Introduction: Overexpression of somatostatin receptors (SSTRs), particularly SSTR2, is found in gastroenteropancreatic neuroendocrine tumors (GEP-NETs), and subsets of other solid tumors such as small-cell lung cancer (SCLC). β-emitting radiopharmaceutical therapy Lu-177 DOTATATE has been approved for SSTR-expressing (SSTR+) GEP-NETs, but the relatively low objective response rate and frequent post-treatment progression represent an unmet medical need. Based on preliminary clinical anti-tumor efficacy, RYZ101 (Ac-225 DOTATATE) is being developed for inoperable SSTR+ well-differentiated GEP-NETs with disease progression following Lu-177-somatostatin analogue (SSA) therapy (ACTION-1, NCT05477576). SCLC accounts for approx. 13% of lung cancer and lacks effective therapeutic options. Immunohistochemistry analysis indicates that ~50% of SCLC tumors are SSTR2+, with a substantial subset showing high and homogenous expression. The goal of this study was to provide preclinical support for RYZ101, as a single-agent or in combination with carboplatin and etoposide, for SSTR+ SCLC. Methods: RYZ101 is comprised of the α-emitting radioisotope actinium-225, chemical chelator DOTA, and SSA octreotate (TATE). Binding affinity to human SSTR1-5 was determined using a competitive radioligand binding assay in engineered cell lines expressing individual SSTR. Internalization was measured by PathHunter SSTR2 Activated GPCR Internalization Assay. The preclinical biodistribution of RYZ101 was carried out in non-tumor-bearing BALB/c mice of both sexes. The efficacy studies were performed in cell line-derived and patient-derived xenograft (PDX) models of SCLC. Results: In preclinical studies where Lanthanum (La3+) was used as a surrogate for Ac-225, RAYZ-10001-La (La-DOTATATE) had high binding affinity (Ki=0.057nM) to human SSTR2 (>600-fold more potent than other SSTR subtypes) and exhibited efficient internalization. In a preclinical biodistribution study, RYZ101 performed similarly to Lu-177 DOTATATE. In multiple SCLC xenograft models, RYZ101 significantly inhibited tumor growth and prolonged survival, with deeper responses, including sustained regression, observed in the models with higher SSTR2 level. The anti-tumor effect was further enhanced when combining RYZ101 with carboplatin and etoposide at clinically relevant doses. Conclusions: In summary, RYZ101 is a first-in-class, highly potent, α-emitting radiopharmaceutical therapy being developed for the treatment of SSTR+ solid tumors. Preclinical data demonstrate the potential of RYZ101 for the treatment of patients with SSTR+ SCLC. Citation Format: Guangzhou Han, Eunmi Hwang, Fanching Lin, Renee Clift, Daniel Kim, Eric Bischoff, Susan Moran, Gary Li. Anti-tumor activity of RYZ101 (Ac-225 DOTATATE) in somatostatin receptor-expressing preclinical models of small-cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5042.

Abstract Overexpression of somatostatin receptors (SSTR), particularly SSTR2, is found in gastroenteropancreatic neuroendocrine tumors (GEP-NET), and subsets of other solid tumors such as small-cell lung cancer (SCLC). SCLC accounts for approximately 13% to 15% of lung cancer and lacks effective therapeutic options. IHC analysis indicates that up to 50% of SCLC tumors are SSTR2-positive, with a substantial subset showing high and homogenous expression. Peptide receptor radionuclide therapy with radiolabeled somatostatin analogue, Lu-177 DOTATATE, has been approved for GEP-NETs. Different strategies aimed at improving outcomes, such as the use of alpha-emitting radioisotopes, are currently being investigated. RYZ101 (Ac-225 DOTATATE) is comprised of the alpha-emitting radioisotope actinium-225, chemical chelator DOTA, and octreotate (TATE), a somatostatin analogue. In the cell-based competitive radioligand binding assay, RAYZ-10001-La (lanthanum surrogate for RYZ101) showed high binding affinity (Ki = 0.057 nmol/L) to human SSTR2 and >600-fold selectivity against other SSTR subtypes. RAYZ-10001-La exhibited efficient internalization to SSTR2-positive cells. In multiple SSTR2-expressing SCLC xenograft models, single-dose intravenous RYZ101 3 μCi (0.111 MBq) or 4 μCi (0.148 MBq) significantly inhibited tumor growth, with deeper responses, including sustained regression, observed in the models with higher SSTR2 levels. The antitumor effect was further enhanced when RYZ101 was combined with carboplatin and etoposide at clinically relevant doses. In summary, RYZ101 is a highly potent, alpha-emitting radiopharmaceutical agent, and preclinical data demonstrate the potential of RYZ101 for the treatment of patients with SSTR-positive cancers.

