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Estrogen receptor (ER) α is expressed in the vast majority of breast cancers and is one of the most successfully prosecuted drug targets in oncology, with multiple classes of endocrine therapies approved for the treatment of ER+ breast cancer. These existing agents are highly active, both as single agents and as combination partners for other targeted therapies, and have significantly benefited patients. However, each of these standard-of-care (SOC) therapies has liabilities that allow for the reengagement of ER signaling as a mechanism of resistance. Data supporting the continued dependence of tumors on ER signaling following exposure to SOC agents have underpinned an extraordinary reenergizing of academic, biotechnology, and pharmaceutical groups pursuing next-generation ER-targeted therapies. The hypothesis that there remains an opportunity to bring further meaningful benefit to patients through fully optimized ER-targeted therapies is currently being investigated in the clinic.

The progression of HR+ breast cancers is governed by ER signaling which makes the development of optimized ER-targeting agents a high priority. Among current therapeutics, selective ER antagonists and degraders (SERDs) have emerged as a particularly attractive class. Fulvestrant is the only SERD that is currently approved, but its clinical potential is hypothesized to be somewhat limited by poor physicochemical properties, dosage and exposure. We identified giredestrant as an orally bioavailable, highly potent non-steroidal ER antagonist, immobilizer and degrader with robust anti-proliferative activity. Giredestrant is currently being evaluated in phase III clinical trials for the treatment of ER+ breast cancer, after showing evidence of clinical activity in phase I and II trials. Here, we set out to investigate the impact of giredestrant on the transcriptome and chromatin landscape of ER+ breast cancer cells in vitro and in vivo. RNA-seq data from MCF-7 cells in vitro and three patient-derived xenograft (PDX) models in vivo showed that giredestrant inversely regulates the RNA levels of E2-regulated genes. Transcriptome wide, the expression of genes affected by giredestrant are strongly correlated (r2=0.8) with those affected by siRNA-mediated silencing of ESR1, consistent with on-target ER antagonism. Performing ATAC-seq in the three PDX models, including one ESR1 mutant model, reveals that giredestrant reduces accessibility in up to 18,173 chromatin regions that are associated with genes induced by E2. These regions are also enriched in the ER binding motif. In addition, we found up to 17,127 chromatin regions with increased accessibility upon giredestrant treatment. These regions are associated with genes suppressed by E2, and are enriched in the FOXA1 binding motif. To probe giredestrant’s role in functional modulation of cis-regulatory elements (CREs), we performed ChIP-seq targeting FOXA1 and GATA3, as key pioneer factors for ER, as well as ER itself and H3K27ac as a marker of active enhancers in MCF-7 cells. We identified 3,195 regions with significantly reduced H3K27ac signals that also exhibit significant reduction of both ER and FOXA1 binding upon giredestrant treatment, indicating that giredestrant deactivates these CREs concomitantly with suppressing ER and FOXA1 binding. ChIP-seq analysis further revealed 2,292 regions with a significant increase in H3K27ac signals upon giredestrant treatment. Interestingly, the GATA3 motif was identified as the most enriched motif in these activated CREs. Further, GATA3 and FOXA1 ChIP-seq signals were stronger in the activated CREs compared to the deactivated ones. Similar profiling of both tamoxifen, an approved selective ER modulator (SERM), as well an GNE-858, a SERM from the same chemical series as giredestrant, showed their epigenetic effects to be highly distinct from those observed for giredestrant, suggesting that the high degree of chromatin remodeling is likely related to giredestrant’s mechanism of action. Our studies here show that giredestrant profoundly alters ER binding and remodels the chromatin landscape, thus changing the transcriptional profile of cells in a manner that is associated with an anti-proliferative response. While deactivation of CREs with ER binding is perhaps in line with expectations, giredestrant treatment also leads to the activation of a number of regions associated with pioneer factors, potentially through their redistribution. This raises the question of the functional consequences of CRE activation and if this is relevant for the anti-proliferative effects of giredestrant. Citation Format: Musaddeque Ahmed, Jane Guan, Wei Zhou, Xiaosai Yao, Ciara Metcalfe, Marc Hafner. The ER antagonist giredestrant induces profound chromatin remodeling including activation of cis-regulatory elements bound by FOXA1 and GATA3 in HR+ breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-04.

Background: Endocrine therapy (ET) is the mainstay of ER+ BC treatment. However; up to 20% of ER+ BC tumors progress into metastatic disease and develop ET resistance, underscoring the need for combination therapies. Preclinical data suggest that combining phosphatidylinositol 3-kinase (PI3K) inhibitors with ET may overcome resistance. OPPORTUNE, a preoperative phase II window trial, evaluated whether the combination of the PI3K inhibitor pictilisib with anastrozole (PIC+ANA) can increase the antitumor effect of ANA in newly diagnosed operable ER+ BC. Early results showed greater suppression of tumor Ki67 in patients treated with PIC+ANA versus ANA alone. Here, we present gene expression analysis from tumors collected pre- and post-treatment, and their associations with Ki67 outcomes. Methods: Postmenopausal women with newly diagnosed operable ER+/HER2-negative BC were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with ANA 1 mg once per day (n = 47) or the combination of ANA 1 mg with PIC 260 mg once per day (n = 89). The primary end point was inhibition of tumor cell proliferation measured by change in Ki67 protein expression via IHC between tumor samples taken pre- and post-treatment. Samples were analyzed by RNA-sequencing—ER pathway activity, PAM50 intrinsic subtypes, and pathway analyses were assessed by Ki67 outcomes. Elastic net regression analysis and transcription factor activity inference with Dorothea were performed to identify features strongly associated with Ki67 outcomes. Results: 124 patients (ANA, n=43; PIC+ANA, n=81) had paired tumor samples at baseline/week 2 that were evaluable for both Ki67 and RNA-seq. PIC+ANA showed improved suppression of Ki67 compared to ANA alone (-83.78% vs -73.85%, p=0.012) and a greater proportion of patients achieved complete cell cycle arrest (CCCA, as defined by Ki67< 2.7%) in the PIC+ANA arm (45.68% vs 36.59%). ER pathway activity suppression was comparable between treatments. PAM50 classification based on RNA-seq showed that 83.0% of tumors were luminal (Lum) A at baseline and 12.9% were LumB, with the remainder being classified as Normal-like. PIC+ANA showed greater suppression of Ki67 in LumB tumors compared to LumA (-92.29% vs -81.62%). This effect was much greater in the PIC+ANA arm compared to LumB tumors treated with ANA alone (-92.29% vs -37.87%); however, given the low number of LumB tumors in the ANA arm (n=3), we could not determine statistical significance. Bioinformatic analysis of RNA-seq from baseline specimens showed that MYBL2 activity was associated with resistance to ANA (as defined by Ki67≥7.5% at week2). Tumors in the top quartile of MYBL2 activity at baseline showed improved Ki67 outcomes in the PIC+ANA arm compared to ANA alone (-91.71% vs -51.44%), while no differences were observed between treatments for tumors with lower MYBL2 activity (-79.16% vs -76.72%). Single-nucleus (sn)-RNA-seq from untreated ER+ BC tumors showed that MYBL2 was enriched in a single cluster of tumor cells, which also had the highest expression of a subset of actionable targets (including CDK1, CDK2, CDK4, EZH2, and AKT1), as compared to other tumor cells. Expression data from DepMap and drug-sensitivity data from ER+ BC cell lines show a positive trend between high MYBL2 expression and sensitivity to the ER degrader, giredestrant. Conclusions: PIC exhibited greater antiproliferative effects in combination with ANA in ER+/HER2- early BC compared to ANA alone, particularly in LumB and MYBL2-high tumors. Furthermore, the transcriptional profile and in vitro response of tumor cells with high MYBL2 expression suggest potential sensitivity to other combination therapies. Citation Format: Alejandro Martinez Chibly, Alice Shia, Radia Johnson, Michael S. Hwang, Marc Hafner, Ciara Metcalfe, Kalpit Shah, Michal Slyper, Chris Bolen, Sarah E. Pinder, Alastair M. Thompson, Steven Gendreau, Peter Schmid. PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-06.

