ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response

Abstract

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.

Extended Data Fig. 1. Enrichment of BAF and P-BAF components in the CRISPR screen

a. Scatterplot of CRISPR screening data, showing enrichment of BAF components following 26 days of different drug treatment, relative to DMSO treated control cells. n= 3 independent viral infections. b. Log2 fold changes showing gRNA enrichment/depletion against all BAF, P-BAF and ncBAF components in the CRISPR screen. Treatment conditions are compared to DMSO control. More proliferative changes represent enriched gRNA after treatment, indicating genes that contribute to drug resistance. c, e. Validation of ARID1A perturbation effect on proliferation and drug response using ARID1A siRNA on MCF7 (c) and ZR-75-1 (e), representative experiments shown from 2 similar independent experiments each cell line. p-values calculated by One way ANOVA test. * denote p < 0.05, *** denotes p < 0.001. Sample size mentioned in S4. Measure of centre represents mean ± SEM (c) and mean ± SD (e). d. Western blot of ARID1A protein levels after siRNA transfection in MCF7 cells. A representative image is shown from 3 similar independent experiments. Unprocessed Western blot in Source Data Fig. 2.

Extended Data Fig. 2. ARID1A co-binds ER and FOXA1-bound regulatory elements, but is depleted with estrogen treatment.

a-c. Single gene profiles showing the binding of ER, FOXA1 and ARID1A on overlapping sites in MCF7 cells. ChIP-seq was performed using three independent biological cell cultures. d. Overlap of binding sites for ER, FOXA1 and ARID1A binding sites in ZR-75-1 cells. e. Boxplots showing the normalized ChIP-seq tag density around 400 bp window around the center of ARID1A binding on DiffBind-defined estrogen independent (constant) and dependent (reduced with estrogen) sites in MCF7. Both classes show reduced ARID1A binding upon estrogen. p-values were calculated by Welch’s t-test, two-sided. Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). Statistical test details are mentioned in Supplementary Table 5e.

Extended Data Fig. 3. Enrichment of SWI/SNF factors with ER and FOXA1 in RIME

a. ARID1A and BRG1 RIME were conducted on asynchronous MCF7 cells on two biological cell cultures. Label free quantification was performed to show the log 2 scaled normalized intensities of the BAF, P-BAF, ncBAF and common subunits of SWI/SNF complex. Rabbit polyclonal IgG is used as the negative control. b. ER qPLEX-RIME was performed on five primary tumours from ER+ breast cancer patients and the ER interactors are shown as enrichment over IgG vs -log10 p-value, corrected by Benjamini and Hochberg multiplicity correction, two-sided. c, d. Boxplots illustrating the more enrichment of HDAC1 (c) and less enrichment of random factors (d) in ERα RIME in five patients compared to IgG negative control in human breast tumours. The values are scaled to the median of IgG and log2 transformed. e. Boxplots illustrating the enrichment of selected known ERα interactors from the RIME experiment in MCF7 cells at a representative timepoint (4-hydroxytamoxifen- 24 hrs) comparing to IgG negative control. The values are scaled to the median of IgG and log2 transformed. n = 5 independent biological cell cultures. For all boxplots, Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range).

Extended Data Fig. 4. Enrichment of SWI/SNF factors during Tamoxifen and Fulvestrant in ChIP-seq experiments

a-d. Asynchronous MCF7 cells were treated with vehicle or Fulvestrant, an ER degrader and ChIP-seq was conducted for ARID1A (b), BRG1 (c) or SNF5 (d). Triplicate independent cell cultures were conducted. d. Single gene profile showing the induction of SWI/SNF complex binding during Fulvestrant treatment. e. Overlap of ARID1A lost sites during estrogen treatment with gained sites during Tamoxifen and Fulvestrant from three independent biological cell cultures. f. Overlap of ARID1A gained sites during Tamoxifen treatment with Fulvestrant and Tamoxifen downregulated genes.

Extended Data Fig. 5. FOXA1 promotes the binding of ARID1A and BRG1.

