Evaluation of the pharmacological activities of RAD1901, a selective estrogen receptor degrader

Abstract

Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains a first-line treatment for estrogen receptor 1 (ESR1) positive breast cancer. However, tumor resistance limits the duration of response. The clinical efficacy of fulvestrant, a selective ER degrader (SERD) that triggers receptor degradation, has confirmed that ESR1 often remains engaged in endocrine therapy resistant cancers. Recently developed, selective ER modulators (SERMs)/SERD hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and reproductive tissues have been advanced as an alternative treatment for advanced breast cancer, particularly in the metastatic setting. RAD1901 is one SSH currently being evaluated clinically that is unique among ESR1 modulators in that it readily enters the brain, a common site of breast cancer metastasis. In this study, RAD1901 inhibited estrogen activation of ESR1 in vitro and in vivo, inhibited estrogen-dependent breast cancer cell proliferation and xenograft tumor growth, and mediated dose-dependent downregulation of ESR1 protein. However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to result in the activation of ESR1 target genes and in the stimulation of xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in breast cancer models, having dose-dependent agonist/antagonist activity displayed in a tissue-selective manner. It remains unclear how this unique pharmacology will impact the utility of RAD1901 for breast cancer treatment. However, being the only SERD currently known to access the brain, RAD1901 merits evaluation as a targeted therapy for the treatment of breast cancer brain metastases.

Keywords: RAD1901; SERM; endocrine-resistant breast cancer; selective estrogen receptor degrader.

Fig. 1. RAD1901 inhibits ESR1 activity in vitro and in vivo

A) Chemical structure of SERM RAD1901. B) SKBR3 cells were plated in phenol red free media supplemented with charcoal stripped FBS (CFS) 24 hours prior to transfection with an ERE-luciferase reporter together with ESR1 or ESR2 expression vectors. 24 hours after transfection, cells were treated with E2 (10 nM) together with RAD (10⁻¹⁰–10⁻⁶ M) for 24 hours prior to harvest and analysis of luciferase activity normalized to co-transfected β-galactosidase control. B) MCF7 cells were plated in phenol red free media supplemented with CFS 48 hours prior to treatment with 10⁻⁹ M E2 together with ICI 182,780 (ICI), RAD1901 (RAD), GW7604, or 4-hydroxytamoxifen (4OHT) (10⁻¹¹–10⁻⁶ M) for 24 hours. mRNA levels of ESR1 target gene trefoil factor 1 (TFF1) were assessed using RT qPCR following RNA isolation. mRNA expression was normalized to the similarly detected 36B4 housekeeping gene, and expression levels are presented as fold change as compared to the vehicle-treated control. D) MCF7 cells were plated in phenol red free media supplemented with CFS 24 hours prior to treatment, and were treated with 10⁻⁹ M E2 as well as with the indicated ligands (10⁻¹¹ – 10⁻⁶ M) on days 1, 4, and 6 of an 8 day proliferation assay. DNA content as assessed by fluorescence was measured as a surrogate for cell proliferation. The relative increase in DNA fluorescence was calculated by normalizing to baseline values detected in a duplicate plate of cells that was harvested on day 1 prior to the initial treatment. Data are representative of at least 3 independent experiments. E–F) MCF7 cell derived tumors were implanted into ovariectomized estrogen-treated nu/nu mice. When tumor volume reached ~0.1 cm³, animals (n = 9–10) were randomized to receive daily treatment with vehicle, tamoxifen (Tam, 20 mg/kg sc) or RAD (20 mg/kg sc). E) Mean tumor volume +/− SEM per day of treatment is presented. Significance (2-way ANOVA of matched values followed by Bonferroni comparison) as compared to the vehicle control is indicated (* p < 0.0001). F) Expression of ESR1 target genes in tumors was analyzed essentially as in (C).

Fig. 2. RAD1901 downregulates ESR1 expression through receptor degradation

A) MCF7 cells were treated for 24 hours with ICI (10⁻¹³–10⁻⁷ M) or RAD (10⁻¹¹–10⁻⁵ M). Expression of ESR1 and loading control cytokeratin 18 (CK18 – Supplementary figure 1A) in whole cell extracts were detected by immunoblot (top). ESR1 levels relative to CK18 were quantitated by densitometry using Adobe Photoshop (bottom). B) MCF7 cells were plated as in Fig. 1B prior to 1 hour pre-treatment with vehicle or MG132 (10 μg/ml), followed by 6 hours of treatment with 10⁻⁷ M vehicle, ICI, Ral or RAD (10⁻⁸ or 10⁻⁶ M). ESR1 expression was detected as in (A). C–D) LTED MCF7 cells were plated in phenol red free media supplemented with FBS that was stripped of growth factors twice using charcoal. C) After 48 hours, cells were treated for 24 hours with E2 (10⁻⁷ M) or SERDs (10⁻⁶ M) and ESR1 was analyzed as in (A). D) LTED MCF7 cells were treated with ICI or RAD (10⁻¹¹ – 10⁻⁶ M) on days 1, 4, and 6 of an 8 day proliferation assay and analyzed as in Fig. 1.

