SRA

Global CRISPR screens provide an unparalleled, longer-term experimental approach for the identification of essential genes in drug resistance. We used an ~18,000 gene deletion screen to discover ARID1A and other BAF complex components as the most critical factors required for response to two classes of Estrogen Receptor (ER) antagonists, namely ER degraders and Selective Estrogen Receptor Modulators (SERMs). Unexpectedly, ARID1A was also the top candidate for response to the BET inhibitor JQ1, but in the opposite direction, where loss of ARID1A sensitised breast cancer cells to BET inhibition. We show that ARIDA binds chromatin at ER cis-regulatory elements and can physically associate with ER in model systems and primary tumour samples. ARID1A binding to ER enhancer elements, can occur in the absence of ER, suggesting that its repressive activity occurs in an enhancer-specific, but ER-independent manner. Specific targeting of ARID1A validated the CRISPR screen and shows that depletion of BAF activity, does not result in redundancy from P-BAF, the other ATP-dependent chromatin remodelling complex, but instead results in loss of HDAC1 binding, increased Histone 4 lysine acetylation and subsequent BRD4-driven growth. ARID1A and the BAF complex therefore function as a critical mechanism of antiestrogen activity and mutation or depletion in BAF activity drives a BRD4-mediated proliferative program that is refractory to ER targeted agents. Since ARID1A is mutated in a subset of treatment-resistant disease, these findings provide mechanistic insight and treatment strategies for patients, based on BAF complex fidelity status. Overall design: Examination of ARID1A, BRG1 and SNF5 binding sites in asynchronous MCF7 cells treated with vehicle (DMSO) or 100 nM 4-Hydroxytamoxifen, 250 nM JQ1 and 10 nM Fulvestrant for 6 hours or double times charcoal stripped serum (DCDT)- treated cells for 72 hours and treated with Vehicle or 10 nM b-estradiol for 6 hours. Examination of ER, H3K27ac, ARID2, BRG1, HDAC1 and BRD4 in MCF7 parental cell lines and ARID1A knockout clones 11 and 14 treated with vehicle (ethanol) or 100 nM 4-Hydroxytamoxifen. Please note that the processed data was generated from merged replicates and is linked to the corresponding rep 1 sample records. Examination of ARID1A and BRG1 binding in hormone-deprived synchronous MCF7 and ZR-75-1 cells with FOXA1 siRNA. Examination of histone H4 acetylation marks on MCF7 ARID1A knockout cells treated with vehicle (DMSO) or 100 nM 4-Hydroxytamoxifen.