Abstract 3440: Targeting the mutant estrogen receptor in metastatic breast cancer

Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection