Abstract
Chromatin remodeling complexes, such as the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, play key roles in regulating gene expression by modulating nucleosome positioning. The core subunit SMARCB1 is essential for these functions, as it anchors the complex to the nucleosome acidic patch, enabling effective chromatin remodeling. While biallelic inactivation of SMARCB1 is a hallmark of several aggressive pediatric malignancies, the functional implication of missense mutations is not fully understood. Current diagnostic approaches focus on detecting the presence or absence of SMARCB1 by immunohistochemistry often without consideration of mutation status. Here, we present a comprehensive deep mutational scanning of SMARCB1, encompassing 8418 alterations, to assess their functional impact. We show that RPT2 missense mutations disrupt SMARCB1 antiproliferation function by destabilizing the SWI/SNF complex and impairing chromatin remodeling and transcriptional regulation comparable to nonsense mutations. These functional defects occur despite maintaining detectable protein expression thereby challenging current diagnostic reliance on IHC. These findings provide deeper understanding of the role of SMARCB1 in chromatin remodeling and cancer biology, highlighting limitations of mutation classification approaches.
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Data availability
The plasmids generated in this study have been deposited in the Addgene database [https://www.addgene.org/Andrew_Hong/]. The SMARCB1 DMS library is available through the Broad Institute’s Genomic Perturbations Platform [https://www.broadinstitute.org/genetic-perturbation-platform]. The WGS, RNA-seq, ATAC-seq, and CUT&RUN data generated in this study have been deposited in the dbGaP database under accession code phs003896.v1.p1 [https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs003896.v1.p1]. These data are available under restricted access to protect patient privacy in accordance with the informed consent under which samples were collected. Access can be obtained by submitting a request through dbGaP [https://grants.nih.gov/policy-and-compliance/policy-topics/sharing-policies/accessing-data/dbgap]. The mass spectrometry data generated in this study has been deposited in the PRIDE database under the accession code PXD062226. The molecular dynamics simulation coordinates (initial and final configurations) generated in this study have been deposited in Zenodo and are available at https://doi.org/10.5281/zenodo.1892942958. The SMARCB1 protein structure used in this study is available in the UniProt database under accession code Q12824. Source data generated in this study are provided in the Supplementary Information/Source Data file. Source data are provided with this paper.
Code availability
All custom code used in this study is available on GitHub (https://github.com/thehonglab/SMARCB1_DMS) and archived on Zenodo (https://doi.org/10.5281/zenodo.18716542).
References
Mittal, P. & Roberts, C. W. M. The SWI/SNF complex in cancer—biology, biomarkers and therapy. Nat. Rev. Clin. Oncol. 17, 435–448 (2020).
Nakayama, R. T. et al. SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters. Nat. Genet. 49, 1613–1623 (2017).
Wang, X. et al. SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation. Nat. Genet. 49, 289–295 (2017).
Valencia, A. M. et al. Recurrent SMARCB1 mutations reveal a nucleosome acidic patch interaction site that potentiates mSWI/SNF complex chromatin remodeling. Cell 179, 1342–1356.e23 (2019).
Versteege, I. et al. Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394, 203–206 (1998).
Cooper, G. W. & Hong, A. L. SMARCB1-deficient cancers: novel molecular insights and therapeutic vulnerabilities. Cancers 14, 3645 (2022).
Parker, N. A., Al-Obaidi, A. & Deutsch, J. M. SMARCB1/INI1-deficient tumors of adulthood. F1000Research 9, 662 (2020).
Margol, A. S. & Judkins, A. R. Pathology and diagnosis of SMARCB1-deficient tumors. Cancer Genet. 207, 358–364 (2014).
Hoot, A. C., Russo, P., Judkins, A. R., Perlman, E. J. & Biegel, J. A. Immunohistochemical analysis of hSNF5/INI1 distinguishes renal and extra-renal malignant rhabdoid tumors from other pediatric soft tissue tumors. Am. J. Surg. Pathol. 28, 1485–1491 (2004).
Hong, S. H. et al. Nucleoporin 210 serves a key scaffold for SMARCB1 in liver cancer. Cancer Res 81, 356–370 (2021).
Wang, L. et al. High-throughput functional genetic and compound screens identify targets for senescence induction in cancer. Cell Rep. 21, 773–783 (2017).
Ahmadzadeh, A. et al. BRAF mutation in hairy cell leukemia. Oncol. Rev. 8, 253 (2014).
AACR Project GENIE Consortium AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discov. 7, 818–831 (2017).
