Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

Abstract

Background: Addition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance.

Methods: Elacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and "de novo" CDK4/6i resistance.

Results: Elacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant's anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo.

Conclusions: We demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.

Keywords: Abemaciclib; Breast cancer; CDK4/6; Elacestrant; Estrogen receptor; Palbociclib; RAD1901; Resistance; Ribociclib; SERD.

Fig. 1

Characterization of palbociclib resistance models developed in ESR1 wild-type and ESR1 mutant (D538G and Y537S) backgrounds. CellTiter-Glo assay, colony formation assay, and western blot analysis of cell cycle proteins of a ESR1^wt-Palbo^S and ESR1^wt-Palbo^R cells, b ESR1^mut: D538G-Palbo^S and ESR1^mut: D538G-Palbo^R cells, and c ESR1^mut: Y537S-Palbo^S and ESR1^mut: Y537S-Palbo^R cells, treated with controls and palbociclib at the indicated doses. d Pathway perturbations in ESR1^wt-Palbo^R vs ESR1^wt-Palbo^S cell lines; red bar represents upregulated genes, blue bar represents downregulated genes

Fig. 2

CDK4/6i-resistant cell lines retain ER and ER signaling. a Western blot analysis of ER and downstream ER target genes (GREB1/PR) in the CDK4/6i-sensitive and CDK4/6i-resistant ESR1 wild-type and ESR1 mutant cell lines. Palbo^S/Palbo^R, Ribo^S/Ribo^R, and Abema^S/Abema^R cells were treated with either control or the indicated CDK4/6i for 24 h. b Table summarizing the changes in ER and ER target genes in the resistant cells when compared to the sensitive cell lines

Fig. 3

Elacestrant inhibits the growth of CDK4/6i-resistant breast cancer cell lines in vitro. a CellTiter-Glo assay of ER+ (wild-type and mutant) CDK4/6i-sensitive and CDK4/6i-resistant breast cancer cells treated with elacestrant at the indicated doses for 7 days. Relative EC₅₀ values were calculated by using a log(inhibitor) vs response curve fit. b Colony formation assay of ER+ (wild-type and mutant) CDK4/6i-sensitive and CDK4/6i-resistant breast cancer cells treated with elacestrant at the indicated dose for 3–5 weeks. c Western blot analysis of ER and downstream ER target genes (GREB1/PR) in the CDK4/6i-sensitive and CDK4/6i-resistant ESR1 wild-type and ESR1 mutant cell lines. CDK4/6i-sensitive, Palbo^R, Ribo^R, and Abema^R cells were treated with either control or elacestrant (300 nM). d qRT-PCR of PGR, TFF1, and GREB1 in palbociclib-sensitive and palbociclib-resistant cells treated with elacestrant at the specified doses

Fig. 4

Elacestrant inhibits the growth of ER+ patient-derived xenograft (PDX) models that represent CDK4/6i resistance. Mean tumor volumes (n = 6–10/arm) ± SEM of PDX-R1 (a, top) and WHIM43 (b, top). Percent change in tumor volumes from baseline for individual tumors from PDX-R1 at the end of study (a, bottom) and WHIM43 at day 55 (b, bottom). For the PDX-R1 model, asterisks represent significant differences between drug-treated and vehicle-treated groups at the end of the study. For the WHIM43 model, statistical analysis of drug-treated groups vs vehicle-treated groups was performed on the day the vehicle-treated groups were taken down (day 55) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). c Western blot analysis of indicated proteins from tumors harvested 4 h post-last dose in the PDX-R1 model. d Western blot analysis of indicated proteins from tumors harvested 4 h post-last dose in the WHIM43 model. e Mean tumor volumes of ST941-HI PDX palbo-naive (P0) and ST941-HI PDX palbo-treated for > 150 days (P3) treated with a combination of fulvestrant and palbociclib, and single-agent elacestrant (in P3 only)

Fig. 5

Elacestrant demonstrates anti-tumor activity as a single agent and in combination with PIK3CA pathway inhibitors. Mean tumor volumes (n = 10/arm) ± SEM of ST3932 (a) and CTG-2432 (b) PDX models in mice treated with indicated treatments. For the ST3932 model, asterisks represent significant differences between vehicle-treated and the indicated groups on the day the first vehicle-treated animal was taken down (day 28). For the CTG-2432 model, asterisks represent significant differences between the indicated groups at the end of the study. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Elacestrant exhibits anti-tumor activity and downregulates key cell cycle proteins in multiple models of CDK4/6i resistance. a Pictorial representation of elacestrant activity in multiple models of CDK4/6i resistance. b Elacestrant activity, represented as IC₅₀ and TGI, as a single agent and in combination in multiple models of CDK4/6i resistance. c Western blot analysis of indicated cell cycle proteins in in vitro models of palbociclib resistance. d Western blot analysis of indicated cell cycle proteins from tumors harvested 4 h post-last dose in the PDX-R1 model. e Western blot analysis of indicated cell cycle proteins from tumors harvested 4 h post-last dose in the WHIM43 model. f PDX tumors were harvested 4 h post-last dose in the PDX-R1 model. FFPE tumor sections were prepared and subjected to IHC with Ki67 antibody (Code# IR626, Agilent). The percent and their staining intensity were assessed by an expert breast pathologist blinded to the treatment to generate an H-score (t test). Representative images for each IHC were shown. Magnification, × 40