Fig. 3. The in vivo pharmacology of RAD1901 is influenced by SERD activity

Ovariectomized C57Bl/6 mice (n = 5) were treated daily for 3 days with (A) vehicle or (B) estradiol (10 μg/kg) together with vehicle, Ralox (10 mg/kg) or RAD (0.3–100 mg/kg). At euthanasia, body weight as well as uterine wet weight were measured prior to cryopreservation of the uterus. Significant changes in uterine weight observed in animals not receiving estrogen treatment (A) were determined by ANOVA followed by Bonferroni comparison to the vehicle control and are indicated (* p < 0.05). C) ESR1 and β-actin expression in extracts made from pulverized uterine tissues were analyzed by immunoblot as in Fig. 1 (left). ESR1 expression relative to β-actin was quantitated as in Fig. 2 (right). Significant downregulation (* p < 0.05) of ESR1 was determined by ANOVA followed by Bonferroni multiple comparison.

Fig. 4. RAD1901 exhibits dose dependent growth stimulation of MCF7 xenograft tumors

MCF7 xenograft tumors were initiated in ovariectomized female nu/nu mice as in Fig. 1. Estrogen pellets were surgically removed when tumors reached ~0.1 cm³ volume, and animals (n = 6–10) then received daily treatment with vehicle or RAD (0.3–10 mg/kg sc). Mean tumor volume +/− SEM per day of treatment is presented. Significance as compared to the vehicle (2-way ANOVA of matched values followed by Bonferroni comparison) is indicated (* p < 0.05, ** p < 0.0005). B) Uterine wet weight at sacrifice (measured as in Fig. 3) and % change in tumor volume (as compared to size at randomization) calculated using the final measurement recorded for mice in (A) are graphically presented. C–D) Expression of ESR1 target genes in tumors was analyzed essentially as in Fig. 1. Estrogen only samples from Fig. 1D were included for comparison. E) ESR1 levels in tumor tissues were analyzed as in Fig. 3 and were normalized to similarly detected Lamin-A. Significant downregulation (* p < 0.05) of ESR1 was determined by ANOVA followed by Bonferroni comparion.

Fig. 5. RAD1901 exerts biphasic agonist/antagonist activity on ESR1 in a dose dependent manner

A) MCF7 cells were treated for 24 hours with 10⁻⁷ M vehicle (Veh), ICI, 4OHT, raloxifene (Ralox), E2 (10⁻⁹ M) or RAD1901 (10⁻⁹–10⁻⁶ M). The expression of ESR1 target genes responsive to (A) agonists, (B) primarily SERMs, or (C) SERMs and agonists was analyzed as in Fig. 1. Relative changes of these and additional target genes designed to evaluate dose dependent response to RAD1901 are presented in Supplementary figure 2. Significant target gene regulation (* p < 0.05) as compared to the vehicle control was detected by 2 way ANOVA followed by Fisher’s LSD test. D) Interaction between ESR1 and conformation-specific peptides in a mammalian two-hybrid system. Triplicate wells of SKBR3 cells were transfected with plasmids expressing ESR1 fused to VP16 together with Gal4DBD alone (control) or Gal4DBD fused to ESR1 interacting peptides noted on the horizontal axis. Cells were then treated with the indicated ESR1 ligands (10⁻⁷ M unless otherwise indicated). Interaction of ESR1 with the Gal4DBD peptide constructs was detected through activation of a Gal4-responsive luciferase reporter construct and was normalized to detected β-galactosidase activity expressed in a constitutive manner using a second vector. Normalized response is expressed as fold increase over the detected level of interaction between Gal4DBD alone and ESR1-VP16 in the absence of ligand (Veh). E) The effect of SERMs and SERDs (10⁻⁶ M) on the proliferation of MCF7 cells in response to E2 (10⁻⁹ M) or insulin (2 × 10⁻⁹ M) was evaluated as in Fig. 1. Statistically similar treatments (2-way ANOVA followed by Bonferroni multiple comparison) are indicated by letters.