Abstract P6-04-13: Preclinical and clinical analyses of estrogen receptor alpha mutant metastatic breast cancer
Authors: Richer Jennifer K , Williams Michelle M , Spoelstra Nicole S , Arnesen Spencer , Christenson Jessica L , O’Neill Kathleen , Reese Jordan M , Blanchard Zannel D , Hanamura Toru , Jacobsen Britta M , Gertz Jason , Elias Anthony 
Published: March 2020

Abstract
Background: Estrogen receptor alpha (ER) positive breast cancer (BC) that recurs as metastatic disease in patients on aromatase inhibitor therapy often harbors mutations in the last exon of the ESR1gene. Such mutations result in amino acid alterations in the ligand binding domain that confer a selective advantage because they render ER constitutively active under estrogen depleted conditions. Hypothesis: We hypothesized that the selective survival advantages gained by metastatic ER mutant (ERmut) tumor cells would become evident in preclinical models simulating the clinical context in which they arise (estrogen deprivation, anchorage independence or metastatic outgrowth) that could then be confirmed in ER+ patient derived xenografts (PDX) and biopsies of metastatic disease. Methods: MCF7 and T47D cells expressing flag-tagged ERmut Y537S or D538G or flag-tagged ER wild type (ERw/t) at the endogenous locus were generated using CRISPR/Cas9. These cells were studied in standard culture conditions as compared to anchorage independent soft agar or poly-HEMA coated plate assays and under long-term estrogen deprivation (LTED) conditions in vitro as well as in vivo in ovariectomized (ovx) mice in late stage metastasis models. ER+ patient derived xenografts (PDX) with naturally occurring ERmutY537S (HCI-013EI) and ERmutL536P (HCI-007) (Dr. Alana Welm) were also studied in vivo. Colony formation assays and tumor growth were evaluated in the context of treatments targeting ER (fulvestrant), androgen receptors (AR) (enzalutamide), steroid hormone synthesis (CYP17 lyase inhibitor seviteronel), or BRD4 Bromo- and Extra-terminal Domain (BET) inhibitor (BETi: JQ1). Drug synergy was determined in vitro using CalcuSyn software or in vivo by tumor caliper measurement and in vivo imaging of tumor luciferase activity. Biopsies of metastatic ER+ BC from clinical trial NCT02953860 were assayed for ESR1 exon 8 mutations using a modified Archer VariantPlex Solid Tumor Assay and immunohistochemistry performed for the four main steroid hormone receptors. Results: ERmutY537S or D538G containing MCF7 and T47D cells exhibited significantly increased anchorage independent survival, a condition in which AR is uniquely upregulated, while ER, progesterone receptors (PR), and glucocorticoid receptors (GR) are not. Furthermore, anti-androgens abrogated the ERmut survival advantage in soft agar. LTED ERmut lines expressed significantly more AR than their non-LTED predecessors or ERw/t counterparts as did ERmut PDX as compared to ERw/t PDX. MCF7 and T47D ERmutY537S and D538G demonstrated a significant increase in AR protein upon anchorage independent survival as compared to ERw/t, with ERmutD538G expressing particularly elevated AR. Biopsies of metastases from consenting patients with ER+ BC confirmed significantly higher AR protein in ERmut metastases as compared to ERw/t by IHC. Treatment of mice with ERmut PDX with combination fulvestrant and anti-androgen decreased tumor growth as compared mice given vehicle control or fulvestrant only. The BET inhibitor JQ1 synergized with anti-androgen to significantly decrease proliferation of ERmut cells. In ovx mice, only ERmut metastases were detectable, while ERw/t did not thrive at metastatic sites. ERmut cells differentially expressed AR-regulated, metastasis-promoting genes and proteins, including chitinase 3-like 1 (CHI3L1), when compared to those with ERw/t. Conclusions: AR is induced by stress encountered during disease progression and treatment, such as anchorage independence and estrogen deprivation. Targeting AR may improve the efficacy of agents such as fulvestrant that inhibit ERmut metastatic disease or delay recurrence, as may agents such as BETi that affect the activity of both ER and AR.
Citation Format: Jennifer K Richer, Michelle M Williams, Nicole S Spoelstra, Spencer Arnesen, Jessica L Christenson, Kathleen O’Neill, Jordan M Reese, Zannel D Blanchard, Toru Hanamura, Britta M Jacobsen, Jason Gertz, Anthony Elias. Preclinical and clinical analyses of estrogen receptor alpha mutant metastatic 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 P6-04-13.