Abstract 742: The integration of a structure-function rule and a transcriptional signature to assign ESR1 fusion activity in metastatic breast cancer

Background: In-frame chimeric ESR1 fusion proteins can cause endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) metastatic breast cancer (MBC) (PMC6171747). Here, we further investigated multiple new examples of ESR1 fusions in order to differentiate between transcriptionally active “driver” ESR1 fusions from inactive, likely “passenger” events. Methods: ESR1 fusions were identified by RNA-sequencing (RNA-seq) or whole genome sequencing in MBC (PMC5913625, PMC6872491 and unpublished). ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines. Cell proliferation and cell motility were assessed. RNA-seq followed by qPCR validation was conducted to examine individual transcriptional profiles. Results: All ESR1 fusions studied contained the first six exons of ESR1 (e6) fused in-frame to diverse partner gene sequences that replaced the ESR1 drug/ligand binding domain. Fusions with a transcription factor (TF) or coactivator (CoA) gene partner, for example ESR1-YAP1, ESR1-ARNT2-e18 and ESR1-SOX9 activated fulvestrant-resistant cell growth and hormone-independent cell motility. Other ESR1-e6 fusions, including ESR1-DAB2, ESR1-GYG1, ESR1-PCMT1 and ESR1-ARID1B did not show these phenotypes. We thus conclude that in-frame ESR1-e6 fusions arising from inter-chromosomal translocations with 39 TF/CoA partners are likely to be active. However, the active ESR1-PCDH11X fusion involves a proto-cadherin and is therefore an exception to this rule. This outlier emphasizes the need to establish additional approaches to determine ESR1 fusion activity. RNA-seq of T47D cells expressing active and inactive ESR1 fusions defined a gene signature associated with active ESR1 fusions, with activation of estrogen response and epithelial-to-mesenchymal transition (EMT) genes. This transcriptional signature was present in a patient-derived xenograft bearing the ESR1-YAP1 fusion, and thus potentially identifies the presence of an active ESR1 fusion protein. We subsequently identified two new ESR1 fusions involving recurrent TF/CoA partners, ESR1-ARNT2-e2 and ESR1-LPP. Both demonstrated tumor cell growth and motility activation, as predicted by the functional rule. The gene activation patterns were similar to the three other active fusions suggesting that despite marked diversity in the 39 partners, the transcriptional activities were similar and potentially diagnostic. Conclusion: The integration of the structure-function rule and the stereotypic transcriptional signature distinguishes pathogenic ESR1 fusions from non-active passenger events, thus prioritizing patients bearing ESR1 translocation-driven tumors for targeted therapeutic approaches. Citation Format: Xuxu Gou, Meenakshi Anurag, Jonathan T. Lei, Sinem Seker, Adrian V. Lee, Dan R. Robinson, Matthew J. Ellis. The integration of a structure-function rule and a transcriptional signature to assign ESR1 fusion activity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 742.