PDF file - 401K, Supplementary Figure S1. Parental and 2nd generation anti-androgen resistant cell line proliferation. Supplementary Figure S2. AR levels in parental and 2nd generation anti-androgen resistant cell lines. Supplementary Figure S3. AR levels in transfection assays. Supplementary Figure S4. Transcriptional reporter assay of AR mutants. Supplementary Figure S5. Ligand binding domain of the AR bound to DHT (1T7T;(1)). Supplementary Figure S6. Competitive binding assay of wild-type AR vs. F876L AR. Supplementary Figure S7. AR levels in AR overexpressing cell lines. Supplementary Figure S8. LNCaP/AR(cs), LNCaP/SRF876L and LNCaP/pCDNAF876L cell proliferation. Supplementary Figure S9. Transcriptional activity of ARN-509 and enzalutamide in LNCaP/pCDNAF876L cells. Supplementary Figure S10. AR ChIP analysis of AR target genes. Supplementary Figure S11. PSA response for 3 patients with detectable AR F876L mutation.

PDF file - 183K, Supplementary Table S1. 1st and 2nd generation AR antagonist activity on F876L-AR. Supplementary Table S2. LNCaP/SRF876L Xenograft Pharmacokinetics Supplementary Table S3. AR nucleotide changes monitored by BEAMing assay Supplementary Table S4. Primary F876L BEAMing of ARN-509-001 Patients Supplementary Table S5. Transcriptional Real-time PCR Oligonucleotide Sequence Supplementary Table S6. ChIP Real-time PCR Oligonucleotide Sequence

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.

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.

Abstract The majority of breast cancers express estrogen receptor alpha (ERα) and thus are treated with anti-hormonal therapies that directly block ER function (e.g.Tamoxifen) or hormone synthesis (Aromatase Inhibitors). While these therapies are initially effective, acquired resistance emerges and disease progression ensues. Importantly, the majority of these tumors continue to depend on ERα for growth and survival via both ligand-dependent and ligand-independent pathways. The emerging evidence that ERα can be activated in the absence of estrogens via point mutations in ERα or cellular signaling pathways supports the development of agents that are not only competitive ERα antagonists but also reduce steady state levels of the receptor and thus limit both ligand dependent and independent signaling. Here we disclose the discovery of ARN-810, also known as GDC-0810. ARN-810 is an oral, potent antagonist of ER that also induces degradation of ERα at picomolar concentrations. ARN-810 treatment results in significant reduction in steady state ERα protein levels in breast cancer cell lines. Using peptide-based conformational profiling, we show ARN-810 induces ERα conformations that are distinct from both fulvestrant and tamoxifen indicating novel mechanism of action. In vitro, ARN-810 is active on wild-type and the constitutively active ERα mutants found in endocrine resistant breast cancer patients. Importantly, ARN-810 is active in cell-line and in vivo models of ESR1 wild-type and mutant, primary and endocrine-resistant breast cancers including patient derived xenograft (PDX) models. These preclinical data indicate that ARN-810, a novel Selective Estrogen Receptor Degrader (SERD), holds promise as a next generation therapy for the treatment of ER+ breast cancer as monotherapy, as well as in combination with agents that target other pathways involved in both intrinsic and acquired endocrine resistance. ARN-810 is in clinical development for the treatment of ER+ breast cancer. Citation Format: James Joseph, Steven Govek, Beatrice Darimont, Daniel Brigham, Anna Aparicio, Eric Bischoff, Mehmet Kahraman, Michelle Nannini, Joshua Kaufman, Andily Lai, Kyoung-Jin Lee, Jason Oeh, Nhin Lu, Wei Zhou, Michael Moon, Jing Qian, John Sensintaffar, Gang Shao, Deepak Sampath, Lori S. Friedman, Peter Rix, Richard A. Heyman, Nicholas Smith, Jeffrey H. Hager. Discovery of GDC-0810 a novel, non-steroidal selective estrogen receptor degrader with robust activity in pre-clinical models of endocrine-resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2015-5053

