Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics
Authors: Guan Jane , Zhou Wei , Daemen Anneleen , Hartman Steven J. , Blake Robert A. , Heidersbach Amy , Yu Mamie , Liang-Chu May , Martin Scott , Chalouni Cecile , Chen Irene , Friedman Lori S. , Wang Xiaojing , Metcalfe Ciara 
Published: August 2018

At least eight new molecular entities targeting the Estrogen Receptor (ER) have recently entered clinical development for ER+ breast cancer, highlighting the profound resurgence of interest in next generation ER therapeutics. The discovery of highly prevalent mutations in ESR1, the gene that encodes ERα, in recurrent ER+ disease, adds weight to the hypothesis that many patients fail ER-targeted agents because those therapeutic agents fail to fully disable ER, and underscores the relevance of ER as a drug target on which tumors depend. Strategies for the development of this new series of therapeutics have primarily centered around creating orally bioavailable ER ligands that belong to the same class of endocrine agent as fulvestrant, a selective ER downregulator (SERD), so named because of its ER degradation function. Though the clinical activity of fulvestrant is believed to be limited by its poor drug-like properties, a recent study demonstrated superiority of fulvestrant over the aromatase inhibitor, anastrozole. This phase III trial result supports the notion that therapeutics mechanistically similar to fulvestrant, but with improved bioavailability, may have best-in-disease potential. Thus, along with attention to pharmacokinetic properties, optimization of ER degradation has emerged as a key parameter in drug discovery. In contrast, the original drug discovery campaign that identified fulvestrant focused on the identification of ER ligands that, as well as antagonizing estrogen, lacked any ER agonist potential under estrogen-deprived conditions (i.e. were full ER antagonists), using the rodent uterus as a sensitive tool to screen out ligands with estrogenic potential. The rationale at the core of this strategy was that ER ligands that achieve full suppression of ER signaling would potentially drive superior efficacy versus the ER ligands available at the time, such as tamoxifen, which although suppressive relative to estrogen, promote weak ER agonism (i.e. partial ER agonist). The observation that fulvestrant promoted ER protein degradation was a retrospective discovery that provided a compelling explanation for how it might achieve full ER suppression. GDC-0810 and AZD9496 were the first ER ligands prospectively optimized for ER degradation in breast cancer cells, to enter clinical trials. Intriguingly though, both molecules display weak ER agonism in the uterus, suggesting that ER degradation in one context does not preclude ER agonism in another. A careful evaluation of GDC-0810 and AZD9496 across a collection of ER+ breast cancer cell lines revealed that despite optimization for ER degradation in MCF7 cells, both fail to promote ER turnover in several other cell lines. Transcriptional analysis of cells treated with GDC-0810 in hormone-deprived conditions further revealed that GDC-0810 can weakly activate ER signaling in breast cancer cells in which it does not induce ER turnover; thus partial ER agonism is not restricted to the uterus. We hypothesized that ligands that inhibit the ER ligand-binding domain (LBD), but do not induce robust ER turnover would trigger weak activation of ER; keeping in mind that in addition to the ligand-activated transactivation domain within the LBD, ER contains transactivation potential in a distinct N-terminal activation function 1 (AF1) domain. To explore this idea we leveraged GDC-0927 and GNE-274. GDC-0927 is in Phase I clinical studies, and like GDC-0810 and AZD9496 was optimized for ER degradation in MCF7 cells. However, GDC-0927 displays no ER agonism in the uterus. GNE-274 is a tool compound that is structurally related to GDC-0927 but does not induce ER turnover. GDC-0927 results in ER degradation in all ER+ cell lines tested, and retains a full antagonist profile even in lines where GDC-0810 displays partial agonism. As predicted, GNE-274 which does not result in ER turnover, functions as a partial ER agonist in breast cancer cell lines and in the uterus. Despite divergent transcriptional activity, GDC-0927 and GNE-274 are both highly/equi-potent inhibitors of the LBD, measured by displacement of co-activator peptides. These data suggest that inhibition of the LBD is not sufficient to suppress activity of the entire estrogen receptor. We propose that ER ligands that inhibit the LBD but fail to increase ER turnover enable ER signaling through activation of the AF1 domain. This paradigm has been proposed for 4OH-tamoxifen, and we extend this model. GDC-0927 and fulvestrant retain ER antagonist and degradation activity against the constitutively active ER.Y537S and ER.D538G variants commonly found in metastatic breast cancer. We speculated that other ER mutations might more dramatically influence the ER antagonist and/or degradation profile of these ligands, and thus performed an ER mutagenesis screen. We discovered a series of single amino acid substitutions that switch GDC-0927 and fulvestrant from full antagonists to weak agonists. Importantly, these mutations prevent GDC-0927- and fulvestrant-mediated degradation of ER, concomitant with their functional switch. This series of alterations appear to converge on disabling helix 12 (H12) - a key functional region of the LBD responsible for co-factor recruitment - since deletion of H12 had the same consequences as the single amino acid substitutions. These data suggest that, 1) H12 plays a key role in ER degradation mediated by GDC-0927 and fulvestrant, and 2) partial agonist activity likely arises from the AF1 domain, since the key functional region of the LBD required for recruitment of co-activators is disabled. Conventional views of ER degradation imply that loss of ER protein would drive the loss of all ER activities, based on observations of SERD-mediated ER protein loss in whole cell lysates. Intriguingly though, cellular fractionation assays demonstrate that ER ligands, regardless of degradation/full antagonist status, trigger rapid engagement with the DNA. In the case of fulvestrant and GDC-0927, re-localization of ER to DNA precedes loss of ER protein evident in whole cell lysates. Even at later time-points, however, when ER protein levels in whole cell lysates are low, ER is retained at the chromatin. A surprising observation we made is that although all ER ligands assessed promote ER DNA engagement, we could distinguish the behavior of the partial agonists from full antagonists prior to loss of ER protein, using chromatin immunoprecipitation (CHIPseq) assays as well as live imaging of cells expressing mNeon-ER. Specifically, FRAP (fluorescence recovery after photobleaching) demonstrated that while partial agonists 4OH-tamoxifen and GNE-274 maintain high mobility of ER, the full antagonists fulvestrant and GDC-0927 profoundly decrease ER mobility, suggesting that the immobilization of ER is likely a general property of full antagonists. In summary, we find that full ER antagonism, defined by suppression of both the LBD and the AF1 domain, is accompanied by robust ER degradation. Notably though, ligands capable of promoting ER degradation in some cell lines are not necessarily full antagonists in all cellular contexts. We argue that the designation of “SERD”, which is ascribed to molecules that show even context-dependent ER degradation activity, does not imply that a molecule belongs to the same mechanistic/therapeutic class as fulvestrant. ER ligands that are truly devoid of partial agonism - the key driver behind the identification and development of fulvestrant - remain a small class. The observation that full ER antagonism invariably associates with ER degradation could be interpreted as degradation driving full antagonism. However, we have discovered that full antagonists can be distinguished from partial agonists prior to the loss of ER protein, suggesting that ER degradation may be a consequence of full suppression of ER, rather than a driver of full suppression.

Citation Format: Jane Guan, Wei Zhou, Anneleen Daemen, Steven J. Hartman, Robert A. Blake, Amy Heidersbach, Mamie Yu, May Liang-Chu, Scott Martin, Cecile Chalouni, Irene Chen, Lori S. Friedman, Xiaojing Wang, Ciara Metcalfe. Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr NG05.