Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER+) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER+ breast cancer. An epigenome CRISPR–CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the ER degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER–FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER+ breast cancer.
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We thank the Center for Epigenetics Research at MSKCC for help with the ATAC-seq and ChIP-seq assays, and E. de Stanchina and the Antitumor Assessment Core Facility for help with xenografts establishment. We also thank the Baselga and Scaltriti laboratory members for helpful advice and discussions. We thank A. Del from the Department of Pathology at MSKCC for the procurement of the FFPE slides. This work has been supported by National Institutes of Health grant nos. P30 CA008748 and RO1CA190642-01A1, the Breast Cancer Research Foundation grant no. BCRF-17-013. E.T. and M. Scaltriti are supported by a kind gift from B. Smith. E.L. is supported by grant no. NCI K00CA212478. This work was also supported by grants from Stand Up to Cancer (Cancer Drug Combination Convergence Team) grant no. SU2C 2015-004, the V Foundation grant no. D2015-036 and the National Science Foundation grant no. PHY-1545853 (G.X. and M. Scaltriti). This work was also funded by a U54 award grant no. CA209975-01 to C.S.L. E.C. is a recipient of an MSK Society Scholar Prize.
M.Scaltriti has received research funds from Puma Biotechnology, Daiichi Sankyo, Immunomedics, TargImmune Therapeutics and Menarini Ricerche, is a cofounder of Medendi Medical Travel and is on the advisory board of Menarini Ricerche. C.K. is a scientific founder, fiduciary Board of Directors member, Scientific Advisory Board member, shareholder and consultant for Foghorn Therapeutics. R.L.L. is on the supervisory board of QIAGEN and is a scientific advisor to Loxo Oncology, Imago, C4 Therapeutics and Isoplexis, each including an equity interest. He receives research support from and consulted for Celgene and Roche, has received research support from Prelude Therapeutics and has consulted for Incyte, Novartis, MorphoSys and Janssen. He has received honoraria from Eli Lilly and Amgen for invited lectures and from Gilead Sciences for grant reviews. J.B. is an employee and shareholder of AstraZeneca, Board of Directors member of Foghorn Therapeutics and is a past board member of Varian Medical Systems, Bristol‐Myers Squibb, Grail, Aura Biosciences and Infinity Pharmaceuticals. He has performed consulting and/or advisory work for Grail, PMV Pharma, ApoGen Biotechnologies, Juno, Eli Lilly, Seragon Pharmaceuticals, Novartis and Northern Biologics. He has stock or other ownership interests in PMV Pharma, Grail, Juno, Varian Medical Systems, Foghorn Therapeutics, Aura Biosciences, Infinity Pharmaceuticals and ApoGen Biotechnologies, as well as Tango Therapeutics and Venthera, of which he is a cofounder. He has previously received honoraria or travel expenses from Roche, Novartis and Eli Lilly. P.R. has received consultation fees from Novartis and institutional research funds from Grail and Illumina. J.S.R. is a consultant of Goldman Sachs and Repare Therapeutics, a member of the Scientific Advisory Board of VolitionRx and Paige (Artificial Intelligence) and an ad hoc member of the Scientific Advisory Board of Ventana Medical Systems, Roche, Genentech, Novartis and InviCRO, outside of the scope of the submitted work. E.T. has received honoraria from AstraZeneca for invited lectures. No potential conflicts of interests were disclosed by the other authors.
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Extended Data Fig. 1 Enrichment of mutations of core subunits of the SWI/SNF complex in HR+ HER2- breast cancer.
(a) Mutation enrichment based on IMPACT study. (b) Mutation enrichment based on TCGA and METABRIC studies.