Supplementary Figure S7: MCF-7 DOX-inducible ER-alpha overexpressing cells without DOX induction respond similarly to taselisib and/or fulvestrant as MCF-7 parental cells.

Supplemental Fig 1,2,3. Tumor growth rates, overall survival and single agent GDC-0973 in vitro response

Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer. Mol Cancer Ther; 14(1); 40–47. ©2014 AACR.

PDF file - 77K, GNE-317, but not vismodegib, reduces the levels of pAKT in the PPM model. Western blot analysis of lysates from allografts, treated as indicated.

APC mutations cause activation of Wnt/β-catenin signaling, which invariably leads to colorectal cancer. Similarly, overexpressed Dvl proteins are potent activators of β-catenin signaling. Screening a large tissue microarray of different staged colorectal tumors by immunohistochemistry, we found that Dvl2 has a strong tendency to be overexpressed in colorectal adenomas and carcinomas, in parallel to nuclear β-catenin and Axin2 (a universal transcriptional target of Wnt/β-catenin signaling). Furthermore, deletion of Dvl2 reduced the intestinal tumor numbers in a dose-dependent way in the ApcMin model for colorectal cancer. Interestingly, the small intestines of Dvl2 mutants are shortened, reflecting in part a reduction of their crypt diameter and cell size. Consistent with this, mammalian target of rapamycin (mTOR) signaling is highly active in normal intestinal crypts in which Wnt/β-catenin signaling is active, and activated mTOR signaling (as revealed by staining for phosphorylated 4E-BP1) serves as a diagnostic marker of ApcMin mutant adenomas. Inhibition of mTOR signaling in ApcMin mutant mice by RAD001 (everolimus) reduces their intestinal tumor load, similarly to Dvl2 deletion. mTOR signaling is also consistently active in human hyperplastic polyps and has a significant tendency for being active in adenomas and carcinomas. Our results implicate Dvl2 and mTOR in the progression of colorectal neoplasia and highlight their potential as therapeutic targets in colorectal cancer. Cancer Res; 70(16); 6629–38. ©2010 AACR.

PDF file - 286K,Proliferation is suppressed in PPM tumors. Phosphorylation of histone H3 (pH3) is tightly correlated with chromosome condensation during mitosis. PM tumors have a high frequency of pH3 positive cells, while in PPM and PPwt/loxpM tumors, pH3 positive cells tend to occur in restricted regions.

PDF file - 2507K, Tumors in PPwt/loxpM mice display MBEN histology, similar to PPM tumors, demonstrated by H&E staining. Scale bars represent 1 mm in the low magnification images, and 100 microm in the high magnification images.

Supplementary Figures 3-6 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

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Supplementary Figure 2 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figure 8 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

PDF file - 5347K, Illustration of CC3 IHC image analysis. Bright-field images of CC3 IHC were imported into ImageJ. Automated local thresholding (Niblack method) and watershed segmentation were used to identify all nuclei, and total nuclear area was calculated. Dark CC3 positive areas were subsequently identified using a predetermined intensity-based threshold, and the area representative of these regions was calculated, enabling us to calculate % CC3 positive area as a function of total nuclear area.

Supplementary Figure 1 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

PDF file - 1233K, Loss of PTEN in Math1-expressing granule neuronal precursor cells results in ectopic positioning of enlarged NeuN-expressing cells.

Supplementary Figure 7 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

ESR1 mutations contribute significantly to resistance against standard-of-care endocrine therapies and are associated with poor outcomes for estrogen receptor-positive (ER+) breast cancer patients. Structural, biochemical and cellular data all support a model in which such mutations trigger estrogen-independent ER activity, consistent with their emergence in patients who have been treated with Aromatase Inhibitors. Engineered cell line models have further suggested that the ESR1 mutations display gain-of-function effects that extend beyond constitutive canonical ER activity by influencing, for example, epithelial to mesenchymal transition and metastatic potential. However, concerns over potential engineering artifacts and limitations of in vitro conditions have challenged interpretation. Importantly, the mechanisms by which ER mutants might broadly impact cell behavior and whether the latest candidate ER therapeutics will inhibit the full spectrum of ESR1 mutant phenotypes are currently unknown. To address these questions, we generated conditional knock-in Esr1-mutant mice, allowing us to study the fundamental properties of the mutations under physiological conditions (equivalent to those experienced by wildtype ESR1) in the mammary gland, as a hormone-sensitive tissue and the origin of breast tumors. We find that ER mutations profoundly re-wire the transcriptional and cellular response of the mammary gland to hormones, including over the normal estrus cycle. In particular, mutant ER cells display hyper-sensitivity to progesterone, which triggers a proliferative program reminiscent of pregnancy (in nulliparous/virgin mice)- a phenotype that is entirely distinct from ESR1 wildtype mammary glands, both under physiological conditions and when stimulated with high levels of exogenous hormones. The ESR1 mutant-specific PR-mediated proliferative program does not reflect elevated Pgr expression but rather correlates with an altered PR cistrome and transcriptome. We found that this PR program also appears important for human tumor biology because progesterone stimulates the growth of ESR1-mutant patient-derived xenograft tumors (PDXs), in contrast to previous work showing progesterone to be generally tumor-suppressive in ESR1 wildtype tumor models. Importantly, Giredestrant (GDC-9545), a latest-generation orally bioavailable ER antagonist and degrader (SERD), counters the PR-mediated proliferative program, both in the mouse mammary model and in human PDXs. Taken together, these data suggest that in addition to promoting estrogen-independence, mutant ER collaborates with PR to drive a pro-tumorigenic proliferative program, perhaps co-opting a relationship between the hormone receptors established to be key for mammary gland development. Citation Format: Jackson Liang, Ciara Metcalfe. Constitutively active estrogen receptor mutants enhance breast cancer pathogenesis by co-opting progesterone receptor activity, which can be countered by Giredestrant [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-09.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