Hormone-deprived ZR-75-1 cells were transfected with control or FOXA1 siRNA and ChIP-seq was conducted for ARID1A (a) and BRG1 (b). n = 3 independent biological cell cultures. MA plots are shown with the average intensity of binding vs log2 fold change with FOXA1 siRNA relative to control siRNA. c. Scatterplot showing the association of the loss of ARID1A and BRG1 binding upon FOXA1 knockdown. PCC – Pearson Correlation coefficient, two-sided. d. Heatmaps shown on ARID1A and BRG1 FOXA1 independent (common) and dependent (lost sites with FOXA1 knockdown) sites in ZR-75-1 cells. e. Boxplots showing the normalized ChIP-seq tag density around 400 bp window of ARID1A and BRG1 on FOXA1 independent (constant, n=70,429 sites) and dependent (lost sites with siFOXA1, n=17,357 sites) sites in ZR-75-1. p-value calculated by Welch’s test, two-sided. n = 3 independent biological cell culture samples. Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). Statistical test details are mentioned in Supplementary Table 5f.

Extended Data Fig. 6. FOXA1 promotes the binding of ARID1A and BRG1.

Hormone-deprived MCF7 and ZR-75-1 cells were transfected with control or FOXA1 siRNA and ChIP-seq was conducted for ARID1A and BRG1. n = 3 independent biological cell cultures. (a-b) Single gene profiles of CCND1 (a) and CDH1 (b) showing the effect on SWI/SNF complex binding with FOXA1 knockdown on MCF7 and ZR-75-1 cells. ER and FOXA1 binding overlap is shown. (c-d) ChIP-qPCR analyses on specific sites (CCND1 and CDH1 ER binding sites) showing ARID1A and BRG1 binding with FOXA1 knockdown in hormone-deprived MCF7 and ZR-75-1 cells (c) or ARID1A binding following Tamoxifen treatment in asynchronous MCF7 cells (d). n = 3 independent biological cell cultures. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001. Precise p-values are mentioned in Fig. S10. Mean is measured as centre shown with standard deviation. Details of the statistical tests are mentioned in Fig. S10.

Extended Data Fig. 7. ATAC-seq analyses shows a negligible regulation of ARID1A on transcription-associated chromatin opening.

a. Heatmap showing ATAC-seq analysis in ARID1A KO clones 11 and 14 following Tamoxifen treatment. Common, gained and lost sites defined by DiffBind analysis. n = 4 independent biological cell cultures. FDR ≤ 0.05 corrected by Benjamini-Hochberg multiplicity correction, two-sided. b. Association of ARID1A KO upregulated and downregulated genes with ATAC-seq gained and lost sites.

Extended Data Fig. 8. ARID1A perturbation regulates ARID2 binding.

a. ARID2 ChIP-seq was conducted in wild type cells or the two ARID1A knock-out clonal cell lines and heatmaps are shown on ARID2 binding sites after Tamoxifen treatment. Also included was ARID1A ChIP-seq from wild type cells treated with vehicle or Tamoxifen. ARID2 binding overlapped with ARID1A binding and was dependent on ARID1A. n = 3 independent biological cell cultures. b. Signal intensity plot showing changes in ARID2 binding in wild type control cells or ARID1A knock-out cells at ARID2 binding sites. n = 3 independent biological cell cultures.

Extended Data Fig. 9. ARID1A promotes BRG1 and HDAC1 binding without affecting ER and H3K27ac occupancy

a, b. BRG1, H3K27Ac, HDAC1 and ER (b) ChIP-seq were conducted in asynchronous wild type cells treated with vehicle or tamoxifen or in the two ARID1A knock-out clones (Clones 11 and 14) following tamoxifen treatment. The binding is shown on regions where HDAC1 is lost in ARID1A knockout cells relative to wild type cells. n = 3 independent biological cell cultures. c, d. Scatterplot showing the correlation of ER (c) or H3K27Ac (d) and HDAC1 binding in ARID1A knockout clone 11 versus wild type cells. n = 3 independent biological cell cultures. PCC – Pearson Correlation coefficient. p-values were calculated by Pearson correlation test, two-sided. e. Principal Component Analysis (PCA) of normalised peptide intensities of PDX tumours after ER qPLEX-RIME. n= 2 PDX each group. f. Details of ARID1A mutations observed within ER+ PDX tumours used in ER qPLEX-RIME.

Extended Data Fig. 10. ARID1A regulates histone H4 acetylation.

