Abstract Background. Dysregulation of the estrogen receptor gene (ESR1) is an established mechanism of inducing endocrine therapy resistance. We previously discovered a chromosomal translocation event generating an estrogen receptor gene fused in-frame to C-terminal sequences of YAP1 (ESR1-YAP1) that contributed to endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer models. This study compares functional, transcriptional, and pharmacological properties of additional ESR1 gene fusion events of both early stage (ESR1-NOP2) late stage (ESR1-YAP1 and ESR1-PCDH11x) breast cancers to gain a better understanding of therapeutic resistance and metastasis. Understanding the role of ESR1 fusions in inducing metastasis is critical, since the primary cause of death in breast cancer patients is through metastasis to distant sites. Methods. RNA-seq screens identified ESR1 fusions from early and late stage, endocrine therapy resistant breast tumor samples. Functional experiments were conducted using ER+ breast cancer cell lines, xenograft, and PDX models to test the ability of ESR1 fusions to induce therapeutic resistance and metastasis. ChIP-seq and RNA-seq were performed to examine transcriptional properties and differential gene expression induced by the fusions which directed subsequent pharmacological experiments with a CDK4/6 inhibitor. Results. ESR1-YAP1 and ESR1-PCDH11x promoted estrogen-independent and fulvestrant-resistant growth in vitro and induced greater tumor growth and increased metastatic capacity to the lungs of xenografted mice. In contrast, the ESR1-NOP2 fusion was sensitive to low estrogen conditions in vitro, and did not promote tumor growth. RNA-seq profiling revealed E2F targets pathway as the most highly enriched pathway induced by the ESR1 fusions. IHC revealed higher levels of pRb in ESR1-YAP1 and ESR1-PCDH11x xenograft tumors and subsequent CDK4/6 inhibition completely blocked tumor growth in an ESR1-YAP1 PDX model. Integrating RNA-seq with ChIP-seq data, we discovered a set of EMT and metastasis genes bound by all ESR1 fusions and WT-ER, but whose expression was strongly and uniquely up-regulated only by the ESR1-YAP1 and ESR1-PCDH11x fusions. These studies also revealed gained sites bound only by the ESR1-YAP1 and ESR1-PCDH11x fusions, not bound by WT-ER nor ESR1-NOP2. Genes mapping to these sites have a role in metastatic biology and were highly up-regulated by the YAP1 and PCDH11x fusions, potentially mediated by long range transcriptional activation. Conclusion. ESR1-YAP1 and ESR1-PCDH11x are driver fusions that occur in drug-resistant, advanced stage breast cancer and are a new class of recurrent somatic mutation that can cause acquired endocrine therapy resistance, yet can be treated with CDK4/6 inhibition. These driver fusions also confer increased metastatic ability through their ability to drive expression of genes that contribute to EMT and metastasis. In contrast, ESR1-NOP2 did not produce functional protein and appears to be a passenger event. These studies may provide pre-clinical rationale for targeting ESR1 translocated breast tumors, since the presence of an ESR1 driver fusion places a patient in a therapeutic category where none of the currently available endocrine therapies are likely to be effective. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Chan DW, Cao J, Anurag M, Singh P, Haricharan S, Kavuri SM, Matsunuma R, Schmidt C, Kosaka Y, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Rodrigues-Peres RM, Lai W-C, Hampton O, Rogers A, Tobias E, Parikh P, Davies S, Ma C, Suman V, Hunt K, Watson M, Hoadley KA, Thompson A, Perou CM, Creighton CJ, Maher C, Ellis MJ. ESR1 gene fusions drive endocrine therapy resistance and metastasis in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-03.