Abstract P5-05-01: Novel mechanisms of action of selective androgen receptor modulator RAD140 in AR+/ER+ breast cancer models
Authors: He Suqin , Conlan Maureen G , Arlt Heike , Morris Charles , Yu Ziyang 
Published: March 2020

Abstract
Background: Recurrence after standard-of-care estrogen receptor α (ER)-targeted therapy presents challenges in management of metastatic ER positive (ER+) breast cancer (BC). The high prevalence of androgen receptor (AR) in ER+ BC, along with preclinical and clinical evidence showing anti-tumor activity of AR agonists in AR+/ER+ BC, suggest an opportunity to address resistance to ER-targeted agents by targeting AR. Significant anti-tumor activity has been reported with RAD140, an oral selective AR modulator (SARM), in estrogen-dependent and -independent AR+/ER+ BC models. Mechanistic investigation revealed that inhibition of tumor growth by RAD140 was in part through AR-mediated reduction of ESR1 mRNA and subsequent suppression of ER signaling, independent of ESR1 alterations. Here, we present new observations on RAD140 mechanisms of action in in vitro and in vivo models of endocrine-independent BC models. Methods: HCC1428-long term estrogen deprived (LTED) line was established by passaging cells in steroid-depleted media for &gt; 6 months. HCC1428-LTED-palbociclib resistant (-palboR) BC cell line was generated from LTED by long-term passaging in the presence of 500 nM palbociclib. Fluorescence-microscopy was used to assess annexin V/propidium iodide (PI) and phospho- H2A.X expression. The endocrine-independent BC PDX model was established in nude mice without supplemental estradiol. Prior anti-cancer therapy of the donor patient included tamoxifen, exemestane, fulvestrant and multiple lines of chemotherapy. RAD140 (3 mg/kg twice daily), elacestrant (30 mg/kg once daily), and palbociclib (10 mg/kg once daily) were administered via oral gavage for the duration of study. Fulvestrant (3 mg/mouse) was given once weekly subcutaneously. Results: In a hormone-independent BC PDX model harboring the ESR1 D538G mutation, RAD140 treatment led to 80% tumor growth inhibition (TGI) compared to the vehicle. The selective ER degrader (SERD) fulvestrant had TGI of 7%, while the oral SERD elacestrant (RAD1901) had TGI of 60%. The combination of RAD140 with elacestrant exhibited enhanced anti-tumor activity greater than that of each agent alone (93% TGI). These in vivo findings suggest the activity of the AR-targeting RAD140 may be mediated through mechanisms beyond its AR-mediated inhibition of ER signaling. We then sought to identify additional mechanisms of action of RAD140 in two hormone-independent AR+/ER+ BC cell line models, HCC1428-LTED and HCC1428-LTED-palboR. After prolonged treatment (2 weeks) at 100 nM, RAD140 induced morphological changes related to apoptosis, along with increased number of annexin V/PI-positive cells. This was further supported by increases in cleaved PARP/caspase3/caspase7 proteins by western blot analysis. A decrease in the pro-survival protein BCL-2 was also seen. These results collectively suggest that RAD140 induced apoptosis in endocrine-resistant BC cells. In line with these findings, an increase in phospho-H2A.X, a sensitive marker for DNA damage, was also observed in RAD140-treated cells. Similar changes were observed with the positive control AR agonist dihydrotestosterone (DHT), but not with fulvestrant. The AR antagonist apalutamide was found to partially reverse the apoptosis induced by RAD140 and DHT, further supporting the on-target effect. Conclusion: Our data suggest the anti-tumor activity of RAD140 may be mediated through suppression of ER signaling as well as additional mechanisms. The effect of RAD140 on DNA damage and apoptosis implies the potential for combination therapy with agents targeting cell survival and DNA damage repair pathways. RAD140 is being studied in a Phase 1, first-in-human trial in postmenopausal women with hormone receptor positive BC (NCT03088527).
Citation Format: Suqin He, Maureen G Conlan, Heike Arlt, Charles Morris, Ziyang Yu. Novel mechanisms of action of selective androgen receptor modulator RAD140 in AR+/ER+ breast cancer models [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 P5-05-01.