Upregulation of histone H4 acetylation in ARID1A knock-out clone 11 and 14 in Vehicle (a) or Tamoxifen (b) treated cells comparing to wild type cells. Heatmap representing the changes in histone H4Ac marks upon ARID1A knockout with Vehicle or Tamoxifen treatment on ER binding sites close to ARID1A repressed genes. n =3. (c) Empirical cumulative probability distribution plots of H4K8Ac and H4K12Ac ChIP-seq signals showing upregulation in intensity (y-axis) with ARID1A knockouts clones 11 and 14. Plots were made on ER sites close to ARID1A repressed genes (n=686 sites) with more than 75% contribution to the variance in intensity. Window – 2 kb around the center of binding.

Fig. 1. CRISPR screens reveal ARID1A and BAF components as essential genes for treatment response.

Log2 fold of gRNA counts change as a function of time per gene (red lines) and on average (black line) based on a sample of n=3 for three categories of genes: the ones showing a rapid growth depletion (a), the ones showing a longer-term growth depletion (b) and the ones showing increased proliferation (c). For each category, example genes are shown in red and ARID1A is shown in blue. d. Heatmap representing log2 fold change of significant genes (n=1915) in non-treated conditions (day 3 to day 20 of infection comparing to uninfected gRNA pool). Rows were ordered according to hierarchical clustering. e. Heatmap representing log2 fold change of genes after 26 days of treatment with Fulvestrant (Fulv, initiated with 300 nM and reduced to 100 nM gradually), 100 nM 4-hydroxytamoxifen (Tamox) or BETi (JQ1 – 1µM reduced to 250 nM) comparing to DMSO treatment (DMSO control after day 9 of infection). Rows were ordered according to hierarchical clustering. f. ARID1A and other BAF components were enriched, but in different directions depending on the specific drug treatment. The values show changes in gRNA levels for these genes, using a log2 fold change relative to DMSO control. g. Frequency of single gRNAs in log 2 scale against BAF complex subunits ARID1A, ARID1B, SMARCB1 and SS18, comparing 4-hydroxytamoxifen or JQ1 with non-treated conditions. h. Example of ARID1A ChIP-seq binding overlap with ER and FOXA1 from MCF7 cells grown in media containing 10% fetal bovine serum containing estrogen, from three independent biological ChIP-seq samples per group. i. Global overlap between ARID1A, ER and FOXA1 ChIP-seq data from MCF7 cells grown in media containing 10% fetal bovine serum containing estrogen (n=3 independent biological ChIP-seq samples per group). j. Heatmaps representing ARID1A binding in hormone-deprived cells treated with vehicle or 10 nM estrogen (n = 3) on the constant sites (n=24,754 sites) defined by DiffBind without any significant change with estrogen treatment and the DiffBind-defined significant sites (n=3,023) which show reduced ARID1A binding during estrogen treatment. Also shown are the relative ER and FOXA1 binding intensities at these regions.

Fig. 2. ARID1A knock-out clonal cells show loss of response to ER antagonists, but responsiveness to BET inhibitors.

a. ARID1A was knocked-out of MCF7 cells using CRISPR deletion. Western blots of ARID1A or ER confirm effective gene deletion in clones 11 and 14, with no change in total ER levels. This figure shows the data of one representative experiment (Source Data Fig. 2) out of the three independent experiments. b. Percentage confluence as a function treatment time, in an in-vitro proliferation assay using Incucyte conducted in asynchronous MCF7 cells treated with vehicle or 1 µM 4-hydroxytamoxifen. This figure shows the data of one representative experiment out of the four independent experiments. Each experiment considered n=3 replicated per group. Mean ± Standard error of the mean is shown in the graph. c. Xenograft tumour volume of MCF-7 (n=13 animals), ARID1A K.O clone 14 (n=8 animals), ARID1A K.O clone 11 (n=12 animals) as a function of time since day of enrolment. The dots and arrows respectively show the average tumour volume and corresponding 95% confidence intervals of mice at risk. Tumour size of animals at different time-points were fitted by means of a linear mixed model on the cubic root scale, with time and group as fixed effect and random intercepts and slopes for mice (Full details are provided in Supplementary Note). The colored curves and shaded areas correspond to the fitted growth curves for each group and 95% confidence intervals, and the p-values to the mixed model difference in growth rate tests. Test statistics in Fig. S5d. p-values were calculated by two-sided Wald test. d. RNA-seq was conducted on the ARID1A knockout cells treated with Vehicle, 10 nM Fulvestrant, 100 nM 4-hydroxytamoxifen or 250 µM JQ1 (n=4 independent biological samples). As controls, both parental MCF7 cells and three wild type clonal lines were included. The plot shows fold change of Fulvestrant-regulated genes (n=1094) (ordered by means of a hierarchical clustering) in wild type cells. Highlighted gene cluster (with a star) shows the maintained downregulated effect of JQ1 regardless of ARID1A status, but upregulation with Vehicle and 4-hydroxytamoxifen upon ARID1A loss. e. Survival rate as a function of time-to-event for 2 groups of ER+ cancer patients: patients showing up- (red) (n=104 for Vehicle and 72 for 4-hydroxytamoxifen) and down- (blue) (n=101 for Vehicle and 61 for 4-hydroxytamoxifen) regulation according to a gene signature defined by ARID1A targeted genes shown to be repressed by vehicle or 4-hydroxytamoxifen. p-values correspond to log-rank tests (two-sided) (estimated test statistics available in Supplementary Fig. 6) respectively comparing the survival distribution of patients with up and down - regulated genes. Total METABRIC cases: 1181.

