Proximity-inducing compounds that modulate target protein homeostasis represent an emerging therapeutic strategy. While the inherent complexity of these bifunctional compounds presents certain challenges, their unique composition offers opportunities to co-opt specific cellular effectors to enhance therapeutic impact. In this study, we systematically evaluate a series of bifunctional degrader compounds engineered with the estrogen receptor-alpha (ERα) inhibitor endoxifen linked to various bioactive ubiquitin ligase ligands. Notably, ERα degraders containing pan-IAP antagonist ligands significantly reduced the proliferation of ERα-dependent cells compared to clinical-stage ERα degraders. These pan-IAP antagonist-based ERα degraders leverage distinct effector ligases to achieve dual therapeutic effects: They utilize XIAP within tumor cells to promote ERα degradation and activate cIAP1/2 in both tumor and immune cells to induce TNFα, which drives tumor cell death. Our findings illustrate a broader concept that co-opting the discrete functions of selected cellular effectors, while simultaneously modulating therapeutic target protein homeostasis, are dual strategies that can significantly enhance the efficacy of induced proximity therapeutics.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.

Targeted protein degradation with proteolysis targeting chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway. Here we identified RNF146 as a ubiquitin ligase (E3) that can catalyze TEAD ubiquitination and negatively regulate their function in cells. We show that this ubiquitin of TEADs is governed by their PARylation state and validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Furthermore, we demonstrate that pharmacologically induced ubiquitination of TEADs by heterobifunctional chemical inducers of protein degradation (CIDE) molecules can promote potent pan-TEAD degradation. These TEAD-CIDEs can effectively suppress activation of TEAD target genes in a dose-dependent manner and exhibited significant anti-proliferative effects in Hippo-dependent tumor cells, thus phenocopy the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates a post-translational mechanism of TEAD protein regulation and provides a proof-of-concept demonstration that pharmacological induced TEAD ubiquitination could be an effective therapeutic strategy to target Hippo-driven cancers.

β-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drives the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging deeming this transcription factor as "undruggable." Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify a monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 through targeting four distinct cysteines within the armadillo repeat domain-C439, C466, C520, and C619-leading to a destabilization of CTNNB1. Using covalent chemoproteomic approaches, we show that EN83 directly engages CTNNB1 in cells with a moderate degree of selectivity. We further demonstrate that direct covalent targeting of three of these four cysteines--C466, C520, and C619--in cells contributes to CTNNB1 degradation in cells. We also demonstrate that EN83 can be further optimized to yield more potent CTNNB1 binders and degraders. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through a destabilization-mediated degradation.

Modulation of proteolysis is an emerging therapeutic mainstay. The clinical success of thalidomide and analogs has inspired development of rationally-designed therapeutics that repurpose endogenous degradation machinery to target pathogenic proteins. However, it is unknown whether target removal is the critical effect that drives degrader-induced efficacy. Here we report that proteasome-generated peptides actively initiate degrader-induced cell death. Utilizing BET family degraders as exemplars, we find that induced proteasomal degradation of the BRD4-long isoform (BRD4-L) generates neo-amino-terminal peptides that neutralize Inhibitor of Apoptosis (IAP) proteins to precipitate cell death. Depletion of BRD4-L paradoxically suppresses caspase activation induced by numerous BET degraders. An unbiased screen revealed that other degrader compounds, including clinical CELMoDs, rely on the same mechanism to potentiate caspase activation and apoptosis. Finally, in the context of constitutive immunoglobulin proteostasis within multiple myeloma cells, we report that therapeutic proteasomal protease inhibition alters the peptide repertoire to neutralize IAPs, thus contributing to the clinical efficacy of bortezomib. Together, these findings clarify the counterintuitive clinical benefit achieved by combining thalidomide analogs with proteasome inhibitors. Our study reveals a previously unrealized pro-apoptotic function of the peptides generated by a variety of proteolysis-modulating compounds, that provide design considerations to maximize therapeutic benefit.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN’s thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo–electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)–CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

