Abstract Approximately 75% of breast cancers are classified as Estrogen Receptor positive (ER+). Tamoxifen, a selective estrogen receptor modulator (SERM), is the standard of care for many of these ER+ breast cancer patients. Unfortunately, tamoxifen resistance occurs in almost 50% of patients within 5 years of treatment, and endocrine-independence accompanying resistance also negates the effects of aromatase inhibitors. Combination therapy is increasingly used in non-cytotoxic therapeutic approaches in many types of cancer. The potential exists that endocrine resistance can be lessened, eliminated, or overcome through targeted therapy in combination with endocrine therapy. Inhibition of kinase signaling (e.g. via CDK4/6 or PI3K) and other pathways (e.g. HSP90) are expected to be effective in combination with endocrine therapy. We have discovered a variety of ER ligands with potential as endocrine therapeutic agents, based upon a single chemical scaffold with a diverse set of pharmacological responses: including SERMs, selective ER downregulators (SERDs), selective estrogen mimics (SEMs), and selective human ER partial agonists (ShERPAs). To predict which agents in combination with these endocrine-targeted ligands would be of potential therapeutic benefit it was necessary to develop 3D spheroidal cultures of ER+ breast cancer cell lines: including endocrine-dependent lines; and cell lines made endocrine-independent either by extended exposure to tamoxifen or extended deprivation of estradiol. In contrast to 2D cultures, drug response in 3D spheroidal cell cultures was predictive of response to treatment in mouse xenograft studies. Growth of endocrine-dependent cell lines was, as expected, inhibited by SERDs; and endocrine-independent, tamoxifen-resistant cell lines were also sensitive to SERD treatment, although one cell line was largely resistant. Growth of all three tamoxifen-resistant cell lines was inhibited by SEMs/ShERPAs. Importantly, regardless of the type of endocrine therapeutic agent studied, concentrations leading to saturation of the target (ER) did not cause cell death. Equally, all endocrine therapies studied benefited from combination treatment with other agents, leading to enhanced cell death. Citation Format: Gutgesell LM, Xiong R, Thatcher GRJ, Tonetti DA. Combination therapy of targeted anticancer pathways and estrogen receptor ligands and their responses in de novo andtamoxifen resistant cell models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-23.