Abstract P5-04-11: Non-canonical, clinical ESR1 mutations promote resistance to antiestrogen therapies

Abstract

ESR1 hotspot mutations have been identified in 30-40% of patients with ER+ MBC and promote resistance to aromatase inhibitors (AIs). Identification of these mutations has been aided by the use of plasma DNA for their detection, however many such tests only survey for hotspot mutations. In this study, we examined the prevalence, biologic and clinical significance of mutations in ESR1 that lie outside previously described hotspots (E380Q, Y537, D538G). Using next generation sequencing of tumor DNA from over 4000 patients with breast cancer, we have identified numerous somatic alterations in ESR1. Among the somatic alterations were mutations detected in the transcription activation function-1 (AF-1), DNA binding domain, dimerization interface and C-terminus of ER. We characterized the functional significance of these non-canonical mutations alongside hotspot mutations, starting with assays of ER driven transcription and deduced several classes of mutations: (1) mutations that weakly promote ligand-independent activity, (2) mutations that led to ligand-independent activity comparable to estradiol stimulation, and (3) mutations that resulted in impaired transcriptional activity. Class 2 mutations remain localized at amino acids 536-538, while the class 1 mutations are observed in various domains of ESR1, including the DNA binding domain and dimerization interface. Several Class 3 mutations were found in proximal to Helix 12, highlighting the critical role of this region. Clinically, non-canonical mutations were not exclusively observed among patients treated with AI, as there were several mutations from SERM/SERD treated patients. We thus examined the effects of different mutants on their sensitivity to ER antagonists, such as fulvestrant or tamoxifen. The data revealed key differences between the different classes of mutants; with majority of the class 2 mutants exhibiting reduced sensitivity to the antagonists compared to wild type. This also correlated with the relative binding affinities (RBA) of the mutants to fulvestrant and 4-hydroxytamoxifen, in which the RBA of class 2 mutants (Y537S and D538G) were significantly lower than wild type, perhaps accounting for their reduced sensitivities. Nevertheless, it appeared that all mutants could be effectively inhibited either by higher concentrations or more potent ER antagonists, implying a continued ability to distort ER into the antagonist conformation. Interestingly, several Class 1 mutants showed weak agonism in response to specific antagonists raising the possibility of their role in SERM/SERD resistance. Taken together, the data demonstrated that not all ESR1 mutations affect receptor function or respond to antiestrogen therapies similarly. These data also imply the importance of more broad sequencing coverage of ESR1 in the clinic to effectively capture the spectrum of biologically relevant alleles. Citation Format: Toy W, Carlson KE, Martin TA, Razavi P, Berger M, Baselga J, Greene G, Katzenellenbogen J, Chandarlapaty S. Non-canonical, clinical ESR1 mutations promote resistance to antiestrogen therapies [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 P5-04-11.

Unperturbed dormancy recording reveals stochastic awakening strategies in endocrine treated breast cancer cells

“…Conversely, the Tꞵ carbon copy offered a glimpse of the temporal order connecting nongenetic and genetic events. In this particular case, cells acquire a non-canonical ESR1 H524L mutation, which has been previously shown to promote Tamoxifen resistance (Chung et al, 2017;Toy et al, 2019) after nongenetic awakening (Fig. 4C-D).…”

Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells

“…S15A). Two of these, p.K268N and p.A64D, are neutral with p.K268N possibly already present in the POTs, whereas the p.H524L mutation might be associated with TAM resistance ( 31 ) but emerged exclusively in the TAMβ carbon copy before becoming subclonal in TAMβ TEP (terminal endpoint or late progression, obtained from awakening samples kept in culture 1 more month with the introduction of serial passaging), raising doubts on its link to the awakening lineage ( Fig. 2E ).…”

Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

“…It is now becoming evident that the mutational landscape of ESR1 in refractory metastases is complex, with several lower frequency mutations being enriched in the metastatic setting (naïve or treatment-related) for which the function is unclear ( 29 ). It is conceivable that like H524L/Y, other low-frequency mutations may similarly impact interactions with ligands to drive resistance to existing therapies or more generically influence ERα conformation/function to influence disease progression/resistance.…”