Abstract The majority of breast cancers express and rely on ERα for tumor growth, thus endocrine therapy is the mainstay of ER-positive breast cancer treatment. Despite the effectiveness of current therapies, many patients relapse with tumors still dependent on ER for growth via both estrogen-dependent and estrogen-independent mechanisms. Acquired mutations in ESR1 were recently identified in the ligand-binding domain of ERα in patients who progressed after aromatase inhibitors and tamoxifen. It is estimated that more than 20% of relapsed ER-positive breast cancer patients acquire activating mutations in ESR1. We created a variety of model systems to study the functional consequences of ESR1 mutations. In stable overexpressing cell lines, the mutant ER protein eventually becomes down-regulated, thus we generated cell lines with dox-inducible and CRISPR engineered ESR1 hotspot mutations. We confirmed ligand-independent transcriptional activity of ESR1 hotspot mutations including Y537S and D538G. Cell lines and patient derived xenograft (PDX) models were utilized in ER ChIP-seq studies. Additionally, we investigated therapeutic response and resistance in the context of ESR1 activating mutations for tamoxifen, fulvestrant, and next generation oral SERDs including ARN-810, also known as GDC-0810. ARN-810 is efficacious in MCF7 ESR1 Y537S xenografts and WHIM20 Y537S PDX tumors, indicating that SERDs may be effective for ER mutants. Citation Format: Wei Zhou, Robert A. Blake, Jim Nonomiya, Jing Qian, Lorna Kategaya, Ingrid Wertz, Anneleen Daemen, Thomas O'Brien, John Sensintaffar, Michael Moon, Michelle A. Nannini, Jason Oeh, Deepak Sampath, Xiaojing Wang, Nicholas Smith, Daniel Brigham, James Joseph, Jeffrey H. Hager, Lori S. Friedman. Selective estrogen receptor degrader (SERD) activity in ESR1 mutant models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2015-1864

Selective estrogen receptor degraders (SERDs) have shown promise for the treatment of ER+ breast cancer. Disclosed herein is the continued optimization of our indazole series of SERDs. Exploration of ER degradation and antagonism in vitro followed by in vivo antagonism and oral exposure culminated in the discovery of indazoles 47 and 56, which induce tumor regression in a tamoxifen-resistant breast cancer xenograft.

Approximately 80% of breast cancers are estrogen receptor alpha (ER-α) positive, and although women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, resistance often emerges. Although a variety of resistance mechanism may be at play in this state, there is evidence that in many cases the ER still plays a central role, including mutations in the ER leading to constitutively active receptor. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and is active in patients who have progressed on antihormonal agents. However, fulvestrant suffers from poor pharmaceutical properties and must be administered by intramuscular injections that limit the total amount of drug that can be administered and hence lead to the potential for incomplete receptor blockade. We describe the identification and characterization of a series of small-molecule, orally bioavailable SERDs which are potent antagonists and degraders of ER-α and in which the ER-α degrading properties were prospectively optimized. The lead compound 11l (GDC-0810 or ARN-810) demonstrates robust activity in models of tamoxifen-sensitive and tamoxifen-resistant breast cancer, and is currently in clinical trials in women with locally advanced or metastatic estrogen receptor-positive breast cancer.

Abstract 60–75% of all breast cancers express the estrogen receptor (ER) and thus are treated with anti-hormonal therapies that directly block ER function (e.g. Tamoxifen) or hormone synthesis (Aromatase Inhibitors). While these therapies are initially effective, acquired resistance invariably emerges. Importantly, the majority of these tumors continue to express and depend on ER for growth and survival, suggesting that novel approaches to target ER signaling have tremendous potential to treat endocrine-resistant disease. We have identified novel ER antagonists that induce degradation of ER at picomolar concentrations resulting in significant reduction in steady state ER protein levels in breast cancer cell lines. These compounds yield regression in both Tamoxifen-sensitive and - resistant models of breast cancer in vivo. Based on their unique in vitro profile, good pharmacokinetics and oral bioavailability, these compounds represent a novel class of Selective Estrogen Receptor Degraders (SERDs) that hold tremendous promise as a next generation therapy for the treatment of ER+ breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A133.