e15113 Background: Ephrin type-A receptor 2 (EphA2) is a glycoprotein of the ephrin receptor subfamily. EphA2 is primarily involved in tissue patterning during embryonic development, and its expression levels are low or absent in normal adult tissues. However, EphA2 overexpression has been observed in multiple malignant tumors such as bladder, cervical, ovarian, colorectal, lung and esophageal cancers. In addition to being a tumor biomarker, EphA2 plays an active role in tumor survival, metastasis and neo-angiogenesis, which can lead to poor prognosis for cancer patients. The broad overexpression in solid tumors and relatively low expression in normal adult tissues make EphA2 attractive for targeted radiopharmaceutical therapy (RPT). Methods: RAYZ-6114 is comprised of a macrocyclic peptide binder to EphA2, a linker, and DOTA chelator which can be complexed with different radiometals. RAYZ-6283 shares the same peptide binder and chelator with RAYZ-6114 but differs in the linker. The binding affinity, selectivity and cross-species reactivity to EphA2 and other Ephrin proteins were determined by surface plasma resonance (SPR). Target-mediated internalization was measured using flow cytometry. In vivo biodistribution and anti-tumor efficacy studies were performed in tumor-bearing athymic nude mice. A coagulopathy study was performed in Sprague Dawley rats. For tumor type identification, EphA2 immunohistochemistry (IHC) was performed on tumor microarrays (TMA) representing diverse tumor types. Results: IHC analyses of TMAs confirmed the expression of EphA2 in a multitude of solid tumors, with the highest positivity rates in cervical, pancreatic, bladder, colorectal, esophageal, and non-small cell lung cancers. RAYZ-6114 showed high binding affinity to human EphA2 with a K D of 0.03 nM. High-affinity binding was conserved across mouse, cynomolgus monkey, and human EphA2. No binding to other Ephrin type-A or Ephrin type-B proteins was detected, nor to EphA2-knockout cells. The binder rapidly and efficiently internalized in EphA2-positive H1299 cells upon target engagement, with ~75% internalized by 1 hour. In PC3 xenograft mice, 177 Lu-RAYZ-6283 showed sustained tumor uptake (~25% ID/g) for up to 48 hours and tumor/kidney ratios of 2.7, 3.3, and 5.9 at 24h, 48h, and day 7, respectively. Low uptake was seen in other normal tissues. Both 177 Lu- and 225 Ac- labelled RAYZ-6114 significantly inhibited tumor growth. Particularly, durable tumor regression and survival benefit were achieved by a single dose of 225 Ac-RAYZ-6114 (3 uCi), out-performing 177 Lu-RAYZ-6114 dosed at 3 mCi. All treatments were well tolerated. Conclusions: RAYZ-6114 and RAYZ-6283 are first-in-class, highly potent and selective macrocyclic peptide binders. Preclinical pharmacodynamic, pharmacokinetic, biodistribution and efficacy data demonstrated their potential for treatment of patients with EphA2-positive tumors.

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 While endocrine therapies are initially effective to treat estrogen receptor (ERα) positive breast cancer tumors, acquired resistance invariably emerges. Although resistant tumors typically display changes in PI3K or other kinase pathways, the majority of these tumors continue to express and depend on ERα for growth and survival. Exploring the hypothesis that endocrine resistance might involve hormone-independent activities of ERα, we have developed two distinct classes of potent, non-steroidal ERα modulators that antagonize hormone-mediated transcriptional activities of ER and lower ERα steady state levels. These compounds showed robust efficacy in pre-clinical models of endocrine-resistant breast cancer, including some with up-regulated PI3K pathways. In contrast to their activity as full antagonists/ inverse agonists in the breast, the two classes of ERα modulators displayed distinct abilities to oppose the activity of estradiol in the uterine endometrium, another well-characterized ERα target tissue. Similar to endocrine-resistant breast cancer, a large percentage of endometrial cancers have altered PI3K pathways and typically respond poorly to endocrine therapy. In mouse xenograft models one of our compounds inhibited the growth of ECC-1 endometrial cancer tumors with substantially higher efficacy than the selective ER modulator arzoxifene, which in a phase II clinical trial exhibited a 30% response rate in patients with recurring ERα-positive endometrial cancers. Hence, beyond their promise as a next generation therapy for the treatment of endocrine resistant ER+ breast cancer, these novel classes of selective estrogen receptor degraders might also open new opportunities for endocrine therapies targeting endometrial cancer.

