Abstract Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy 1, 2. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors 3. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells. Bibliography 1 American Cancer Society, Cancer Facts & Figures 2014. 2 Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618. 3 Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51. Citation Format: Raquel Torres, Bruna Calsina, Ana Hermoso, Carmen Baquero, Cecilia Mur, Karsten Boehnke, Joaquín Amat, Alfonso De Dios, Xueqian Gong, Sean Buchanan, Richard Paul Beckmann, Maria Jose Lallena. Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2836.