Abstract The CDK4/6 inhibitor palbociclib was recently approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib (Novartis). In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 21 and 17 kinases, respectively, which induced a >30% increase in cell viability compared to control cells; 11 of these kinases overlapped in both treatment groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive ‘hits’ for treatment with fulvestrant/ribociclib. Five of 11 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these five genes showed only FGFR1 to be amplified/overexpressed in 17% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib, each alone, modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regressions in all mice without associated toxicity. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides or kinase inhibition with erdafitinib restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and fulvestrant ± palbociclib. At this time, we are examining whether FGFR1 amplification measured by FISH correlates with maintenance of proliferation (Ki67) in 110 patients with ER+/HER2- breast cancer treated with palbociclib for 14 days immediately before surgery (Arnedos et al. ASCO 2016). In summary, using a gain-of-function ORF kinome screen, we identified FGFR1 amplification as a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. Based on these data we propose FGFR inhibitors should be tested in combination with ER antagonists and CDK4/6 inhibitors in patients with ER+/FGFR amplified breast cancer. Citation Format: Luigi Formisano, Yao Lu, Valerie M. Jansen, Joshua A. Bauer, Ariella B. Hanker, Melinda E. Sanders, Paula González-Ericsson, Sunkyu Kim, Monica Arnedos, Fabrice André, Carlos L. Arteaga. Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1008. doi:10.1158/1538-7445.AM2017-1008