Satish, S., Bone, M., Ritter, D. & Plon, S. E. Evaluating the consistency of SMARCB1 variant classification and assertions of genotype-phenotype relationships in ClinVar. Cancer Genet. 296–297, 84–87 (2025).
Fowler, D. M. & Attendees, C. M. 2024 Clinical Atlas of Variant Effects meeting summary. https://doi.org/10.5281/zenodo.13900941 (2024).
Giacomelli, A. O. et al. Mutational processes shape the landscape of TP53 mutations in human cancer. Nat. Genet. 50, 1381–1387 (2018).
Findlay, G. M. et al. Accurate classification of BRCA1 variants with saturation genome editing. Nature 562, 217–222 (2018).
Weng, C., Faure, A. J., Escobedo, A. & Lehner, B. The energetic and allosteric landscape for KRAS inhibition. Nature 626, 643–652 (2024).
Hayes, T. K. et al. Comprehensive mutational scanning of EGFR reveals TKI sensitivities of extracellular domain mutants. Nat. Commun. 15, 2742 (2024).
Hong, A. L. et al. Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition. eLife 8, e44161 (2019).
Mikhailenko, D. S. et al. Germline nonsense mutations in the SMARCB1 gene in Russian patients with rhabdoid renal tumors. Cancer Urol. 13, 14–19 (2017).
Borja, N. A., Schrier Vergano, S. A. & Tekin, M. Coffin-Siris syndrome and cancer susceptibility. Genet. Med. Open 1, 100818 (2023).
Cheng, J. et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science 381, eadg7492 (2023).
Ioannidis, N. M. et al. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. Am. J. Hum. Genet. 99, 877–885 (2016).
Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J. & Kircher, M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 47, D886–D894 (2019).
Mittal, K. et al. Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin. Commun. Biol. 7, 426 (2024).
Yang, X. et al. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software. bioRxiv 2021 06, 448102 (2021).
Cooper, G. thehonglab/SMARCB1_DMS: initial Release—SMARCB1 missense mutants disrupt SWI/SNF complex stability and remodeling activity. Zenodo https://doi.org/10.5281/ZENODO.18716542 (2026).
Allen, M. D., Freund, S. M. V., Zinzalla, G. & Bycroft, M. The SWI/SNF subunit INI1 contains an N-terminal winged helix DNA binding domain that is a target for mutations in schwannomatosis. Struct. Lond. Engl. 23, 1344–1349 (2015).
Hadfield, K. D. et al. Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis. J. Med. Genet. 45, 332–339 (2008).
Boyd, C. et al. Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis. Clin. Genet. 74, 358–366 (2008).
He, S. et al. Structure of nucleosome-bound human BAF complex. Science 367, 875–881 (2020).
Yuan, J., Chen, K., Zhang, W. & Chen, Z. Structure of human chromatin-remodelling PBAF complex bound to a nucleosome. Nature 605, 166–171 (2022).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Karczewski, K. J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581, 434–443 (2020).
Kadoch, C. & Crabtree, G. R. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Cell 153, 71–85 (2013).
Mashtalir, N. et al. A structural model of the endogenous human BAF complex informs disease mechanisms. Cell 183, 802–817.e24 (2020).
Dixit, U. et al. INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication. Nat. Commun. 12, 2743 (2021).
Tsuboyama, K. et al. Mega-scale experimental analysis of protein folding stability in biology and design. Nature 620, 434–444 (2023).
Marabotti, A., Scafuri, B. & Facchiano, A. Predicting the stability of mutant proteins by computational approaches: an overview. Brief. Bioinform. 22, bbaa074 (2021).
Randles, L. G. et al. Using model proteins to quantify the effects of pathogenic mutations in ig-like proteins *. J. Biol. Chem. 281, 24216–24226 (2006).
Liu, N. Q. et al. SMARCB1 loss activates patient-specific distal oncogenic enhancers in malignant rhabdoid tumors. Nat. Commun. 14, 7762 (2023).
Langer, L. F., Ward, J. M. & Archer, T. K. Tumor suppressor SMARCB1 suppresses super-enhancers to govern hESC lineage determination. eLife 8, e45672 (2019).
Mashtalir, N. et al. Modular organization and assembly of SWI/SNF family chromatin remodeling complexes. Cell 175, 1272–1288.e20 (2018).
Alver, B. H. et al. The SWI/SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers. Nat. Commun. 8, 14648 (2017).
Michel, B. C. et al. A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation. Nat. Cell Biol. 20, 1410–1420 (2018).
