PROJECT SUMMARY Approximately 70% of breast cancers express estrogen receptor (ER) and are treated typically with endocrine therapy. Despite the clinical benefit obtained from several types of endocrine therapies, emergence of resistance to these agents eventually develops in all patients with metastatic disease. We have sequenced 2,752 ER-positive breast cancer samples for which detailed clinical information on response to endocrine therapy is available. These analyses have also shown a correlation between emergence of endocrine therapy resistance and the presence of inactivating mutations in the chromatin remodeler ARID1A. In parallel, we have performed an epigenome-wide CRISPR knockout screen on MCF7 ER-positive breast cancer cells and have identified ARID1A to be the top candidate gene whose loss results in resistance to the Selective ER Degrader (SERD) fulvestrant followed by the loss of additional SWI/SNF subunits, namely SMARCB1, SMARCE1. When we interrogated our internal patient cohort, we found that ARID1A loss is enriched in the metastatic setting and correlates with resistance to SERDs (e.g. fulvestrant). These findings indicate an important role for ARID1A in mediating resistance to endocrine therapy. Mechanistically, our published and preliminary data demonstrate that loss of ARID1A leads to widespread changes in the chromatin landscape of breast cancer cells, resulting in loss of motifs for TFs involved in ER-dependent transcription and luminal (ER+) cell identity. This was accompanied by increase expression of basal (ER-) markers in cell line models and patient samples harboring ARID1A inactivating mutations. In this proposal, we will seek to define the mechanistic basis by which ARID1A acts as a mediator of resistance to endocrine therapy in breast cancer cell lines, patient-derived xenografts and samples from patients with ARID1A mutant breast cancers, and to determine how ARID1A loss results in trans-differentiation from a luminal to a basal program in breast cancers and normal cells. We will perform RNA-seq utilizing samples from patients with hormone therapy-refractory breast cancers that are either wild-type or null for ARID1A. We will then investigate the impact that the knockout of ARID1A has on chromatin recruitment of the SWI/SNF complex and on the binding of the SWI/SNF complex at dominant transcription factors that regulate gene expression programs critical for the cellular differentiation state in breast cancer. Finally, we will study the effects of Arid1a loss in mammary-gland morphogenesis utilizing a mammary epithelium-specific conditional Arid1a null mouse model we have developed and normal mammary and breast cancer 3D organoids. Our results will shed light on the role of ARID1A in determining cell fate/lineage and how ARID1A and the SWI/SNF complex plays a causative role in limiting the sensitivity to endocrine therapy.