The ubiquitin system is complex, multifaceted, and is crucial for the modulation of a vast number of cellular processes. Ubiquitination is tightly regulated at different levels by a range of enzymes including E1s, E2s, and E3s, and an array of DUBs. The UPS directs protein degradation through the proteasome, and regulates a wide array of cellular processes including transcription and epigenetic factors as well as key oncoproteins. Ubiquitination is key to the dynamic regulation of programmed cell death. Notably, the TNF signaling pathway is controlled by competing ubiquitin conjugation and deubiquitination, which governs both proteasomal degradation and signaling complex formation. In the inflammatory response, ubiquitination is capable of both activating and dampening inflammasome activation through the control of either protein stability, complex formation, or, in some cases, directly affecting receptor activity. In this review, we discuss the enzymes and targets in the ubiquitin system that regulate fundamental cellular processes regulating cell death, and inflammation, as well as disease consequences resulting from their dysregulation. Finally, we highlight several pre-clinical and clinical compounds that regulate ubiquitin system enzymes, with the aim of restoring homeostasis and ameliorating diseases.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Abstract Purpose: Treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge, largely due to the genetic heterogeneity of these malignancies. Our study goal was to design a rational treatment strategy that simultaneously targets multiple disease drivers to provide safe, efficacious, and durable responses in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Experimental procedures and data summary: Venetoclax is a selective BCL-2 inhibitor that induces apoptosis and is efficacious in chronic lymphocytic leukemia; however, single-agent efficacy in other lymphoid neoplasms is limited. Using pharmacologic and genetic studies, we identified the pro-survival BCL-2 protein family member MCL-1 as a venetoclax resistance factor in NHL cell lines. We found that the monomethyl auristatin E (MMAE) payload of the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system, thus providing a strong rationale for combination with venetoclax. Mechanistically, polatuzumab vedotin combined with venetoclax promoted apoptotic NHL cell death. In NHL animal models, venetoclax, polatuzumab vedotin, and the anti-CD20 antibody obinutuzumab, that activates targeted antibody- and complement-dependent cell cytotoxicity, produced durable tumor regressions. In a phase 1b clinical trial, 33 patients with relapsed or refractory follicular lymphoma were evaluable for response over 6 dose levels with 19 complete responses and 6 partial responses (overall response rate 76%). All patients (8 of 8) treated with the recommended phase 2 regimen (venetoclax 800mg + polatuzumab vedotin 1.8mg/kg + obinutuzumab 1000mg) achieved complete responses at end of induction. The median duration of follow-up for all dose levels was 17.7 months. This triplet combination was well-tolerated by most patients and had an expected and acceptable safety profile. Conclusions: Our studies implicate MCL-1 as a key venetoclax resistance factor in NHL that can be overcome by polatuzumab vedotin, which promotes MCL-1 degradation. Pre-clinical NHL animal models and early clinical data confirmed that the combination of venetoclax, polatuzumab vedotin, and obinutuzumab is safe and promotes durable responses. Because MMAE and other anti-tubulin agents are likely not the only therapeutics that antagonize MCL-1, our study provides scientific rationale to pursue the broader strategy of identifying other therapeutics that similarly neutralize MCL-1 function. Such agents could be used in combination with venetoclax in other malignancies where MCL-1 is a venetoclax resistance factor. Our mechanism-based treatment regimen that directly targets oncogenic drivers in NHL patients may serve as a framework for treating other malignancies with complex etiologies. Citation Format: Dhara N. Amin, Rajat Bannerji, Raghuveer Singh Mali, Jason Oeh, Eva Lin, Anuradha Zindal, MaryAnn Go, Shang-Fan Yu, Maxwell Krem, Chris Arthur, Uwe Hahn, Anna M. Johnston, Vinit G. Karur, Nadia Khan, Paula Marlton, Tycel Phillips, Giuseppe Gritti, John F. Seymour, Monica Tani, Sam Yuen Yuen, Scott Martin, Matthew T. Chang, Christopher M. Rose, Victoria C. Pham, Elizabeth A. Lasater, Andrew G. Polson, YiMeng Chang, Jamie Hirata, Lisa Musick, Deepak Sampath, Christopher R. Flowers, Ingrid E. Wertz. Targeting BCL-2 and MCL-1 overcomes treatment resistance in relapsed and refractory non-Hodgkin lymphoma: Pre-clinical rationale and results from an open-label phase 1b study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT133.