Fig. 3. The SWI/SNF complex interacts with ER and is recruited to chromatin following drug treatment.

a. ER, ARID1A or BRG1 RIME was conducted in asynchronous MCF7 cells. IgG was used as a negative control. ER, FOXA1 and HDAC1 were identified as interactors in the ARID1A and BRG1 pull downs and vice versa. Boxplots shows the enrichment of selected known interactors in the pulldown samples compared to IgG controls. Pull downs were performed in two biological cell culture samples and label free quantification was performed using Minora algorithm. The log2 intensities are normalised so that the median of IgGs is zero. Centre line shows the median. n=2 independent biological cell culture samples. b. Five ER+ PR+ primary tumour samples were split for ER or IgG pull downs and the enrichment of known co-factors in the ER compare to IgGs such as HDAC1 and BAF components are shown. Boxplots shows the enrichment of selected known ERα interactors in the ERα RIME samples compared to IgG controls in human breast cancer tissues. The log2 values are normalised so that the median of IgGs is zero. Centre line shows the median. c. ER qPLEX-RIME was conducted in asynchronous MCF7 cells treated with 100 nM 4-hydroxytamoxifen in a 4-point time course (n = 6 independent biological samples per group). Specific BAF proteins are highlighted and the enrichment of the BAF components in the ER complex upon 4-hydroxytamoxifen treatment is shown. Centre line shows the median. d. ChIP-seq of ARID1A, BRG1 or SNF5 (SMARCB1/BAF47) in asynchronous MCF7 cells treated with vehicle (ethanol) or 100 nM 4-hydroxytamoxifen (n = 3 independent biological ChIP-seq samples). The heatmaps represent the 39,214 ARID1A binding events observed after 4-hydroxytamoxifen treatment. Also included are H3K27Ac, ER and FOXA1 binding signal intensity at these regions.

Fig. 4. FOXA1 promotes binding of ARID1A and BRG1 to a subset of potential enhancer elements.

a, b. Hormone-deprived MCF7 cells were transfected with control or FOXA1 siRNA and ChIP-seq was conducted for ARID1A (a) or BRG1 (b). n = 3 independent biological ChIP-seq samples. MA plots are shown with the average intensity of binding vs log2 fold change with FOXA1 siRNA comparing to control siRNA. c, d. Heatmaps (c) and boxplots (d) shown on ARID1A-BRG1 constant (n= 65563 sites) and ARID1A-BRG1 lost sites (n= 9355 sites) defined by DiffBind following FOXA1 silencing in MCF7 cells. ER and FOXA1 overlap are also shown on (c) these sites. n = 3 independent biological cell culture samples. p-values (d) were calculated by Welch’s t-test, two-sided. For boxplot, centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). More statistical details are mentioned in Supplementary Table 5a. e. Scatterplot showing the association of decreased ARID1A and BRG1 binding following FOXA1 silencing. PCC – Pearson Correlation coefficient. p-values were calculated by Pearson Correlation test, two-sided. f-g. Scatterplot showing the association of ARID1A (f) and BRG1 (g) binding following FOXA1 silencing at tamoxifen-induced ARID1A (f) and BRG1 (g) binding sites from Fig. 3d. PCC – Pearson Correlation coefficient. p-values were calculated by Pearson Correlation test, two-sided. h. Boxplots illustrating the effect of siFOXA1 on ARID1A and BRG1 binding on the ER binding sites (n=2,746 sites) close to ARID1A repressed genes in Vehicle conditions. p-values were calculated by Welch’s t-test, two-sided. Window – 400 bp around center of the factor binding. Centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). More statistical details are mentioned in Supplementary Table 5b.

