Dual ARID1A/ARID1B loss leads to rapid carcinogenesis and disruptive redistribution of BAF complexes

Abstract

SWI/SNF chromatin remodelers play critical roles in development and cancer. The causal links between SWI/SNF complex disassembly and carcinogenesis are obscured by redundancy between paralogous components. Canonical BAF (cBAF)-specific paralogs ARID1A and ARID1B are synthetic lethal in some contexts, but simultaneous mutations in both ARID1s are prevalent in cancer. To understand if and how cBAF abrogation causes cancer, we examined the physiological and biochemical consequences of ARID1A/ARID1B loss. In double-knockout liver and skin, aggressive carcinogenesis followed dedifferentiation and hyperproliferation. In double-mutant endometrial cancer, add-back of either induced senescence. Biochemically, residual cBAF subcomplexes resulting from loss of ARID1 scaffolding were unexpectedly found to disrupt a polybromo-containing BAF (pBAF) function. Of 69 mutations in the conserved scaffolding domains of ARID1 proteins observed in human cancer, 37 caused complex disassembly, partially explaining their mutation spectra. ARID1-less, cBAF-less states promote carcinogenesis across tissues, and suggest caution against paralog-directed therapies for ARID1-mutant cancer.

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Fig. 1: Loss of both ARID1A and ARID1B in the liver leads to impaired liver function, increased cell growth and tumor formation.
Fig. 2: Loss of ARID1A and ARID1B in the liver leads to hyperproliferation and dedifferentiation.
Fig. 3: Hyperproliferation and dedifferentiation are immediate effects after ARID1A/1B loss.
Fig. 4: UBC-CreER-induced DKO mice develop invasive SCCs.
Fig. 5: Loss of both ARID1A and ARID1B proteins in endometrial cancer cells is essential for transformation and proliferation.
Fig. 6: Residual cBAF complexes secondary to ARID1A/B loss do not possess neofunctionality.
Fig. 7: The presence of residual cBAF subcomplexes is associated with pBAF disruption.
Fig. 8: Biological consequences of 69 ARID1A/1B mutations in cancer.

Data availability

All sequencing data have been deposited in the Gene Expression Omnibus with the accession nos. GSE147664 for mRNA-seq and GSE140183 for ChIP–seq. Source data are provided with this paper. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank C. Kadoch, S. McBrayer, J. Wu, J. Xu, L. Banaszynski, X. Liu and S. Wang for constructive comments on the manuscript; C. Lewis and J. Shelton for histopathology; Proteomics Core at UTSW (A. Lemoff) for MS; and the CRI Sequencing Core (J. Xu) for genomics. Funding sources: NIH R03ES026397-01 (to T.W.), CPRIT RP150596 (to T.W.), CPRIT RP170267 (to H.Z.), NIH/NIDDK R01DK111588 (to H.Z.) and Stand Up To Cancer Innovative Research Grant (no. SU2C-AACR-IRG 10-16 to H.Z.).

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Authors

Contributions

Z.W. and H.Z. conceived the project, performed the experiments and wrote the manuscript. J-C.C., X.S., Z.W., L.L. and C.C. created and analyzed the mouse models. K.C., Y.J., X.S., F.H., X.L. and T.W. generated and analyzed genomic data. Y.-H.L. assisted with the histology analysis. D.H.C edited the manuscript and provided assistance with disease models.

Corresponding author

Correspondence to Hao Zhu.

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Competing interests

At the time of publication, H.Z. owned Ionis Pharmaceuticals stock worth less than $US10,000 and has active collaboration with Alnylam Pharmaceuticals and Twenty-Eight Seven Therapeutics. The remaining authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Loss of both ARID1A and ARID1B in the liver leads to impaired liver function.

a Kaplan-Meier survival curve of WT and DKO mice within the first month of life. b Body weight of WT and DKO mice at the age of 1 month (n = 12 and 8 mice). c-g. Liver function analysis using plasma (n = 6 and 5 mice for the Arid1af/f group; 6 and 6 for the Arid1bf/f group; 8 and 8 for the Arid1af/f; Arid1bf/f group). h Gross inspection of plasma from AKO, BKO and DKO and their corresponding WT mice. i IHC staining of ARID1A and ARID1B on WT and DKO liver sections. j Western blot showing ARID1A and ARID1B protein levels in WT and DKO livers. k Quantification of Western blot data in j (n = 6 and 6 mice for each group). Data are presented as mean ± s.d. (b-g,k). Statistical significance was determined by two-tailed unpaired Student’s t-tests with Welch’s correction (b-g, k). Source data