Abstract The androgen receptor (AR) plays a central role in the development and progression of prostate cancer. Recent studies demonstrate that AR remains essential in the majority of castration resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed. The clinical efficacies of MDV3100 and abiraterone acetate, both of which target the AR pathway in the castrate resistant setting, support these findings. ARN-509 is a 2nd generation competitive AR antagonist that, unlike bicalutamide, maintains full antagonist activity in preclinical CRPC models. ARN-509 does not robustly induce AR nuclear localization or DNA binding. However, ARN-509 displays maximal efficacy in the LNCaP/AR xenograft model of CRPC at lower dose and steady state plasma concentrations compared to MDV3100, suggesting potential for higher therapeutic index and ability to deliver the maximally efficacious dose in man. To date, ARN-509 has shown promising antitumor activity in mCRPC patients enrolled in a Phase 1 study. Given that approximately 50% of CRPC patients have suboptimal response to MDV3100 and abiraterone acetate as well as the observation that resistance eventually develops in patients who initially respond to therapy, we sought to determine whether AR remains a viable therapeutic target in the MDV3100 and ARN-509 resistant setting. To this end, we generated several MDV3100 and ARN-509 resistant derivatives of the LNCaP and LNCaP/AR cell lines. While work is underway to determine the molecular mechanisms of resistance, a subset of cell lines does not require androgens for growth in vitro. These androgen independent derivatives express AR at levels comparable to LNCaP/AR (approximately 3X LNCaP) or 2-3 fold LNCaP-AR. When representative lines are injected into castrated mice, they demonstrate a decreased latency of tumor formation compared to the parental cell line both in the presence and absence of ARN-509. Importantly, in all lines tested, small interfering RNA mediated reduction in AR levels dramatically impaired the ability of the androgen independent resistant cell lines to proliferate in the absence of androgens. These data support the hypothesis that AR remains a viable therapeutic target for second generation anti-androgen resistant prostate cancer and is the first step toward establishing a platform to screen for next generation anti-androgens. Citation Format: James D. Joseph, Anna Aparicio, Josh Kaufman, Jackie Julien, Celine Bonnefous, Nicholas D. Smith, Peter Rix, Michael E. Jung, Charles L. Sawyers, Richard A. Heyman, Jeffrey H. Hager, Nicola J. Clegg, John Sensintaffar, Nhin Lu, Kate Grillot, Eric Bischoff, Gang Shao, Jing Qian, Beatrice Darimont. Targeting AR in castration-resistant prostate cancer: Development of ARN-509 and second-generation antiandrogen resistance models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C16.

28 Background: ARN-509 is a second-generation anti-androgen discovered in a screen to identify full androgen receptor (AR) antagonists in the context of AR over-expressing prostate cancer cells, a model for castration resistant prostate cancer (CRPC). It has been reported that other second-generation anti-androgens, MDV3100 and BMS-641988, can induce seizures at high dose in pre-clinical species and man and that this is mediated through antagonism of the CNS-based GABAA receptor. To define the clinical potential of ARN-509, we carried out a comprehensive assessment of its in vitro and in vivo activity in validated models of CRPC and assessed its seizure inducing potential.METHODSARN-509 and MDV3100 were profiled in a series of assays to monitor both on- and off-target activity. Comparative in vivo efficacy in the LNCaP/AR mouse xenograft model of CRPC and pharmacokinetics were determined. Seizure inducing potential was assessed in an acute pentylenetetrazol (PTZ) infusion model.RESULTSIn vivo, in the LNCaP/AR model of CRPC, an ARN-509 dose of 10 mg/kg/day exhibited tumors regressions equivalent in frequency and magnitude to a 30 mg/kg/day dose of MDV3100. Tumor re-growth following once daily dosing (30 mg/kg) for 28 days revealed that ARN-509 treated tumors exhibited a more durable response than MDV3100 treated tumors as evidenced by a significantly longer time to re-growth. At doses that yielded equivalent degree of tumor regression, the steady state plasma and brain levels were significantly lower for ARN-509 (10 mg/kg) than MDV3100 (30 mg/kg). ARN-509 and MDV3100 exhibit similar binding affinity to the GABAA receptor; IC50 3.0 and 2.7 mM, respectively. In vivo seizure potential of ARN-509 and MDV3100 was assessed in an acute PTZ infusion model in mice. MDV3100 was found to produce a dose dependant lowering of seizure threshold, while ARN-509 had no effect at any dose tested.CONCLUSIONSARN-509 is a second-generation anti-androgen with significant efficacy and an appropriate safety profile that supports its clinical development in both CRPC and earlier stages of prostate cancer. ARN-509 is currently in a phase I/II study in CRPC patients. [Table: see text].