Fig. 5. Loss of ARID1A results in decreased BRG1 and HDAC1 recruitment and increased histone H4 acetylation.

a, b, c. Quantitative signal from BRG1 (a), HDAC1 (b) and ER (c) ChIP-seq within ARID1A knock-out cells (n=3 independent biological cell culture samples per group). ChIP-seq was conducted in the wild type cells or the two ARID1A knock-out clones, showing decreased binding of the factors in the absence of ARID1A. Average plots were shown on HDAC1 lost sites in the ARID1A knock-out cells. d, e. Scatterplots showing the association of decreased BRG1 and HDAC1 binding in ARID1A knockout clone 11 (d) and clone 14 (e) following 100 nM 4-hydroxytamoxifen treatment. n = 3 independent biological cell culture samples. PCC – Pearson Correlation coefficient. P-values were calculated by Pearson Correlation test, two-sided. f. ER qPLEX-RIME was conducted in four ER+ PDX tumours, two of which had loss of ARID1A via mutation (MT1/2) and two were wild type (WT1/2) for ARID1A. Heatmaps reveals decreased BAF and HDAC1 interactions with ER in ARID1A mutant tumours compare to the wild type tumours. g. We specifically identified ARID1A repressed genes in proximity to the ER-bound regulatory elements (n=686 sites) that display, according to PCA, more than 75% contribution to the variance in intensity of histone H4 acetylation. The data is shown as boxplots. ARID1A dependent genes acquired gained H4 acetylation, especially H4K8Ac and H4K12Ac at adjacent enhancers, coincident with increased gene expression. P-values were calculated by Welch’s t-test, two-sided. Window – 2 kb around center of the binding event. More statistical details are provided in Supplementary Table 5c.

Fig. 6. Loss of ARID1A results in increased BRD4 recruitment and a gain in intrinsic proliferation.

a- c. BRD4 ChIP-seq was conducted in wild type or ARID1A knock-out cells (n=3 independent ChIP-sew samples per group). a. Boxplots were shown on ER bound regions close to ARID1A repressed genes (n=686) that display, according to PCA, more than 75% contribution to the variance in intensity of H4 acetylation. p-values were calculated by Welch’s t-test, two-sided. Window – 400 bp around center of the factor binding. For boxplot, centre line shows the median values with bounds of box corresponding to the first and third quartiles and the upper and lower whiskers extend to the largest or the smallest value no further than 1.5 × IQR (inter-quartile range). More statistical details are mentioned in Supplementary Table 5d. b. Scatterplot showing the association of BRD4 and HDAC1 binding in the ARID1A knockout clone 11 cells. n = 3 independent biological cell culture samples. PCC – Pearson Correlation coefficient. P-values were calculated by Pearson Correlation test, two-sided. c. Heatmap shows the gained BRD4 occupancy and decreased HDAC1 binding on the ER-bound regulatory elements (n=2,746 sites) adjacent to ARID1A target genes. d. Overall patient survival was assessed based on ARID1A mutational status in a cohort of 1,824 breast cancer patients. p-value was calculated by log rank survival test, two-sided (estimated test statistics available in Supplementary Fig. 14). e. Ki67 IHC protein levels stained on an ARID1A mutant PDX cultivated ex vivo and treated with DMSO vehicle (n=10 explant chunks), 250 nM JQ1 (n=9 explant chunks) or 1µM IBET762 (n =10 explant chunks) for 48hr in a single experiment. Median values are shown with p-values calculated using Wilcoxon test, two-sided (Wilcoxon test Statistic W= 17 for both comparisons). f. Representative images of Ki67 IHC in BETi ex vivo tumour tissue, with each image representing a region of 100 μm in length. The explant chunks were treated with DMSO vehicle (n=10), 250 nM JQ1 (n=9) or 1µM IBET762 (n =10). g. Model of FOXA1-ARID1A-HDAC1-BRD4 axis in ARID1A wild type and mutant contexts.