Abstract P6-04-04: Functional characterization of ESR1 fusions in breast cancer
Authors: Gou Xuxu , Lei Jonathan T , Seker Sinem , Kim Beom-Jun , Rehman Saif , Lee Adrian V , Robinson Dan R , Ellis Matthew J 
Published: March 2020

Background: We recently reported two in-frame estrogen receptor alpha gene (ESR1) fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, in endocrine therapy (ET)-refractory, metastatic estrogen receptor positive (ER+) breast cancer patients. Both fusions were causal in driving ET resistance and metastasis in experimental models. CDK4/6 inhibition suppressed tumor growth driven by ESR1 fusions, but did not induce tumor regression (Lei et al., Cell Rep., 2018). Additional ESR1 fusions with diverse partner genes have been recently reported (ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9) (Hartmaier et al., Ann Oncol., 2018), or communicated to us (ESR1-ARNT2, ESR1-PCMT1 and ESR1-ARID1B) (Dr. Dan R. Robinson, University of Michigan). All were identified in ET-refractory tumors. We therefore undertook detailed functional characterization of these new ESR1 fusions to examine their role as disease drivers. We also used mass spectrometry-based kinomics to identify therapeutic targets induced by individual ESR1 fusions.

Methods: Unreported ESR1 fusions were identified by RNA sequencing followed by informatics designed to identify fusion gene reads. Stable ER+ breast cancer cell lines (T47D and MCF7) expressing in-frame ESR1 fusion proteins were generated by lentiviral transduction. ET-resistant cell proliferation was assessed in low estradiol medium and/or in the presence of anti-estrogen. Expression of estrogen responsive and epithelial-to-mesenchymal transition (EMT) genes were measured by mRNA-qPCR. Cell motility driven by these fusions was examined using scratch wound assay. Kinase expression patterns were examined using a Kinase Inhibitor Pulldown (KIP) mass spectrometry-based assay.

Results: All the fusions we studied contain the first 6 exons (ESR1-e6) of ESR1 that provides the first 4 coding exons. In-frame C-terminal sequences were provided by different partner genes. In addition to previously described ESR1-YAP1 and ESR1-PCDH11X fusions, ESR1-SOX9 and ESR1-ARNT2 conferred estrogen-independent and fulvestrant-resistant growth in stable ER+ breast cancer cells (T47D and MCF7). In T47D cells, these ESR1 fusions upregulated not only classical ER targets, including GREB1, PGR, and TFF1, but also EMT-associated genes, SNAI1 (Snail) and VCAN in an estradiol-independent, fulvestrant-resistant manner. In addition, transcriptionally active ESR1 fusions promoted cell motility. KIP profiling of T47D cells expressing transcriptionally active ESR1 fusions and a patient-derived xenograft naturally harboring the ESR1-YAP1 fusion revealed an increase in abundance of multiple receptor tyrosine kinases including RET and insulin like growth factor 1 receptor (IGF1R). Responses to targeting these kinases have been observed in vitro. Other ESR1-e6 fusions produced stable proteins (ESR1-DAB2, ESR1-GYG1, ESR1-PCMT1, ESR1-ARID1B) but had much less clear transcriptional phenotypes and fewer upregulated kinases identified by KIP.

Conclusion: A classification of ESR1 fusions in ET-refractory breast cancer is emerging from this study. About half of ESR1-e6 fusions studied are transcriptionally active, presumably because the 3’ partner in these examples is encoded by transcription factor gene (TF) (SOX9 or ARNT2) or co-activator gene (CoA) (YAP1). These can be classified as ESR1-e6 TF/CoA fusions. The phenotype of the remaining fusions remains under investigation because of the functionally diverse nature of the 3’ partners and the low activity in functional assays. While some of these may be passengers, the example of PCDH11X suggests a non-TF/CoA partner ESR1-e6 fusion can still be active and further studies on additional fusions will be necessary. Downstream targeting of fusion-dependent kinases may be effective.

Citation Format: Xuxu Gou, Jonathan T Lei, Sinem Seker, Beom-Jun Kim, Saif Rehman, Adrian V Lee, Dan R Robinson, Matthew J Ellis. Functional characterization of ESR1 fusions in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-04.