Abstract Despite the impressive clinical activity of the second-generation antiandrogens enzalutamide and ARN-509 in patients with prostate cancer, acquired resistance invariably emerges. To identify the molecular mechanisms underlying acquired resistance, we developed and characterized cell lines resistant to ARN-509 and enzalutamide. In a subset of cell lines, ARN-509 and enzalutamide exhibit agonist activity due to a missense mutation (F876L) in the ligand-binding domain of the androgen receptor (AR). AR F876L is sufficient to confer resistance to ARN-509 and enzalutamide in in vitro and in vivo models of castration-resistant prostate cancer (CRPC). Importantly, the AR F876L mutant is detected in plasma DNA from ARN-509–treated patients with progressive CRPC. Thus, selective outgrowth of AR F876L is a clinically relevant mechanism of second-generation antiandrogen resistance that can potentially be targeted with next-generation antiandrogens. Significance: A missense mutation in the ligand-binding domain of the androgen receptor F876L confers resistance to the second-generation antiandrogens enzalutamide and ARN-509 in preclinical models of AR function and prostate cancer and is detected in plasma DNA from ARN-509–treated patients with progressive disease. These results chart a new path for the discovery and development of next-generation antiandrogens that could be coupled with a blood-based companion diagnostic to guide treatment decisions. Cancer Discov; 3(9); 1020–9. ©2013 AACR. See related commentary by Nelson and Yegnasubramanian, p. 971 This article is highlighted in the In This Issue feature, p. 953

Abstract Greater than 50% of high grade serous ovarian cancers express the estrogen receptor alpha (ERα). This observation, in addition to multiple lines of epidemiological and preclinical data, suggests that, similar to breast and endometrial cancer, estrogen receptor signaling may play a role in the development and progression of ovarian cancer. Unfortunately, unlike in breast cancer, therapeutically targeting ERα signaling in patients with recurrent ovarian cancer typically yields only marginal clinical responses. However, patient selection based solely on ERα expression increases the response rate of aromatase inhibitors suggesting that ERα may be a viable therapeutic target in a subset of ovarian cancer patients. Given the presentation of late-stage disease, the mutational complexity and alteration of multiple signaling pathways known to induce ligand independent ERα activity in ovarian cancer, additional levels of patient stratification as well as novel therapeutics that target both the ligand dependent and independent ERα signaling, have the potential to yield better therapeutic outcomes. We have identified novel, orally bioavailable non-steroidal ERα antagonists that induce ERα degradation at picomolar concentrations in vitro resulting in significant reduction in steady state ERα protein levels in multiple cancer cell lines. Using peptide-based conformational profiling, we show that these ligands induce estrogen receptor conformations that are distinct from both fulvestrant and tamoxifen indicating novel mechanism of action. Importantly, these compounds block the growth of tamoxifen-sensitive and -resistant models of breast cancer and endometrial cancer in vivo. Similar to the breast and endometrial cancer models, these compounds antagonize ER target gene expression and induce ERα degradation in two ER+ ovarian cancer cell lines, OVSAHO and OVKATE, whereas the first generation ER antagonist tamoxifen antagonizes transcription but stabilizes ERα in this setting. Consistent with their transcriptional antagonist and degrader activities, these compounds also inhibit the hormone-dependent growth of these cell lines in vivo. Based on these findings, these compounds represent a novel class of Selective Estrogen Receptor Degraders (SERDs) that may hold promise as a next generation therapy for the treatment of ER+ ovarian cancer as monotherapy and importantly as combination therapy with agents that target the key nodal points critical to malignant progression or new, emergent agents displaying promising activity. Citation Format: James D. Joseph, Beatrice Darimont, Steven Govek, Dan Brigham, Anna Aparicio, Mehmet Kahraman, Andiliy Lai, Kyoung-Jin Lee, Nhin Lu, Johnny Nagasawa, Josh Kaufman, Michael Moon, Rene Prudente, Jing Qian, John Sensintaffar, Gang Shao, Peter Rix, Nick Smith, Jeff Hager. A novel class of selective estrogen receptor degraders display activity in pre-clinical models of ERα+ ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A37.