Kaelin, W. G. Common pitfalls in preclinical cancer target validation. Nat. Rev. Cancer 17, 441–450 (2017).
Yang, X. et al. A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8, 659–661 (2011).
Tan, K.-T. et al. Haplotype-resolved germline and somatic alterations in renal medullary carcinomas. Genome Med. 13, 114 (2021).
Melnikov, A., Rogov, P., Wang, L., Gnirke, A. & Mikkelsen, T. S. Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes. Nucleic Acids Res. 42, e112 (2014).
Soucek, S. et al. The evolutionarily-conserved polyadenosine RNA binding protein, Nab2, cooperates with splicing machinery to regulate the fate of pre-mRNA. Mol. Cell. Biol. 36, 2697–2714 (2016).
Seyfried, N. T. et al. A multi-network approach identifies protein-specific co-expression in asymptomatic and symptomatic Alzheimer’s disease. Cell Syst. 4, 60–72.e4 (2017).
Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6, e21856 (2017).
Meers, M. P., Tenenbaum, D. & Henikoff, S. Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics Chromatin 12, 42 (2019).
Case, D. A. et al. Recent developments in amber biomolecular simulations. J. Chem. Inf. Model. 65, 7835–7843 (2025).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–28 (1996).
Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).
Cooper, G. Molecular dynamics simulation coordinates for SMARCB1 missense variants: initial and final configurations. Zenodo https://doi.org/10.5281/ZENODO.18929429 (2026).
Acknowledgements
Research reported in this publication was supported by the following: NIGMS T32GM008490 (G.W.C.), NCI F31CA278008 (G.W.C.), NCI R01CA289761 (A.L.H.), DOD HT9425-23-0609 (Y.S.), DOD W81XWH1910281 (A.L.H.), ACS MRSG-18-202-01 (A.L.H.), R01-GM148586 (J.C.G), R01-HL168894 (K.N.C), R01-HD102534 (D.U.G). Wong Family Award in Translational Oncology (A.L.H.). Team Lick Cancer (S.N.C. and A.L.H.). Research reported in this publication was supported in part by the Emory University Emory Integrated Proteomics Core Facility (RRID:SCR_023530) and in part by the Emory Integrated Genomics Core (EIGC) (RRID:SCR_023529)—both shared resources of Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292. Additional support was provided by the Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health. The authors would like to acknowledge the American Association for Cancer Research and its financial and material support in the development of the AACR Project GENIE registry, as well as members of the consortium for their commitment to data sharing. Interpretations are the responsibility of the study authors. We thank the patients and their families for their participation. We thank Carlos Moreno, Roger Deal, and David Katz along with the Hong, Gorkin, and Spangle labs for their thoughtful comments and suggestions.
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G.W.C., S.N.C., A.L.H. designed the study. G.W.C. and A.L.H. wrote the manuscript. X.Y., R.E.L., F.P., A.O.G., T.P.H., A.L.H. developed the SMARCB1 DMS pools. W.J.K., A.L.H. performed the pooled screens. G.W.C., E.S., J.C.G. performed molecular dynamic studies. G.W.C., Y.S., V.Z.C. performed cellular experiments and growth assays. G.W.C. and P.B. performed LC-MS/MS experiments and mass-spec processing. G.W.C, B.P.L., and X.Y. performed computational analyses and interpreted results. K.N.C., D.E.R., B.L., W.C.H., D.U.G., J.A.B., S.N.C, and A.L.H. supervised the studies. All authors discussed the results and implications and edited the manuscript.
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G.W.C. completed an internship with GRAIL, Inc in Summer 2024. R.E.L. is currently employed by RAN Biotechnologies. F.P. is a current employee of Merck Research Laboratories. D.E.R. receives research funding from members of the Functional Genomics Consortium (Abbvie, BMS, Jannsen, and Merck), and is a director of Addgene, Inc. W.C.H. is a consultant for Thermo Fischer, Solasta Ventures, KSQ Therapeutics, Frontier Medicines, Jubilant Therapeutics, RAPPTA Therapeutics, Serinus Biosciences, Kestral Therapeutics, Crane Biotherapeutics, Function Oncology, Perceptive, Biotherapeutics, Function Oncology and Calyx. The remaining authors declare no competing interests.
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Cooper, G.W., Lee, B.P., Kim, W.J. et al. SMARCB1 missense mutants disrupt SWI/SNF complex stability and remodeling activity. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71531-8
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DOI: https://doi.org/10.1038/s41467-026-71531-8
