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

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 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.

Abstract Tumor-associated macrophages (TAMs) are thought to be regulators of solid tumor development based on their capacity to enhance metastatic, invasive, and angiogenic programming of neoplastic tissue. Colony stimulating factor-1 (CSF-1) is a key cytokine involved in recruitment and activation of tissue macrophages, exerting these effects through binding to a high-affinity receptor tyrosine kinase, the CSF-1 receptor. We have developed a small molecule CSF1R inhibitor AC708 in an attempt to impact the TAM-related progression of human tumors. Here we demonstrate that AC708 possesses significant specificity for CSF1R relative to the rest of the kinome, and to the closely related PDGFR family receptors PDGFRα and β, FLT3, and KIT. In cell based assays, AC708 potently inhibited CSF1R phosphorylation mediated by CSF-1 (IC50 = 26 nM) and by IL-34 (IC50 = 33 nM). It also inhibited the viability of growth-factor dependent cells cultured in CSF-1 (IC50 = 38 nM) or IL-34 (IC50 = 40 nM), and inhibited the CSF-1-mediated differentiation and survival of primary human osteoclast with an IC50 of 15 nM. In cytokine release experiments where enriched human monocytes were stimulated with either CSF-1 or IL-34, AC708 inhibited MCP-1 release with nearly identical IC50 regardless of which cytokine was used (CSF-1 (93 nM), IL-34 (88 nM)), and with a lower IC50 than that obtained with the benchmark compound GW-2580 (CSF-1 (148 nM), IL-34 (140 nM)). In vivo, AC708 was assessed for its ability to inhibit the intraperitoneal growth of M-NFS-60 cells in mice. In these experiments, AC708 inhibited M-NFS-60 growth in a dose-dependent manner, with a greater than 80% reduction in cell number at 100 mg/kg, similar to that achieved with the benchmark compound Ki-20227. Two assays were employed to assess the ability of AC708 to modulate endogenous CSF1R. In the first model, AC708 inhibited CSF-1-mediated MCP-1 release in vivo by 60% when dosed at 100 mg/kg. In the second model, plasma levels of TRAP5b were determined following injection of recombinant Parathyroid hormone-related protein (PTHrP) with or without co-administration of AC708. PTHrP-induced increases of plasma TRAP5b were reduced by AC708 in a dose-dependent manner, with levels falling to below baseline in the 100 mg/kg dose group. Lastly to assess the ability of AC708 to modulate TAMs we utilized the 4T-1 breast cancer line implanted orthotopically. Although primary tumor growth was relatively unchanged by AC708 treatment in this model, administration of drug for two weeks resulted in a dose-dependent reduction of tumor resident macrophages, with a 70% reduction at the 100 mg/kg dose relative to vehicle control. AC708 impact on tumor angiogenesis and metastatic potential is currently under investigation and will be reported. These studies further validate CSF1R as a potential target in cancer, and support the development of AC708 as a therapeutic in oncology. Citation Format: Robert C. Armstrong, Barbara Belli, Martin W. Rowbottom, Ron R. Nepomuceno, Alan Q. Dao, Allison M. Rooks, Dan Brigham, Craig W. McMannus, Michael D. Hocker, Mark W. Holladay, Gang Liu. AC708 is a potent and selective inhibitor of CSF1R and reduces tumor associated macrophage infiltration in a breast tumor model . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2013-903