(a) In vitro proliferation of ARID1A knockout (KO) MCF7 cells as measured by cell quantification. (b) Cell cycle distributions as measured by FACS analyses of control and ARID1A KO MCF7 cells. Error bars=mean ±SEM, n=2 biologically independent samples, center values are means. P values were calculated using two-way ANOVA test; all P values > 0.2. N.S=non-significant. (c) Cell quantification of ARID1A KO vs. control cells upon fulvestrant treatment (100nM). (d) In vitro proliferation assay in ARID1A KO vs. control cells upon a dose response of the ER degrader GDC0927. The experiments were repeated thrice with similar results. (e) Cell quantification of ARID1A KO vs. control cells under estrogen (E2) depleted media vs. full media. (f) In vitro proliferation assay of ARID1A KO vs. control cells in estrogen depleted media and full media. The experiments were repeated thrice with similar results. (g) Cropped western blot of SMARCB1 or SMARCE1 KO (sg1-sg5) vs. control MCF7 cells. (h) In vitro proliferation assay in SMARCB1 or SMARCE1 KO vs. control MCF7 cells upon treatment with fulvestrant (100nM). The experiments were repeated three times with similar results. (i) The ratio of RFP+ SMARCB1 or SMARCE1 (sg1-sg5) knockout cells to GFP+ control cells (sgNT-GFP) upon DMSO or fulvestrant treatment (100nM) for 8 days as measured by flow cytometry. For (a), (c), (e), (i), error bars=mean ±SEM, n=3 biologically independent samples, center values are means. P values, Student’s two-sided t test. Source data
Extended Data Fig. 3 ARID1A knockout leads to equal chromatin accessibility changes in DMSO or fulvestrant setting.
(a) Cropped western blot with indicated antibodies in MCF7 cells. (b) Pie chart of peak distributions to various genic parts. (c) ATAC-seq analysis revealed 59,000 peaks in total; 33% in intergenic regions, ~30% in promoter regions, and 35% in intron regions. Violin plot shows probability density of peaks across the samples. (d) Heatmap of differential peaks in control vs. ARID1A KO (knockout) upon DMSO or fulvestrant (fulv) treatment (absolute log2 fold change > 0.5, Benjamini-Hochberg adjusted P < 0.05). (e) ChIP-qPCR analyses of TEAD4 binding in control and ARID1A KO MCF7. Error bars=mean ±SEM, n=2 biologically independent samples, center values are means. P values, Student’s two-sided t test. (f) Cropped western blot of TEAD4 in control and ARID1A KO MCF7. (g) Crystal violet assay of TEAD4 knockdown cells in control and ARID1A KO upon DMSO or fulvestrant (100nM). (h) Cropped western blot of GATA3 overexpression in MCF7 (n=3). (i) In vitro proliferation of GATA3 overexpressed cells in control and ARID1A KO setting upon DMSO or fulvestrant treatment (100nM). The experiments were repeated thrice with similar results. (j) Cell quantification of GATA3 overexpressed cells in control and ARID1A KO setting upon DMSO or fulvestrant treatment (100nM). Error bars=mean ±SEM, n=3 biologically independent samples, center values are means. P values, Student’s two-sided t test. (k) Learned coefficients of transcription factors motifs that gain or lose enrichment in control vs. ARID1A KO in DMSO or fulvestrant (n=15 samples). Source data
(a) Volcano plot; x-axis is log2 fold change and y-axis represents -log10(P); n=18 samples, statistical by DESeq2. (b) mRNA levels of luminal and basal-like/stemness markers in control and ARID1A KO cells. Error bars=mean ±SEM, n=2 biologically independent samples, center values are means. *P value<0.05, ** P value<0.01, Student’s two-sided t test. (c) Cropped western blot of indicated antibodies. (d) mRNA levels of aforementioned markers in MCF7 upon addition of doxycycline (DOX) knockdown of ARID1A. Error bars=mean ±SEM, n=2 biologically independent samples, center values are means. *P value<0.05, ** P value<0.01, Student’s two-sided t test. Also shown are ARID1A and vinculin levels. (e and f) Cropped western blot with indicated antibodies in BT474 or MDA-MB-361 cells expressing sgNT and two sgRNAs against ARID1A. (g and h) Enrichment of basal-like signatures in BT474 (Charafe breast cancer luminal vs. basal down) or MDA-MB-361 (Huper breast basal vs. luminal up) upon ARID1A KO. (n=6 per cell type, nominal P values and FDR adjusted P values were calculated using GSEA package.) (i and j) Enrichment of basal-like and estrogen response signatures in MCF7 after SMARCB1 or SMARCE1 knockout; n=8 per gene knockout, nominal P values and FDR adjusted P values were calculated using GSEA package. (k) Enrichment of basal-like signatures in ARID1A wild type vs. biallelic loss of ARID1A of patient sample pairs (*, FDR < 0.25;). n=2 for each patient pair, nominal P values and FDR adjusted P values were calculated using GSEA package). Source data
Extended Data Fig. 5 SWI/SNF binding to chromatin but not complex assembly is lost upon ARID1A loss.
