Abstract PD7-11: Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers

Abstract

Abstract Background: Endocrine therapy is used in estrogen receptor (ER)-positive breast cancers, however, 25% of these patients are at risk of distant relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been validated to be acquired during the development of endocrine resistance. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity and emerges subclonally during aromatase inhibitor treatment. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to a novel checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in steroid deprived conditions. Cell cycle analysis and apoptosis were examined by flow cytometry annnexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar and MTT assays. Replication stress was identified by RPA32 and gamma-H2AX foci formation assay. For in vivo studies, MCF-7 ESR1 Y537S mutant cells were injected into female athymic nude mice with 17β-estradiol (E2) supplemented water. When tumors reached 350 mm3, tamoxifen (20 mg/kg; s.c.; three times a week), fulvestrant (200 mg/kg; s.c; once a week) and/or PF477736 Chk1 inhibitor (7.5 mg/kg; i.p.; twice a day and twice a week) was treated without E2. Results: ESR1 Y537S mutant cells accumulated approximately 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen-deprived (ED) conditions. BrdU incorporation also increased about 2.5-fold, however, apoptosis was decreased about 60 % compared with wild-type ER parental cells. ESR1 Y537S mutant cells induced significant replication stress, showing increased RPA32 foci together with increased gamma H2AX foci, a marker of DNA double-stranded breaks. ChIP-seq analysis revealed binding sites on ATR and CHEK1 genomic locations. ATR/Chk1-mediated checkpoint signaling was activated in ESR1 Y537S mutant cells, and was repressed with fulvestrant, tamoxifen, or ESR1 siRNA treatment. The Chk1 inhibitor, PF477736, sensitized MCF-7 expressing the ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen, and the ER degrader AZD9496 in cell proliferation assays. In MCF-7 ESR1 Y537S mutant xenograft and patient derived mouse models, tamoxifen treatment combined with the Chk1 inhibitor PF477736 repressed primary xenograft tumor doubling times (P=0.038, Wilcoxon test). Treatment of mutant tumors with PF477736 together with fulvestrant significantly inhibited the frequency of distant lung metastases by 80% (P=0.0031, t-test), suggesting that these combinations may be useful in second line treatment of metastatic breast cancer patients resistant to endocrine therapies. Conclusion: These preclinical results suggest that ESR1 mutant tumors have a therapeutic vulnerability to combination endocrine therapy with cell cycle checkpoint kinase inhibitors. These data demonstrate that this new therapeutic approach may be useful to restore endocrine sensitivity in metastatic breast cancer patients with ESR1 mutation driven-endocrine resistance. Citation Format: Kim J-A, Dustin D, Gu G, Corona-Rodriguez A, Edwards D, Coarfa C, Keyomarsi K, Fuqua SA. Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-11.