ESR1 (estrogen receptor 1) hotspot mutations are major contributors to therapeutic resistance in estrogen receptor–positive (ER + ) breast cancer. Such mutations confer estrogen independence to ERα, providing a selective advantage in the presence of estrogen-depleting aromatase inhibitors. In addition, ESR1 mutations reduce the potency of tamoxifen and fulvestrant, therapies that bind ERα directly. These limitations, together with additional liabilities, inspired the development of the next generation of ERα-targeted therapeutics, of which giredestrant is a high-potential candidate. Here, we generated Esr1 mutant–expressing mammary gland models and leveraged patient-derived xenografts (PDXs) to investigate the biological properties of the ESR1 mutations and their sensitivity to giredestrant in vivo. In the mouse mammary gland, Esr1 mutations promote hypersensitivity to progesterone, triggering pregnancy-like tissue remodeling and profoundly elevated proliferation. These effects were driven by an altered progesterone transcriptional response and underpinned by gained sites of ERα-PR (progesterone receptor) cobinding at the promoter regions of pro-proliferation genes. PDX experiments showed that the mutant ERα-PR proliferative program is also relevant in human cancer cells. Giredestrant suppressed the mutant ERα-PR proliferation in the mammary gland more so than the standard-of-care agents, tamoxifen and fulvestrant. Giredestrant was also efficacious against the progesterone-stimulated growth of ESR1 mutant PDX models. In addition, giredestrant demonstrated activity against a molecularly characterized ESR1 mutant tumor from a patient enrolled in a phase 1 clinical trial. Together, these data suggest that mutant ERα can collaborate with PR to drive protumorigenic proliferation but remain sensitive to inhibition by giredestrant.

Estrogen Receptor alpha (ERα) stands as one of the most successfully prosecuted drug targets in oncology, beginning with the approval of tamoxifen for women with ERα positive (ER+) breast cancer over 40 years ago. The field continued to advance with the development of aromatase inhibitors and the pure antiestrogen fulvestrant. With multiple endocrine therapies approved for the treatment of ER+ breast cancer, efforts to generate novel ERα-targeted therapeutics somewhat diminished in the early 2000s. Today however, there are at least eight new molecular entities targeting ERα under active clinical investigation, each with the aim of bringing further benefit to patients. This remarkable re-energizing of the field was spurred in part by the discovery of highly prevalent ERα mutations as a mechanism of resistance to standard-of-care therapies, which provided unequivocal evidence of the continued, and broad, dependence of tumors on ERα, despite relapsing after earlier lines of endocrine therapy. Re-engagement of the pharmaceutical and biotechnology industries with ERα as a drug target has been further underpinned by the impressive advances made in medicinal chemistry, enabling desirable mechanistic features - high potency full ERα antagonism - to be combined with improved drug-like properties - oral bioavailability and optimized pharmacokinetics. In this chapter, we describe the rich history and science behind the currently evolving landscape of ERα targeting in breast cancer.

Background: Endocrine therapy remains the mainstay treatment for ER+ BC. CDK4/6 inhibitors induce cell cycle arrest and decrease tumor cell proliferation, as measured by the biomarker Ki67, when used in combination with aromatase inhibitors (AI) such as anastrozole (A). Giredestrant is an oral, well-tolerated, and highly potent selective ER degrader (SERD) that achieves robust ER suppression and has demonstrated antitumor activity in the metastatic setting either as monotherapy or in combination with the CDK4/6 inhibitor palbociclib. The randomized, phase 2 coopERA Breast Cancer study (NCT04436744) evaluated giredestrant in postmenopausal women with untreated ER+/HER2- early BC and met its primary endpoint, demonstrating superior Ki67 suppression with giredestrant vs A after two weeks of single agent treatment. This suppression was maintained at surgery where giredestrant vs A was evaluated in combination with palbociclib. Here, we present gene expression analysis and associations with Ki67 response. Methods: 221 eligible patients with measurable ER+/HER2– untreated early BC and baseline Ki67 ≥ 5% were randomized 1:1 to receive 30 mg oral daily (PO QD) giredestrant or 1 mg PO QD A on Days 1–14 of a neoadjuvant window-of-opportunity phase, followed by four 28-day cycles of PO QD giredestrant or A with 125 mg PO QD palbociclib on Days 1–21 before surgery. FFPE specimens were collected at baseline, week 2 and surgery; and RNA-sequencing (seq) was performed. Gene expression analysis included ER pathway activity, PAM50 intrinsic subtypes, and other pathway analyses, assessed by Ki67 response. Results: 112 and 92 patients had paired tumor samples at baseline/week 2 and baseline/surgery, respectively, that were evaluable for RNA-seq and Ki67. The trend for greater Ki67 protein suppression by giredestrant vs A from baseline to week 2 was maintained in the RNA-seq evaluable subset. Interestingly, the same subset revealed similar suppression of both proliferation gene signatures and ER pathway activity between A and giredestrant. PAM50 subtyping showed that 69% of tumors were luminal (Lum) A and 29% were LumB at baseline. Less than 1% were classified as basal or HER2. Interestingly, giredestrant (G) showed greater suppression of both Ki67 and ER pathway activity vs A in LumB tumors (Ki67: -82% [G] vs -62% [A]; ER activity: -0.83 [G] vs -0.66 [A]) compared to LumA at week 2 (Ki67: -74% [G] vs -71% [A]; ER activity: -0.60 [G] vs -0.70 [A]). Moreover, at week 2, 83% (13/18) of LumB tumors at baseline transitioned into a LumA subtype after giredestrant treatment compared to 46% (5/11) of A-treated tumors. Giredestrant-treated tumors also achieved lower mean ER pathway activity compared to those treated with A at surgery (p=0.023). Gene set enrichment analysis showed downregulation of cell-cycle and ER-related pathways at week 2 and surgery in both treatment arms. A subset of cytokine signaling and immune response pathways were increased at week 2 compared to baseline after treatment with A but not with giredestrant. These pathways were also associated with Ki67 resistance (Ki67 ≥ 7.4%) in A-treated tumors. This was consistent with differential expression analysis of samples collected at week 2, in which cytokine signaling pathways were enriched in A compared to giredestrant. Notably, IL12 signaling was enriched in tumors resistant to A but not giredestrant. Conclusions: Giredestrant has a greater effect on Ki67 protein suppression in ER+/HER2- early BC compared to A, which is more pronounced in LumB tumors. This benefit may involve differential regulation of cytokine and immune responses. These exploratory findings reveal novel mechanisms that may differentiate the activity of SERDs vs AIs, which warrant further validation. Citation Format: Alejandro M. Chibly, Tharu M. Fernando, Ciara Metcalfe, Marc Hafner, Gilbert Owusu-Manu, Sara Hurvitz, Aditya Bardia, Peter A. Fasching, Yeon H. Park, Vanesa Quiroga, Jutta Steinseifer, Pablo Perez-Moreno, Heather M. Moore. PD13-02 Exploratory gene expression analysis of coopERA Breast Cancer (BC): a study evaluating neoadjuvant giredestrant versus anastrozole alone and in combination with palbociclib in ER-positive, HER2-negative untreated early BC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD13-02.