(a) BAF155-2 and BRG1-2 at BAF155/BRG1 binding sites in control and ARID1A KO MCF7 cells (n=1). (b) Box plot representing mean signal across differential BAF155-2 or BRG1-2 after ARID1A KO at BAF155/BRG1 sites. (c) Cropped western blots of co-immunoprecipitation of BRG1 with subunits of the SWI/SNF complex in control and ARID1A KO MCF7. (d) Plot of the fold change between control and ARID1A KO of ATAC-seq sites vs. similar fold change of BAF155/BRG1 sites; n=14838 peaks, R and P values calculated using spearman correlation from ggpubr package in R. (e) BAF155-2 and BRG1-2 at differential accessible sites in control and ARID1A KO MCF7. (f) Box plot representing mean signal across differential BAF155-2 or BRG1-2 after ARID1A KO at lost accessible sites. (g) ChIP-qPCR analysis of ER, FOXA1, and GATA3 in shared loci in control and ARID1A KO cells. (h) ChIP-qPCR analysis of FOS, JUN, and IgG control. (i) ChIP-seq tracks of BRG1 and BAF155 in control and ARID1A KO cells (n=1). For (g) and (h), error bars=mean ±SEM, n=3 biologically independent samples, center values are means. P values, Student’s two-sided t test. For the box plots P-values, Mann-Whitney U test (Wilcoxon rank-sum test, two-sided) and effect size (rosenthal’s coefficient) are shown. The log2FC which is calculated as log2 (mean KO / mean Control) is also indicated (n=6). Box shows 25th, median and 75th percentiles with whiskers extending to ± 1.5 * IQR. Source data
(a) ECDF plot of log2 fold changes in gene expression between ARID1A knockout and control for genes nearest to the TSS-distal SWI/SNF binding sites at GRHL1, FOXA1, FOS, JUN, GATA3, and ER motifs loci. P values were measured by the Mann-Whitney U test (Wilcoxon rank-sum test, two-sided) and effect size (rosenthal’s coefficients. The log2FC (fold change) values which are calculated as log2 (mean KO / mean Control) are also indicated (n=9). (b) Expression of ER canonical targets in control and ARID1A knockout MCF7 cells. Error bars=mean ±SEM, n=3 biologically independent samples, center values are means. P values, Student’s two-sided t test. (c) Cropped western blot of AR+ TNBC breast cancer cells BT549 and HCC70 with the indicated antibodies. (d) and (e) Cropped western blot with the indicated antibodies of control and ARID1A knockout BT549 or HCC70. (f) and (g) GSEA of androgen response in BT549 and HCC70 after ARID1A knockout; n=8 for each cell line, nominal P values and FDR adjusted P values were calculated using GSEA package. Source data
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Xu, G., Chhangawala, S., Cocco, E. et al. ARID1A determines luminal identity and therapeutic response in estrogen-receptor-positive breast cancer. Nat Genet 52, 198–207 (2020). https://doi.org/10.1038/s41588-019-0554-0