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes.

Azepino[4,5-b]indoles have been identified as potent agonists of the farnesoid X receptor (FXR). In vitro and in vivo optimization has led to the discovery of 6m (XL335, WAY-362450) as a potent, selective, and orally bioavailable FXR agonist (EC(50) = 4 nM, Eff = 149%). Oral administration of 6m to LDLR(-/-) mice results in lowering of cholesterol and triglycerides. Chronic administration in an atherosclerosis model results in significant reduction in aortic arch lesions.

Our laboratory has demonstrated that treatment of MCF-7 breast cancer cells with melatonin (Mlt) followed 24h later with physiological concentrations of all-trans retinoic acid (atRA) results in apoptosis. These studies were extended into trials using the N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model. Initial studies conducted by feeding the animals 9-cis-retinoic acid (9cRA in the chow) and administering melatonin by subcutaneous injection in the late afternoon demonstrated that the combination of Mlt and 9cRA was able to significantly prevent tumor development, and that the combination was more efficacious that either Mlt or 9cRA alone. In this report, we conducted studies to determine if lower doses of 9cRA could be used in combination with Mlt while still maintaining anti-tumor activity and if the route of administration of 9cRA (bolus (gavage) v.s. chronic (chow) routes) affected its interaction with Mlt. The studies presented here demonstrate that significantly reduced doses of 9cRA can be used in combination with Mlt while maintaining anti-tumor efficacy. Furthermore, our studies demonstrate that 9cRA is equally effective when it is administered chronically (chow) or as a bolus (gavage). These data demonstrate that the combined use of Mlt and 9cRA produces additive or synergistic effects, which are more efficacious than 9cRA alone. This combination of Mlt and 9cRA could be a potentially useful clinical treatment regimen for breast cancer since it allows the use of lower doses of retinoic acid, thus, avoiding the toxic side effects associated with the use of high dose retinoids.

The conventional treatment of uterine leiomyomas, or fibroids, with gonadotropin-releasing hormone (GnRH) agonists is often associated with serious side effects, necessitating short-term, palliative use of this therapy. Therefore, we examined a retinoid X receptor (RXR)-selective ligand, LGD1069, as a possible treatment for leiomyoma. LGD1069 has demonstrated efficacy as a chemopreventive agent in the N-nitroso-N-methylurea (NMU)-induced rat mammary carcinoma model and is a therapeutic agent in several epithelial tumor models. Previous studies have shown that it has both antitumor effects and antiestrogenic activity in the rat uterus, suggesting the potential utility of this agent for treatment of hormonally dependent uterine fibroids. The expression of retinoid receptors in tumors and cell lines derived from leiomyomas arising in the Eker rat was confirmed by Northern analysis. After treatment for 4 months with LGD1069, the number of grossly observable tumors was substantially reduced although the total incidence of tumors, including microscopic lesions, remained unaffected, suggesting an effect of the compound on tumor growth kinetics rather than on tumor initiation. Analysis of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and determination of 5-bromo-2-deoxyuridine (BrdU) incorporation indicated that the reduction in grossly observable tumors that occurred in treated animals was mediated by a significant increase in the level of apoptosis rather than a decrease in cell proliferation. These results suggest that LGD1069 may be an effective therapeutic agent for uterine leiomyoma that may inhibit tumor growth and, consequently, alleviate the symptoms associated with this disease.