[Image: see text] Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms—as a full antagonist and selective estrogen receptor degrader (SERD)—but lacks oral bioavailability. Thus, we envisioned a “best-in-class” molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Background: Estrogen (ER) and progesterone (PR) receptor staining with immunohistochemistry (IHC) forms the basis for endocrine therapy (ET) eligibility. However, ER protein level does not necessarily reflect ER functionality and is insufficient as a predictor of response or as a pharmacodynamic (PD) biomarker to assess the impact of ET on ER activity. Furthermore, assessing PD modulation based on a single ER target gene, PR, lacks robustness and cannot be utilized in PR- tumors. We established a transcriptional signature that more comprehensively reflects ER pathway activity. We showcase, in vitro, in vivo, and in patients enrolled in a Phase I trial of GDC-9545 -a novel, potent, orally bioavailable, selective estrogen receptor antagonist / degrader- that this signature may have utility as a predictive and PD biomarker. Methods: A signature of 21 estradiol (E2)-induced and 17 E2-repressed genes was experimentally derived by transcriptional profiling of 8 E2-stimulated ER+ breast cancer (BC) cell lines in vitro, and refined by leveraging TCGA RNA-seq data. The effect of ER inhibition by GDC-9545 on cellular proliferation was determined in an 8 day in vitro viability assay, and correlated with pre-treatment ER pathway activity as determined by the signature. The signature was evaluated in an in vivo efficacy study with GDC-9545 (0.1, 1 or 10 mg/kg) in PDX breast model HCI-013, measured using a Fluidigm® panel. Paired pre- and on-treatment tumor biopsies from 7 patients enrolled in the Phase I dose escalation study of GDC-9545 (10, 30 or 90 mg) in ER+/HER2- advanced or metastatic BC were collected to retrospectively assess ER pathway modulation. ER, PR and Ki67 protein levels were analyzed by IHC, while gene expression analysis from FFPE tissue was performed using Illumina’s RNA Access protocol and HiSeq. The ER pathway activity score was defined as the difference in average expression of the E2-induced versus E2-repressed genes, relative to ER pathway activity in a reference collection of 139 procured ER+/HER2- breast tumors. Results: A panel of 14 ER+/HER2- BC cell lines exhibited a spectrum of in vitro responses to GDC-9545 that strongly correlated with the pre-treatment ER pathway activity score. GDC-9545 had little anti-proliferative effect in cell lines with lowest scores, while having a considerably greater impact on cell lines exhibiting higher scores. Besides potential applicability as a predictive biomarker, we explored the utility of the ER activity score in vivo as a PD biomarker. We observed enhanced transcriptional pathway suppression with increased dose of GDC-9545, and a concomitant greater impact on Ki67 expression, in the in vivo HCI-013 PDX breast model. The ER signature was further evaluated in pre- and on-treatment tumor biopsy pairs from 7 patients enrolled in the GDC-9545 Phase I trial. ER activity scores of tumors from these patients were benchmarked against a cohort of 139 procured ER+/HER2- breast tumors, revealing a range in ER pathway activity prior to treatment with GDC-9545. Two tumors exhibiting profound Ki67 responses (post-treatment IHC Citation Format: Anneleen Daemen, Jill M Spoerke, Wei Zhou, Jane Guan, Ellen Ingalla, Amy Young, Marc Hafner, Junko Aimi, Ching-Wei Chang, Jennifer M Giltnane, Mary Gates, Ingrid A Mayer, Analia Azaro, Eric P Winer, Sherene Loi, Komal Jhaveri, Jennifer Lauchle, Steven Gendreau, Eric W Humke, Ciara Metcalfe. ER pathway activity signature as a biomarker for endocrine agent GDC-9545 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-05.

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.

Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER positive breast cancer. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. Based on preclinical and clinical data, SERDs are expected be effective in patients harboring ESR1 mutations. Biomarker analysis was performed on plasma and tumor samples from the Phase I study of GDC-0927 in metastatic breast cancer (Dickler et al, SABCS 2017) with the goal of evaluating activity in both ESR1 mutant and wildtype tumors, and to assess ER pathway modulation. Methods: Hotspot mutations in ESR1, PIK3CA, and AKT1 were analyzed in baseline, on-treatment and end of treatment plasma derived circulating tumor DNA (ctDNA) using the BEAMing assay in patients treated at multiple dose levels of GDC-0927. A subset of samples was analyzed with Foundation Medicine's next generation sequencing ctDNA assay (FACT), which covers genomic alterations in 62 commonly altered genes. Paired pre- and on-treatment biopsies were collected to assess ER pathway modulation. ER, PR, and Ki67 protein levels were analyzed by immunohistochemistry. Gene expression analysis was performed using Illumina's RNA Access library preparation kit followed by paired-end (2x50b, 50M reads) sequencing on the HiSeq. Results: Baseline and on-treatment plasma samples were available for 40 patients. ESR1 and PIK3CA mutations were observed in 52% and 33% of patient baseline samples, respectively (BEAMing method). Mutant allele frequencies (MAF) generally declined in the first on-treatment samples collected for both ESR1 (16 out of 21 samples) and PIK3CA (7 out of 12 samples). The majority of the reductions were greater than 95% relative to baseline. Increases in ESR1 MAFs were observed in later time-points and were not associated with any particular ESR1 mutation. There were six instances for which an ESR1 mutation was detected in an on-treatment sample that was not detected in the baseline sample, three at L536P and one each at D538G, L536H, and S463P, and four out of six with MAFs close to the limit of detection. The FACT assay also detected alterations in CDH1, NF1, PTEN, and TP53 in baseline samples. The relationship between MAF changes and clinical benefit to GDC-0927 will be presented. A predefined, experimentally-derived set of ER target genes were evaluated in pre- and on-treatment tumor biopsy pairs from six patients. Four of the six patients showed evidence of suppression in ER pathway activity, one patient treated at the 1000 mg dose level and three at the 1400 mg dose. The degree of pathway suppression was associated with pre-treatment pathway levels and decreases of ER and Ki67 protein levels. Conclusions: We report here evidence of consistent reduction of ESR1 and PIK3CA ctDNA in patients treated with GDC-0927. ER pathway suppression was observed at both the transcript and protein level confirming pharmacodynamic activity of the SERD. Citation Format: Spoerke JM, Daemen A, Chang C-W, Giltnane J, Metcalfe C, Dickler MN, Bardia A, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez Martin A, Cortes J, Martin M, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Liu L, Li R, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Lackner MR. Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD) [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 P5-11-01.