Extended Data Fig. 2 AAV mediated deletion of ARID1A and ARID1B in the liver leads to organ failure.

a Gross inspection of plasma from mice injected with AAV-GFP or AAV-Cre. b-f. Liver function tests of Arid1af/f; Arid1bf/f mice injected with AAV-GFP or AAV-Cre (n = 10 and 10 mice for AST; 9 and 10 for ALT; 9 and 11 for TBIL; 10 and 11 for ALKP; 9 and 11 for Albumin. Data are presented as mean ± s.d. Statistical significance was determined by two-tailed unpaired Student’s t-tests with Welch’s correction). g Representative genome browser tracks showing ARID1A binding to the promoter or enhancer regions of differentiation and Cytochrome P450 genes in liver. h IHC staining of EpCAM and CK-19 on AAV-Cre liver sections. Source data

Extended Data Fig. 3 cBAF subunit levels showed limited to no decrease in ARID1-less cells or DKO livers.

a Western blot analysis of cBAF subunit levels in WT and ARID1-less H2.35 cells (n = 3 and 3 independent clones). b Colony formation assay for control and ARID1-less H2.35 cells. 0.1 million H2.35 cells were seeded in each well of 6-well plate and cultured for 10 days in the presence of Dox. c Western blot analysis of cBAF subunit levels in WT and DKO livers (n = 6 and 6 mice). Same batch of western blots/protein samples as in Extended Data Fig. 1j. d Quantification of western blot data in c (Data are presented as mean ± s.d. Statistical significance was determined by two-tailed unpaired Student’s t-tests with Welch’s correction). Source data

Extended Data Fig. 4 ChIP-seq analysis of SWI/SNF complexes binding to genomic DNA in control and ARID1-less H2.35 cells.

a Expression of Ty1 tagged BRD9 and Brg1 in WT and ARID1-less H2.35 cells. BRD9 expression was only examined using the Ty1 antibody due to the lack of a commercial anti-mouse BRD9 antibody. b Heatmap displaying ChIP-seq peaks of intact SWI/SNF complexes in WT H2.35 cells. ARID1A, ARID1B, and BAF45d peaks were used to represent cBAF, ARID2 for pBAF, BRD9 for ncBAF, and Brg1 for all BAF complexes. 3000 bp upstream and downstream of peak centers are shown in this figure (n = 2 independent ChIP experiments for each protein). c Venn diagram showing the shared and unique binding loci among three types of BAF complexes from ChIP-seq data. d Comparison of BRD9 occupancies in control and ARID1-less cells. Heatmap and the corresponding averaged peak map and Venn diagram are shown (n = 2 and 2 independent ChIP experiments). e Representative genome browser tracks showing that ncBAF binding was unaffected in ARID1-less cells (BRD9 peaks in ARID1-less cells). Source data

Extended Data Fig. 5 Mapping of domains, residues, and mutations responsible for ARID1A’s scaffolding role.

a Multiple sequence alignment of ARID1A protein C-terminal regions from human, mouse, dog, bovine, rabbit, chicken, clawed frog, and zebrafish showing two conserved ARID1 scaffolding domains (ASD1 and ASD2). b Secondary structure prediction of ARID1A using LCR-eXXXplorer server. Regions with a score lower than 0.5 (shown as a cyan line for the IUPRED score and a red line for the ANCHOR score) are likely well-folded globular domains. c Alanine scans within ASD1 of ARID1A and IP experiments to assess residues for cBAF subunit interactions. The indicated two residues were mutated to alanine in each construct. d IP experiments showing the influence of ARID1A missense mutations within ASD2 on BAF subunit interactions. e Western blot showing the influence of ARID1A hotspot missense mutations on protein stability in H2.35 cells. f Western blot showing the influence of ARID1A truncations on protein stability in H2.35 cells. Source data

Supplementary information

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Supplementary Table

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Wang, Z., Chen, K., Jia, Y. et al. Dual ARID1A/ARID1B loss leads to rapid carcinogenesis and disruptive redistribution of BAF complexes. Nat Cancer 1, 909–922 (2020). https://doi.org/10.1038/s43018-020-00109-0

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