Abstract The contribution of neoantigen-specific T cells to PD-(L)1 efficacy has largely been inferred from tumor mutational burden. We functionally profiled circulating T cell responses against 7,038 predicted HLA-I–restricted and 21,453 HLA-II–restricted neopeptides in 27 patients with advanced non-small cell lung cancer treated with anti–PD-(L)1. CD4 responses were frequent and correlated with neoantigen availability but not clinical benefit. In contrast, the magnitude and breadth of neoantigen-specific CD8 T cell responses were associated with clinical benefit, progression-free and overall survival, independently of tumor mutational burden. Patients mounting coordinated CD4 and CD8 responses experienced improved progression-free survival. Tumors from CD8 responders displayed immune signatures indicative of both T cell priming and effector functions. Circulating neoantigen-specific CD8 T cells recognized endogenously processed antigens, trafficked to tumors, and selectively expanded under therapy while retaining CD28, CD226, and CXCR3 expression. These findings identify coordinated, functionally engaged neoantigen-specific T cell responses as central determinants of PD-(L)1 efficacy.

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields. ### Competing Interest Statement KMD and AH are current employees of Genentech, and BH and JPF have previously been employees of Genentech, where the CRISPRa mouse libraries presented in this work were developed. A provisional patent application that relates to the work has been filed.

Aberrant activation of the mitogen-activated protein kinase (MAPK) cascade promotes oncogenic transcriptomes. Despite efforts to inhibit oncogenic kinases, such as BRAFV600E, tumor responses in patients can be heterogeneous and limited by drug resistance mechanisms. Here, we describe patient tumors that acquired COP1 or DET1 mutations after treatment with the BRAFV600E inhibitor vemurafenib. COP1 and DET1 constitute the substrate adaptor of the E3 ubiquitin ligase CRL4COP1/DET1, which targets transcription factors, including ETV1, ETV4, and ETV5, for proteasomal degradation. MAPK-MEK-ERK signaling prevents CRL4COP1/DET1 from ubiquitinating ETV1, ETV4, and ETV5, but the mechanistic details are still being elucidated. We found that patient mutations in COP1 or DET1 inactivated CRL4COP1/DET1 in melanoma cells, stabilized ETV1, ETV4, and ETV5, and conferred resistance to inhibitors of the MAPK pathway. ETV5, in particular, enhanced cell survival and was found to promote the expression of the pro-survival gene BCL2A1. Indeed, the deletion of pro-survival BCL2A1 re-sensitized COP1 mutant cells to vemurafenib treatment. These observations indicate that the post-translational regulation of ETV5 by CRL4COP1/DET1 modulates transcriptional outputs in ERK-dependent cancers, and its inactivation contributes to therapeutic resistance.

Tumor-specific T cells play a central role in tumor control, yet the attainment of their full tumor rejection potential is hindered by regulatory mechanisms, including expression of inhibitory immune checkpoints. Although immunotherapy by immune checkpoint blockade (ICB) has transformed cancer care, only a proportion of patients experience clinical benefit. Ancillary studies to clinical trials evaluating ICB have revealed an association between high tumor mutational burden (TMB) and treatment efficacy, suggesting that unleashing T cells targeting neoepitopes encoded by somatic mutations could underlie ICB-mediated tumor control or regression. Nevertheless, TMB or tumor neoantigenic burden (TNB) are not consistently related to clinical response, potentially because most studies have addressed the influence of TMB and TNB by in silico prediction without systematic analysis of actual neoantigen-specific T cells. We hypothesized that the presence of actual neoantigen-specific T-cell responses would predict response to ICB, rather than the mere presence of a high TMB or TNB. Using whole exome and transcriptome sequencing in a cohort of 29 non-small cell lung cancer patients receiving anti-PD-1/PD-L1, we assessed expressed non-synonymous exonic mutations in each patient's tumor and predicted the encoded neoepitopes restricted by each patient's HLA-I and -II alleles. In our cohort, HLA-I TNB was better correlated to clinical response and progression free survival (PFS) than TMB and HLA-II TNB. We developed a workflow allowing for the assessment of circulating CD8 T-cell responses to more than 400 prioritized neoepitopes, and to 50 long peptides harboring each several predicted CD4 neoepitopes containing the same mutated amino acid. We showed, in 28 patients from whom PBMCs were available, that circulating CD8 T-cell responses to predicted neoantigens were of higher magnitude and breadth in clinical responder compared to non-responder patients. The presence of a CD8 T-cell response to neoantigens was associated with better PFS. No correlation was observed between expressed TMB or TNB (HLA-I or -II) and the magnitude of CD8 or CD4 T-cell responses. Longitudinal ex vivo enumeration of neoantigen-specific CD8 T-cells using HLA/peptide tetramers showed that they had a memory phenotype and that they experienced significant proliferation under therapy. Our results ascertain the contribution of neoantigen-specific CD8 T-cells in therapeutic tumor rejection. They also provide an opportunity, through the analysis of tumor molecular signatures, to unveil the mechanisms supporting or restraining the priming of spontaneous T-cell responses targetable by ICB. This research was a collaborative effort by sites within the imCORE network, made possible through support from F.Hoffmann-La Roche. Célia Ramade, Noémie Thébault, Clara-Maria Scarlata, Françoise Lauzéral-Vizcaino, Caroline Fournier, Victor Sarradin, Anna Salvioni, Marie Michelas, Daniel Oreper, Suchit Jhunjhunwala, William Thrift, Adric Quade Broadwell, Nicolas Lounsbury, Kai Liu, Meng Xiao He, Martine Darwish, Catherine Ross, Hang Xu, Milena Hornburg, Amy Heidersbach, Fanny Bouquet, Catia Fonseca, Laetitia Tudeau, Nicolas Congy-Jolivet, Carlos Gomez-Roca, Thomas Filleron, Lélia Delamarre, Julien Mazières, Jean-Pierre Delord, Maha Ayyoub. Circulating CD8 T-cell responses to neoantigens predict responsiveness to PD-(L)1 axis blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6037.