Targeted therapies have dramatically improved progression-free survival (PFS) in hormone-receptor positive (HR+) breast cancers, however, achieving durable responses remains a challenge. In particular, mutations in PIK3CA and ESR1 are common in advanced settings and can potentially drive resistance to aromatase inhibitors, including when combined with CDK4/6 inhibitors. With the goal of improving patient outcomes, next-generation endocrine agents and PIK3a-specific inhibitors are being developed and are in active clinical studies. Importantly however, the molecular underpinnings of how such agents combine with one another, and with CDK4/6 inhibitors, have not yet been fully explored. Here we profiled the latest generation molecules, inavolisib (GDC-0077), a PIK3a-specific inhibitor, and giredestrant (GDC-9545), a full estrogen receptor (ER) antagonist and selective ER degrader, in combination with palbociclib, a CDK4/6 inhibitor. Specifically, we evaluated the transcriptional and phenotypic effects of these molecules on PIK3CA mutant MCF7 cells expressing either ESR1 wild-type (WT) or ESR1-Y537S. Cells were treated with single-, double-, or triple-agent inavolisib, giredestrant, and palbociclib and were harvested at 24 and 96 hours for transcriptional profiling by both bulk and multiplexed scRNA-sequencing, or assayed for viability. In both WT and mutant (MUT) cells, expression of cell cycle-related genes was reduced at 24h upon treatment with single-agent palbociclib, but rebounded to baseline by 96h. Combination with giredestrant induced the expected downregulation of ER activity at both 24 and 96h. Notably, the combination further reduced the expression of cell cycle-related genes compared to palbociclib alone. Surprisingly, the repression of cell cycle-associated genes driven by the combination of giredestrant and palbociclib was sustained through 96h in WT cells but not in MUT cells, despite sustained repression of ER activity. Addition of PIK3a-inhibitor inavolisib to the palbociclib/giredestrant combination further reduced expression of cell cycle-related genes in both WT and MUT cells to an extent not achieved by any other treatment variation in this study. Most importantly, the triple-combination also prevented the rebound of cell cycle genes in the ESR1-MUT, consistent with a sustained response. Analysis of the scRNA-seq data revealed a substantial heterogeneity in the vehicle-treated population. While giredestrant treatment results in a particularly profound impact on transcriptional programs, heterogeneity is maintained. Together, our data provide mechanistic support for ongoing triple-combination studies in HR+ breast cancers. Citation Format: Taylor Marohl, Jenille Tan, Wei Zhou, Jane Guan, Sandra Melo Carlos, Shiqi Xie, Marc Hafner, Ciara Metcalfe. Triple-threat: Inavolisib and giredestrant combine with palbociclib to achieve sustained cell cycle arrest in ER-positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5388.

It has been 40 years since the US Food and Drug Administration approved the estrogen receptor (ER) antagonist tamoxifen for the treatment of ER-positive breast cancer, ushering in the era of targeted therapy coupled with a companion diagnostic. The prostate cancer field quickly followed suit with the approval of the androgen receptor (AR) antagonist bicalutamide. In the years since, there has been sustained scientific interest in understanding these hormone-dependent signaling pathways and in drug discovery efforts to identify novel hormone-directed therapeutic agents. Recently, there have been breakthrough discoveries relating to mechanisms that enable reactivation of ER and AR signaling in the presence of antihormonal agents and that enable loss of hormone dependency, providing multiple routes of acquired resistance to hormone therapy. This review discusses parallels between breast and prostate cancer, including their pathobiologies, existing therapeutic modalities, acquired resistance to such therapeutics, and novel therapies being developed to target distinct states of resistance.

Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER+ BC. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. ER antagonists that are efficacious against ligand-dependent and ligand-independent, constitutively active ESR1 mutant tumors may be of substantial therapeutic benefit. GDC-0927 (formerly known as SRN-927) is a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader (SERD) that induces tumor regression in ER+ BC patient-derived xenograft models. Methods: A phase I dose escalation study with 3+3 design was conductedin postmenopausal women with ER+ (HER2-) metastatic BC (progressing ≥ 6 months on endocrine therapy and with ≤ 2 prior chemotherapies in the advanced or metastatic setting) to determine the safety, pharmacokinetics (PK) and the recommended Phase 2 dose (RP2D) of GDC-0927. Pharmacodynamic (PD) activity was assessed with [18F]-fluoroestradiol (FES)-PET scans. Plasma PK samples (after single dose and at steady state), CT scans, and when feasible, pre and on-study tumor biopsies were obtained Results: From March 16, 2015 to March 17, 2017 patients (pts) with a median age of 53 years (range 44-69) and a median number of prior therapies for MBC 4 (range 1-7) were enrolled at 3 total daily dose levels (600, 1000, 1400 mg) once daily (QD) given orally with fasting (n = 12). Increases in GDC-0927 exposure were approximately dose proportional. Treatment related adverse events (AEs) were all grade 1 or 2. The most common treatment-related AEs were nausea (54%, n = 7), diarrhea (46%, n = 6), elevated aspartate aminotransferase (39%, n = 5) and anemia, constipation, (each 31%, n = 4). Treatment interruption was required for 2 pts due to nausea and vomiting. Of those pts with FES-PET avid disease at baseline (9 of 12), all post-therapy scans showed complete or near complete (> 90%) suppression of FES uptake to background levels, including pts with ESR1 mutations. Evidence of reduced ER levels and Ki67 staining was observed in on-treatment biopsies. Five of 12 pts (1 at 600 mg and 4 at 1400 mg) were on study ≥ 24 weeks (CBR = 41.6 %) with the best overall response of stable disease with 1 patient (ESR1 mt+ D538G) on study for over 490 days. There were no dose limiting toxicities and no SAEs related to study drug. R2PD was 1400 mg and was selected for single arm dose-expansion which is now complete with last patient enrolled on March 17, 2017. Updated results from dose-escalation and dose-expansion will be presented at the meeting (N = 43). Conclusions: GDC-0927 appears well-tolerated to date with PK exposure supporting QD dosing, evidence of robust PD target engagement, and encouraging anti-tumor activity in heavily pretreated pts with advanced or metastatic ER+ BC, including pts with ESR1 mutations. Citation Format: Dickler MN, Villanueva R, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez-Martin A, Cortes J, Martin M, Giltnane J, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Spoerke JM, Metcalfe C, Liu L, Li R, Morley R, McCurry U, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Bardia A. A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-0927, in postmenopausal women with estrogen receptor positive (ER+) HER2-negative metastatic breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-10.

