PROJECT SUMMARY Tamoxifen is the most prescribed cancer drug in the world. Tamoxifen is a selective estrogen receptor modulator (SERM) that is used to treat patients with breast tumors that express estrogen receptor alpha (ER), acting as a partial antagonist that blocks ER’s growth-promoting activity. The use of tamoxifen has greatly benefited breast cancer patients by significantly reducing the risk of recurrence. Unfortunately, several severe side effects accompany the recommended 5-10-year course of tamoxifen treatment, including increased risk of endometrial cancer. It has been shown that tamoxifen acts as an ER agonist in the endometrium and in endometrial cancer cells. However, despite the initial observation of tamoxifen-associated endometrial cancer more than 30 years ago, the molecular mechanisms remain poorly understood. The leading hypothesis is that different cofactors interact with ER upon tamoxifen binding and these cofactors differ between breast cancer cells and endometrial cells. In this proposal, we will address the decades-old question of how tamoxifen acts as an agonist in endometrial cells using cutting edge techniques. We will use two approaches to determine key factors that underlie tamoxifen’s differential actions in breast and endometrial cancer. In specific aim 1, we will focus on the cofactor hypothesis by applying RIME, which identifies co-occurring factors on chromatin, to breast cancer cells and our unique collection of endometrial cancer and normal endometrial samples that have undergone treatment with estradiol and 4-hydroxytamoxifen. In specific aim 2, we will cast a broader net by using CRISPR approaches to identify genes essential for tamoxifen’s ER agonist role in endometrial cells and compare results to similar studies in breast cancer cells. The successful completion of this project will lead to a mechanistic understanding of how tamoxifen has dichotomous roles, being both an effective breast cancer treatment and an endometrial cancer risk factor. This knowledge will aid in identifying alternative breast cancer treatment strategies that reduce the chance of developing a deadly side effect and will help in discovering new therapeutic targets for endometrial cancer patients.