Based on the success of cancer immunotherapy, personalized cancer vaccines have emerged as a leading oncology treatment. Antigen presentation on MHC class I (MHC-I) is crucial for the adaptive immune response to cancer cells, necessitating highly predictive computational methods to model this phenomenon. Here, we introduce HLApollo, a transformer-based model for peptide-MHC-I (pMHC-I) presentation prediction, leveraging the language of peptides, MHC, and source proteins. HLApollo provides end-to-end treatment of MHC-I sequences and deconvolution of multi-allelic data, using a negative-set switching strategy to mitigate misassigned negatives in unlabelled ligandome data. HLApollo shows a 12.65% increase in average precision (AP) on ligandome data and a 4.1% AP increase on immunogenicity test data compared to next-best models. Incorporating protein features from protein language models yields further gains and reduces the need for gene expression measurements. Guided by clinical use, we demonstrate pan-allelic generalization which effectively captures rare alleles in underrepresented ancestries.

CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with an optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.

Summary Strategies for maximizing the potency and specificity of cancer immunotherapies have sparked efforts to identify recurrent epitopes presented in the context of defined tumor-associated neoantigens. Discovering these “neoepitopes” can be difficult owing to the limited number of peptides that arise from a single point mutation, a low number of copies presented on the cell surface, and variable binding specificity of the human leukocyte antigen (HLA) class I complex. Due to these limitations, many discovery efforts focus on identifying neoepitopes from a small number of cancer neoantigens in the context of few HLA alleles. Here we describe a systematic workflow to characterize binding and presentation of neoepitopes derived from 47 shared cancer neoantigens in the context of 15 HLA alleles. Through the development of a high-throughput neoepitope-HLA binding assay, we surveyed 24,149 candidate neoepitope-HLA combinations resulting in 587 stable complexes. These data were supplemented by computational prediction that identified an additional 257 neoepitope-HLA pairs, resulting in a total of 844 unique combinations. We used these results to build sensitive targeted mass spectrometry assays to validate neoepitope presentation on a panel of HLA-I monoallelic cell lines engineered to express neoantigens of interest as a single polypeptide. Altogether, our analyses detected 84 unique neoepitope-HLA pairs derived from 37 shared cancer neoantigens and presented across 12 HLA alleles. We subsequently identified multiple TCRs which specifically recognized two of these neoantigen-HLA combinations. Finally, these novel TCRs were utilized to elicit a T cell response suggesting that these neoepitopes are likely to be immunogenic. Together these data represent a validated, extensive resource of therapeutically relevant neoepitopes and the HLA context in which they can be targeted.

Antigen presentation on MHC class I (MHC-I) is key to the adaptive immune response to cancerous cells. Computational prediction of peptide presentation by MHC-I has enabled individualized cancer immunotherapies. Here, we introduce HLApollo, a transformer-based approach with end-to-end modeling of MHC-I sequence, deconvolution, and flanking sequences. To achieve this, we develop a novel training strategy, negative set switching, which greatly reduces overfitting to falsely presumed negatives that are necessarily found in presentation datasets. HLApollo shows a meaningful improvement compared to recent MHC-I models on peptide presentation (20.19% average precision (AP)) and immunogenicity (4.1% AP). As expected, adding gene expression boosts the performance of HLApollo. More interestingly, we show that introduction of features from a protein language model, ESM 1b, remarkably recoups much of the benefits of gene expression in absence of true expression measurements. Finally, we demonstrate excellent pan-allelic generalization, and introduce a framework for estimating the expected accuracy of HLApollo for untrained alleles. This guides the use of HLApollo in a clinical setting, where rare alleles may be observed in some subjects, particularly for underrepresented minorities.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

Abstract CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been thoroughly exploited in this context. Here we establish a CRISPRa mouse (dCas9a-SAMKI/+) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI/KI primary lymphocytes, we induced B cell restricted genes in the T cell lineage and vice versa, demonstrating the power of this system. Next, to model double hit lymphoma (DHL), we transactivated pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Lethally-irradiated mice transplanted with these cells rapidly developed lymphomas expressing high BCL-2. Unlike standard Eµ-Myc lymphomas, BCL-2-expressing lymphomas were highly sensitive to the BCL-2 inhibitor venetoclax. Finally, we performed genome-wide activation screens in these lymphoma cells and found a dominant role for the BCL-2 family protein A1 in venetoclax resistance. This demonstrates the power of our CRISPRa model for mimicking disease and provides insights into potential resistance mechanisms towards targeted therapies.

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.

BACKGROUND:Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis.METHODS:APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity.RESULTS:APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes.CONCLUSIONS:Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER “degraders” exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138− cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1α inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1α with kinase-based small-molecule inhibitors in MM.Significance statement Multiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding the ER’s protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1α elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1α as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1α-XBP1s pathway plays a critical role in MM growth. We further show that IRE1α’s kinase domain is an effective and safe potential small-molecule target for MM therapy.

Abstract 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.