ER+ breast cancers can depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to classification as a full ER antagonist, which is thought to be achieved through degradation of ER protein. However, the full clinical potential of fulvestrant is believed to be limited by poor bioavailability, spurring attempts to generate ligands capable of driving ER degradation but with improved drug-like properties. Here, we evaluate three ER ligand clinical candidates that recently emerged from prospective optimization of ER degradation – GDC-0810, AZD9496 and GDC-0927 - and show that they display distinct mechanistic features. GDC-0810 and AZD9496 are more limited in their ER degradation capacity relative to GDC-0927 and fulvestrant, display evidence of weak transcriptional activation of ER in breast cancer cells (i.e. partial agonist activity), and do not achieve the same degree of in vitro anti-proliferative activity as GDC-0927 and fulvestrant. In the HCI-013 (ER.Y537S) and HCI-011 (ER.WT) ER+ patient-derived xenograft models, GDC-0927 drives greater transcriptional suppression of ER, and greater anti-tumor activity relative to GDC-0810. We found that despite their full antagonist phenotype, GDC-0927 and fulvestrant promote association of ER with DNA, including at canonical ERE motifs, prior to ER degradation. Interestingly however, integration of ER ChIP-Seq and ATAC-Seq data revealed that ER complexed with fulvestrant or GDC-0927 fails to increase chromatin accessibility at DNA binding sites, in contrast to partial agonists which result in increased chromatin accessibility at ER binding sites. Thus, although ER contacts DNA when engaged with fulvestrant and GDC-0927, it is functionally inert. To further explore mechanistic features that might account for the differential activity of full antagonists and partial agonists that occurs prior to ER degradation, we used cell-based florescence recovery after photobleaching (FRAP) to measure the kinetics of ER diffusion within the nucleus. We demonstrate that while ER is generally highly mobile, including after engagement with GDC-0810 and AZD9496, GDC-0927 and fulvestrant immobilize intra-nuclear ER. A site saturating mutagenesis screen revealed a series of novel ER mutations that prevent ER immobilization by fulvestrant and GDC-0927. This class of “always mobile” ER variants promotes an antagonist-to-agonist transcriptional switch for fulvestrant and GDC-0927, and simultaneously prevents ER degradation by these molecules, implying that ER immobilization is a key functional determinant of robust transcriptional suppression. We thus propose that ER degradation is not a driver of full ER antagonism, but rather a downstream consequence of ER immobilization, occurring after a suppressive phenotype has been established at chromatin. We additionally argue that evaluating the transcriptional output of candidate ER therapeutics, both pre-clinically and clinically, will be critical for the identification of ER ligands with best-in-class potential. Citation Format: Metcalfe C, Zhou W, Guan J, Daemen A, Hafner M, Blake RA, Ingalla E, Young A, Oeh J, De Bruyn T, Ubhayakar S, Chen I, Giltnane JM, Li J, Wang X, Sampath D, Hager JH, Friedman LS. Prospective optimization of estrogen receptor degradation yields ER ligands with variable capacities for ER transcriptional suppression [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 GS3-05.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

577 Background: Modulation of ER activity and/or estrogen synthesis is the mainstay therapeutic strategy in ER+ BC treatment. Giredestrant is a highly potent, nonsteroidal oral selective ER degrader (SERD) that achieves robust ER occupancy and is effective regardless of ESR1 mutation status. The first short-term preoperative WOO study (NCT03916744) of giredestrant in ER+/HER2– operable BC was designed for dose selection, while providing an early readout of PD as measured by traditional immunohistochemistry (IHC) and transcriptional profiling by assessing treatment effects in paired tumor tissue pre/posttreatment. We present an interim analysis. Methods: Pts were assigned to 14 days’ preoperative treatment with 10, 30, or 100 mg PO giredestrant QD. Pts had newly diagnosed, stage I–III operable, ER+/HER2– untreated BC ≥1.5 cm in diameter (by ultrasound). Modulation of ER signaling and cell proliferation were assessed using paired formalin-fixed paraffin-embedded tumor specimens collected before and after ̃14 days of study treatment. ER, progesterone receptor (PR), and Ki67 protein levels were analyzed by IHC. Change from baseline in tumor cell proliferation by Ki67 was the primary endpoint. Gene expression analysis was performed using the Illumina TruSeq RNA Access method. Results: From Jul 26, 2019 to Oct 15, 2020, 46/75 biomarker-evaluable pts were enrolled across three dose cohorts (10 mg: n = 15; 30 mg: n = 18; 100 mg: n = 13). Pt demographics and tumor characteristics were similar across cohorts. Baseline PAM50 analysis classified tumors as Luminal A (77%) or B (23%). Giredestrant treatment resulted in robust and indistinguishable PD and biologic activity at all doses. Geometric mean posttreatment proportional reduction of Ki67 was 79% (95% CI: 69–89; 10 mg: 80%; 30 mg: 76%; 100 mg: 80%), and 51% of tumors exhibited complete cell cycle arrest, defined as Ki67 ≤2.7%. Mean posttreatment proportional reductions of ER and PR H-scores were 71% (95% CI: 67–75) and 60% (95% CI: 51–70), respectively. An analysis of a predefined, experimentally derived set of 38 ER target genes (the ‘ER activity signature’), was completed for 42 paired tumor specimens. Forty-one of 42 pts (98%) showed a posttreatment reduction in ER activity with a mean proportional decrease of 79% (95% CI: 70–88). A wide range of baseline ER activity was observed with no correlation to baseline ER or PR H-score, or Ki67. There were no discontinuations due to adverse events (AEs). A single grade 3 serious AE was reported in each cohort (all assessed as unrelated to giredestrant). No grade 4 or 5 AEs were reported. Conclusions: Giredestrant was well tolerated in the preoperative setting in ER+/HER2– operable BC, and PDs were consistent with the 30 mg dose achieving maximal ER inhibition. Clinical trial information: NCT03916744.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Real-world data (RWD) are potentially a highly valuable resource to accelerate drug discovery and improve clinical practice. In particular, datasets combining clinical outcomes and comprehensive genomic profiling (CGP) could uncover new mechanisms of resistance and identify new patient populations for targeted therapies. Here, we used a de-identified clinico-genomic database (CGDB) from routine clinical care based on a clinical EHR-derived data from Flatiron Health linked to tumor sequencing results from Foundation Medicine to assess how prevalence of genomic alterations changes following progression on targeted therapies in the metastatic setting. One of the main challenges we faced is the lack of systematic and longitudinal genomic testing: among the patients receiving a specific treatment, some had a CGP performed prior to treatment, others only around the end of the line of therapy, but only a handful had multiple tests. We therefore established a methodology to assess changes in alteration prevalence and tested how our results are sensitive to cohort building parameters such as timing of testing, duration of therapy, or prior lines of treatment. Our approach successfully recovered three established cases of therapeutic resistance: (1) EGFR T790M in non-small cell lung cancer patients treated with erlotinib or gefitinib; (2) AR alterations following hormonal therapies in prostate cancers; and (3) ESR1 mutations in hormone-receptor positive breast cancer patients progressing on endocrine therapies other than fulvestrant. We delved into the latter case and identified differences in the type of ESR1 mutation that occurred following endocrine therapies if CDK4/6 inhibitors were co-administered or not. On one hand, RWD lack randomization and can thus be subject to confounding variables but on the other hand, RWD have the advantage of including a larger number of patients in a real-world setting compared to clinical trials. Our analysis shows that real-world CGDB can recapitulate the expected emergence of mutations following specific lines of treatment. Our study is a proof of concept of usage of RWD to assess how prevalence of genomic alterations changes following treatment. Our method allowed us to assess differences between patient cohorts and generate novel hypotheses on treatment-related changes in genomic profiles, which can ultimately lead to new drug indications. Citation Format: Nayan Chaudhary, Patricia Luhn, Gunther Jansen, Ciara Metcalfe, Marc Hafner. Identification of genomic alterations related to treatment progression in RWD [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 6323.

