Conneely, David Root, William Hahn, David Gorkin, Bo Liang, Jaclyn Biegel, Susan Chi, Andrew Hong. SMARCB1 missense mutants destabilize SWI/SNF complex stability and remodeling activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 986.