Abstract PD10-02: PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms
Authors: Yates Megan E. , Liu Tiantong , Hooda Jagmohan , Yang Sichun , Bao Riyue , Atkinson Jennifer M. , LEE ADRIAN V. , Oesterreich Steffi 
Published: March 2023

Breast cancer is a leading cause of female mortality and despite advancements in diagnostics and personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The druggable estrogen receptor (ER, ESR1), overexpressed in two-thirds of all breast cancer, evolves in 30% of tumors exposed to endocrine therapy consequently resulting in treatment resistance. A more recently discovered mechanism of ER mediated endocrine resistance is ER fusion proteins. ER fusions, found predominately in metastatic endocrine resistant disease, harbor ESR1 exons 1-6 fused to an in-frame gene partner due to an ESR1 intron 6 translocation break. Our lab has demonstrated that ER fusion proteins, which lack the C-terminal ligand-binding domain (LBD), recapitulate phenotypes of ER proteins harboring endocrine-resistant point mutations occurring in the LBD. Our research goals aim to 1) determine fusion prevalence and emergence, 2) understand fusion mechanisms of resistance and 3) explore alternative treatment options for our patients. The promiscuous nature of fusion partners hinders fusion detection by traditional sequencing and thus our research team has developed a novel ER fusion detection method, EnRich. The EnRich probe set (4,324 probes in total) is composed of two separate probe pools targeting at 2x tiling intronic and exonic ESR1, the upstream promoter region and select oncogenic genes. To optimize the EnRich pipeline, DNA was extracted from a frozen tumor sample and a PDX model harboring ESR1-DAB2 and ESR1-LPP fusions, respectively. ESR1-DAB2 and ESR1-LPP were accurately detected by quantifying discordant paired-end or split reads mapped to chromosome 6. In addition, liquid biopsies from 15 patients with ER positive advanced breast cancer were also assessed through EnRich to uncover unidentified ESR1 structural variants. Two patient samples were detected to harbor ESR1 fusions (ESR1-CCDC170, ESR1-AKAP12 and ESR1-YAP1) and were further validated in corresponding mRNA. These recurrent and novel fusions were supported with more than 10 reads each, indicating that the EnRich pipeline is an effective and accurate sequencing approach to understanding ER fusion prevalence. Our lab, furthermore, has studied ER fusion proteins mechanistically. We have stably overexpressed ER fusions in transgenic breast cancer cell lines engineered with shRNA targeting the endogenous wildtype ER (ESR1-WT). In this ESR1-WT depleted cellular context, we found that ER fusions (notably ESR1-SOX9 and ESR1-YAP1) demonstrate ER hyperactivation through an estrogen response element (ERE) assay compared to ESR1-WT and a truncated exon 1-6 ESR1 (ESR1∆CTD). Importantly, fusion ERE activity was robust in the absence of the ER ligand, estradiol, as well as in the presence of endocrine therapies, implying ER fusion proteins function in a ligand independent, endocrine resistant mechanism. ER fusion positive cell lines were also enriched in oncogenic phenotypes such as enhanced 3D growth, cell survival via colony formation, and migration in a wound scratch assay. These enhanced metastatic potentials of the ER fusions were observed in both invasive ductal and lobular carcinoma cell lines, albeit at varying magnitudes depending on the 3’ fusion partner and phenotype being assessed. Although the ER fusions harbored unique characteristics due to the C-terminal partner, transcriptomic profiling revealed that enhanced EMT and KRAS signaling signatures were shared among all fusions when compared to the ESR1-WT and ESR1∆CTD cell lines, which may serve as future exploitable drug targets. Comprehensive detection and functional evaluation of ER fusion proteins will provide clinicians and patients with better understanding of tumor endocrine-resistant prevalence and discovery of more effective treatment options.

Citation Format: Megan E. Yates, Tiantong Liu, Jagmohan Hooda, Sichun Yang, Riyue Bao, Jennifer M. Atkinson, ADRIAN V. LEE, Steffi Oesterreich. PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-02.