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.

Background: Modulation of estrogen activity and/or synthesis is a mainstay therapeutic strategy for ER+ breast cancer (BC). However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand-binding domain, that drive ER-dependent transcription and proliferation in the absence of estrogen. ER antagonists that robustly suppress ER signaling in both ESR1 wild-type and mutant tumors may be of substantial therapeutic benefit. GDC-9545 is a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader (SERD) that induces tumor regression in ER+ BC patient-derived xenograft models. Methods: A phase I dose escalation study with 3+3 design was conducted in postmenopausal women with ER+ (HER2−) advanced or metastatic BC (mBC). Patients were eligible if they had advanced or mBC that had progressed while being treated with adjuvant endocrine therapy for ≥ 24 months and/or endocrine therapy in the advanced or mBC setting for ≥ 6 months. No more than 2 prior treatments for advanced or mBC. Primary objective was to determine the safety, pharmacokinetics (PK) and the recommended Phase 2 dose (RP2D) of GDC-9545. Pharmacodynamic (PD) activity was assessed with [18F]-fluoroestradiol (FES)-PET scans and, when feasible, by performing biomarker analysis of pre- and on-study tumor biopsies. Plasma samples were collected for PK analyses and imaging for response assessment (CT and bone scans) were repeated every 8 weeks. Results: From November 27 2017 to February 1 2019 patients (n = 29) with a median age of 58 years (range 42-75) and a median number of prior therapies for mBC of 2 (range 1-4) were enrolled at 4 total daily dose levels (10, 30, 90 and 250 mg) given once daily (QD) while fasting. Increases in GDC-9545 exposure were approximately dose proportional and t1/2 was ~30 hours. Treatment related adverse events (AEs) were all Grade 1 or 2. The most common treatment-related AEs in ≥10% of patients were fatigue (21%, n=6), nausea (21%, n=6), hot flushes (17%, n=5), diarrhea (17%, n=5), arthralgia (17%, n=5), constipation (10%, n=3) and dyspepsia (10%, n=3). No dose-limiting toxicities, treatment-related serious adverse events (SAEs), Grade ≥ 3 events, or fatal events have been reported. Treatment interruption was required for 3 patients: one patient for a short episode of dyspepsia, vomiting, diarrhea, and dizziness; and two patients due to unrelated SAEs. In 14 patients with FES-PET avid disease at baseline and post-baseline scans, 11 (78%) showed complete or near complete (> 90%) suppression of FES uptake to background levels, including patients with ESR1 mutations. Evidence of reduced ER, PR, and Ki67 IHC values and an ER activity signature was observed in paired pre- and on-treatment biopsies (n = 7). Twelve of 27 patients (10 mg [n=2], 30 mg [4], 90 mg [4], 250 mg [2]) had either confirmed partial responses (n = 3) or were on study ≥ 24 weeks (CBR = 44%), including patients pretreated with CDK4/6i, fulvestrant or harboring baseline ESR1 mutations. The R2PD was 100 mg QD and was selected for dose-expansion in post- and pre/peri-menopausal women with and without palbociclib, which is ongoing. Updated results will be presented. Conclusions: GDC-9545 is well-tolerated to date with PK exposure supporting QD dosing, evidence of robust PD target engagement, and encouraging anti-tumor activity in patients with advanced or ER+ mBC, including patients with ESR1 mutations. Dose expansion cohorts of GDC-9545 with or without palbociclib in post-menopausal and pre/peri-menopausal women with mBC are ongoing. Citation Format: Komal Jhaveri, Eric Paul Winer, Elgene Lim, Jose AlejandroPerez Fidalgo, Meritxell Bellet, Ingrid Alina Mayer, Valentina Boni, Jaymin M Patel, Aditya Bardia, Jose Manuel Garcia, Peter Kabos, Mary Gates, Ya-Chi Chen, Jill Fredrickson, Xiaojing Wang, Lori Sickels Friedman, Jill Spoerke, Steven Gendreau, Ciara Metcalfe, Lichuan Liu, Ching-Wei Chang, Sharareh Monemi, Monica Gonzalez, Ursula McCurry, Sandra Milan, Eric William Humke, Sherene Loi. A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-9545, in postmenopausal women with estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-05.

Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer. Mol Cancer Ther; 14(1); 40–47. ©2014 AACR.

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Lgr5 marks mitotically active intestinal stem cells (ISCs). Importantly, intestinal homeostasis can be maintained after depletion of Lgr5(+) cells due to the activation of Lgr5(-) reserve ISCs. The Lgr5(-) ISC populations are thought to play a similar role during intestinal regeneration following radiation-induced damage. We tested this regeneration hypothesis by combining depletion of Lgr5(+) ISCs with radiation exposure. In contrast to the negligible effect of Lgr5(+) ISC loss during homeostasis, depletion of Lgr5(+) cells during radiation-induced damage and subsequent repair caused catastrophic crypt loss and deterioration of crypt-villus architecture. Interestingly though, we found that crypts deficient for Lgr5(+) cells are competent to undergo hyperplasia upon loss of Apc. These data argue that Lgr5(-) reserve stem cells are radiosensitive and that Lgr5(+) cells are crucial for robust intestinal regeneration following radiation exposure but are dispensable for premalignant hyperproliferation.

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.

Aromatase inhibitors (AIs), the Estrogen Receptor (ER) modulator tamoxifen and the full ER antagonist fulvestrant each target ER signaling and constitute the backbone of standard of care for ER+ breast cancer. The growing appreciation of liabilities associated with each of these modalities, which may limit their clinical potential, has triggered a wave of activity around next-generation ER therapeutics, and there are currently more than 10 new molecular entities targeting ER being evaluated in Phase 1 clinical trials. In addition to a collection of ER-targeted therapeutics either approved or under evaluation, the emergence of CDK4/6 inhibitors as combination partners has meaningfully contributed to the oncologist’s tool box for the treatment of breast cancer, while also adding complexity to the treatment landscape. Although direct ER antagonists are expected to be superior to AIs in patients with tumors expressing constitutively activating ER mutations (e.g. ER.Y537S), it is currently unclear if a subset of ER wildtype patients may likewise derive superior benefit from direct ER targeting. It is also unclear which ER+ tumors require CDK4/6 inhibitor/ER antagonist combinations for maximal anti-tumor activity, versus those where single-agent ER antagonist treatment may achieve full cell cycle control. To address these key questions, we used a panel of 22 ER+ cell lines to evaluate the relative anti-proliferative and cytotoxic effects of estrogen (E2) withdrawal (modeling AIs), ER modulation, and full ER inhibition, as single agents and in combination with the CDK4/6 inhibitor Palbociclib. We find a number of cell lines in which direct ER antagonism is superior to E2 withdrawal and is associated with E2-independent ER signaling not attributable to ER mutations. The impact of E2 withdrawal on cell cycle control and proliferation is significantly enhanced by concurrent CDK4/6 inhibition, in line with clinical observations, however, direct ER antagonism in combination with CDK4/6 inhibition remains most efficacious. We are integrating our cell response data with detailed molecular analyses and transcriptional profiling to 1) identify cellular contexts, beyond ER mutations, where direct ER antagonism is superior to targeting E2 synthesis, 2) identify tumors where full cell cycle control and anti-tumor efficacy is achievable through single-agent ER targeting, and 3) better understand the basis for the co-operativity of endocrine suppression with CDK4/6 inhibition. Deconvolution of the ER+ breast cancer response to distinct modes of ER signaling inhibition alone and in combination with CDK4/6 inhibitors has the potential to guide clinical decision-making for personalized patient care. Citation Format: Wei Zhou, Jane Guan, Georgia Hatzivassiliou, Anneleen Daemen, Marc Hafner, Ciara Metcalfe. Leveraging an expanding tool box for ER+ breast cancer: exploring breast cancer dependencies to support optimal therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1004.

ER+ breast cancers depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to its classification as a full ER antagonist, which is thought to be achieved through degradation of ERα protein. However, the full clinical potential of fulvestrant is believed to be limited by poor physiochemical properties and exposure limitations due to its administration by intramuscular injection. Strategies to generate orally bioavailable molecules that retain fulvestrant's full antagonist profile but with considerably improved drug-like properties are thus being widely employed to identify next generation ER therapeutics. However, we find that therapeutic candidates that have recently emerged from prospective optimization of ER degradation, including GDC-0810 and GDC-0927, are not mechanistically equivalent. GDC-0810, GDC-0927, and fulvestrant display unique profiles in terms of ER degradation, transcriptional phenotypes and anti-proliferative potential across a panel of ER+ breast cancer cell lines. In HCI-011 (ER.WT) and HCI-013 (ER.Y537S) ER+ patient-derived breast cancer xenograft (PDX) models, GDC-0927 achieves more robust transcriptional suppression of ER than GDC-0810, and also and greater efficacy. Although displaying a more desirable mechanistic profile than GDC-0810, GDC-0927 has more rapid clearance and poor oral bioavailability, leading to a high pill burden and potential exposure limitation. Here, we describe for the first time GDC-9545, in which the distinct liabilities of GDC-0810, GDC-0927 and fulvestrant are addressed. GDC-9545 is a non-steroidal ER ligand that is highly potent in competing with estradiol for binding and in driving an antagonist conformation within the ER ligand binding domain. Like fulvestrant, and displaying some improvements over GDC-0927, GDC-9545 consistently induces ER turnover and drives deep transcriptional suppression of ER, resulting in robust in vitro anti-proliferative activity. GDC-9545 exhibits reduced metabolism and increased oral bioavailability relative to GDC-0927, resulting in an overall improved oral exposure in multiple species. As a result of both its mechanistic pharmacology and improved oral exposure, GDC-9545 can achieve the same degree of anti-tumor activity as GDC-0927 but at 100-fold lower doses in the HCI-013 PDX model. The in vivo efficacy of GDC-9545 in this model is greater than GDC-0810 and fulvestrant at clinically relevant exposures. The highly potent in vivo efficacy of GDC-9545 likely arises due to the particular combination of high binding potency, full suppression of ER signaling, and an improved DMPK profile when compared to GDC-0927 and fulvestrant. GDC-9545 is currently being evaluated in Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT03332797). Citation Format: Metcalfe C, Ingalla E, Blake RA, Chang J, Daemen A, De Bruyn T, Giltnane JM, Guan J, Hafner M, Hartman S, Kategaya L, Kleinheinz T, Liang J, Mody V, Nannini M, Oeh J, Ubhayakar S, Wertz I, Young A, Zbieg J, Zhou W, Sampath D, Friedman LS, Wang X. GDC-9545: A novel ER antagonist and clinical candidate that combines desirable mechanistic and pre-clinical DMPK attributes [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 P5-04-07.