Estrogen Receptor-positive (ER+) breast cancer is a significant unmet medical need. Tumors expressing ER exhibit evidence of mitogenic ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents. Such continued dependence on ER signaling highlights the need for next generation therapies that more effectively block ER function in tumors. Fulvestrant was first discovered as a “pure antiestrogen”, in contrast to earlier generation ER therapeutic ligands that exhibit weak agonistic activity, such as tamoxifen. After its discovery as a full ER antagonist, fulvestrant was demonstrated to decrease ER protein levels through proteasome-mediated degradation. These observations led to the compelling hypothesis that elimination of ER by fulvestrant drives suppression of ER signaling. Importantly however, we recently demonstrated that fulvestrant and other full ER antagonists (e.g. GDC-0927) dramatically slow the intranuclear mobility of ER [Cell 178:4 (2019)]. We argue that such immobilization prevents ER function, and that increased ER turnover is a downstream consequence of immobilization, rather than a cause of ER inhibition. Here, we describe GDC-9545, our latest generation ER antagonist, currently being evaluated in clinic, that robustly immobilizes ER, and drives profound ER suppression in vivo. In the HCI-011 PDX model, we find that GDC-9545 can achieve greater ER pathway inhibition than can be achieved by tamoxifen. Intriguingly, although tamoxifen exhibits partial ER inhibition, likely through preventing recruitment of co-activators to the ER ligand binding domain, it drives increased accessibility at ~2500 chromatin sites, as determined by ATAC-seq. Chromatin regions exhibiting increased accessibility upon tamoxifen treatment are significantly enriched for the ERE motif, while a smaller number of sites exhibiting decreased accessibility upon treatment are enriched for AP-1 motifs. In contrast, GDC-9545 profoundly decreases chromatin accessibility at both ERE and AP-1 motifs, despite not fully eliminating ER protein. Notably, sites exhibiting decreased accessibility upon GDC-9545 treatment in the PDX in vivo, significantly overlap with sites displaying increased accessibility in estrogen-stimulated MCF7 cells in vitro. GDC-9545-treatment additionally alters accessibility at sites enriched for the FOXA1 motif, though unexpectedly, a sub-set of these sites exhibit increased accessibility while a distinct sub-set of sites exhibit decreased accessibility. We speculate that this particular pattern of accessibility changes may reflect redistribution of FOXA1 upon GDC-9545 treatment, and we will further explore this hypothesis. These data provide further insights into the impact of ER immobilization by the latest generation of pure antiestrogens. Citation Format: Ciara Metcalfe, Wei Zhou, Jane Guan, Robert A. Blake, Tom De Bruyn, Jennifer M. Giltnane, Ellen Ingalla, Tracy Kleinheinz, Jun Liang, Vidhi Mody, Jason Oeh, Savita Ubhayakar, Ingrid Wertz, Amy Young, Jason Zbieg, Xiaojing Wang, Marc Hafner. GDC-9545: A pure antiestrogen clinical candidate that immobilizes the estrogen receptor and profoundly alters chromatin accessibility in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3406.

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.

Abstract Estrogen Receptor-positive (ER+) breast cancer is a significant unmet medical need. Tumors expressing ER exhibit evidence of mitogenic ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents. Such continued dependence on ER signaling highlights the need for next generation therapies that more effectively block ER function in tumors. Fulvestrant was first discovered as a “pure antiestrogen”, in contrast to earlier generation ER therapeutic ligands that exhibit weak agonistic activity, such as tamoxifen. After its discovery as a full ER antagonist, fulvestrant was demonstrated to decrease ER protein levels through proteasome-mediated degradation. These observations led to the compelling hypothesis that elimination of ER by fulvestrant drives suppression of ER signaling. Importantly however, we recently demonstrated that fulvestrant and other full ER antagonists (e.g. GDC-0927) dramatically slow the intranuclear mobility of ER [Cell 178:4 (2019)]. We argue that such immobilization prevents ER function, and that increased ER turnover is a downstream consequence of immobilization, rather than a cause of ER inhibition. Here, we describe GDC-9545, our latest generation ER antagonist, currently being evaluated in clinic, that robustly immobilizes ER, and drives profound ER suppression in vivo. In the HCI-011 PDX model, we find that GDC-9545 can achieve greater ER pathway inhibition than can be achieved by tamoxifen. Intriguingly, although tamoxifen exhibits partial ER inhibition, likely through preventing recruitment of co-activators to the ER ligand binding domain, it drives increased accessibility at ~2500 chromatin sites, as determined by ATAC-seq. Chromatin regions exhibiting increased accessibility upon tamoxifen treatment are significantly enriched for the ERE motif, while a smaller number of sites exhibiting decreased accessibility upon treatment are enriched for AP-1 motifs. In contrast, GDC-9545 profoundly decreases chromatin accessibility at both ERE and AP-1 motifs, despite not fully eliminating ER protein. Notably, sites exhibiting decreased accessibility upon GDC-9545 treatment in the PDX in vivo, significantly overlap with sites displaying increased accessibility in estrogen-stimulated MCF7 cells in vitro. GDC-9545-treatment additionally alters accessibility at sites enriched for the FOXA1 motif, though unexpectedly, a sub-set of these sites exhibit increased accessibility while a distinct sub-set of sites exhibit decreased accessibility. We speculate that this particular pattern of accessibility changes may reflect redistribution of FOXA1 upon GDC-9545 treatment, and we will further explore this hypothesis. These data provide further insights into the impact of ER immobilization by the latest generation of pure antiestrogens. Citation Format: Ciara Metcalfe, Wei Zhou, Jane Guan, Robert A. Blake, Tom De Bruyn, Jennifer M. Giltnane, Ellen Ingalla, Tracy Kleinheinz, Jun Liang, Vidhi Mody, Jason Oeh, Savita Ubhayakar, Ingrid Wertz, Amy Young, Jason Zbieg, Xiaojing Wang, Marc Hafner. GDC-9545: A pure antiestrogen clinical candidate that immobilizes the estrogen receptor and profoundly alters chromatin accessibility in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3406.