G1T48, an oral selective estrogen receptor degrader, and the CDK4/6 inhibitor lerociclib inhibit tumor growth in animal models of endocrine-resistant breast cancer

Abstract

Purpose: The combination of targeting the CDK4/6 and estrogen receptor (ER) signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER-positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here, we report the identification of G1T48, a novel orally bioavailable, non-steroidal small molecule antagonist of ER.

Methods: The pharmacological effects and the antineoplastic mechanism of action of G1T48 on tumors was evaluated using human breast cancer cells (in vitro) and xenograft efficacy models (in vivo).

Results: G1T48 is a potent and efficacious inhibitor of estrogen-mediated transcription and proliferation in ER-positive breast cancer cells, similar to the pure antiestrogen fulvestrant. In addition, G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance including those harboring ER mutations and growth factor activation. In vivo, G1T48 has robust antitumor activity in a model of estrogen-dependent breast cancer (MCF7) and significantly inhibited the growth of tamoxifen-resistant (TamR), long-term estrogen-deprived (LTED) and patient-derived xenograft tumors with an increased response being observed with the combination of G1T48 and the CDK4/6 inhibitor lerociclib.

Conclusions: These data show that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER-positive breast cancer.

Keywords: CDK4/6 inhibitor; Combination therapies; Endocrine-resistant breast cancer; G1T48; Selective estrogen receptor degrader.

Fig. 1

G1T48 is a potent selective estrogen receptor downregulator (SERD). a Chemical structures of G1T48 and benchmark SERMs and SERDs. b G1T48 downregulates the estrogen receptor in breast cancer cells. MCF7 cells were treated with ER ligands (10^–12–10^–6 M) for 18 h prior to fixation and detection of ER levels by In-Cell Western. *For tamoxifen and GW5638, dose response was 10^–11–10^–5 M. Error bars indicate the SD of triplicate samples

Fig. 2

G1T48 is a complete estrogen receptor antagonist. a G1T48 inhibits ER target gene expression in breast cancer cells. MCF7 cells were treated with ER antagonists (10^–10–10^–7 M) plus estradiol (E2; 10^–9 M) for 18 h. TFF1 mRNA expression was analyzed by real-time PCR. GAPDH was used to normalize real-time PCR data. b G1T48 competes for estrogen binding to ER. MCF7 cells were treated with 10^–10 M ³H-17β-E2 and competitor ligand (10^–12–10^–6 M) for 2 h. Cells were collected and radioactive counts were monitored on a Beckman LS 6000SC Scintillation counter. Error bars indicate the SD of duplicate samples. c G1T48 regulates ER target gene pharmacology similar to other SERDs. MCF7 breast cancer cells were treated with ER ligands (E2, fulvestrant, RU, RAL @ 100 nM; G1T48, 810, 9496, Laso, 4OHT, 7604, BZA @ 1.0 μM; 5638, Tam @ 10 μM) for 24 h. mRNA expression was analyzed by real-time PCR. GAPDH was used to normalize real-time PCR data. Heatmaps were generated from real-time PCR data after analysis with JMP pro software (SAS) using the Ward hierarchical clustering algorithm. d G1T48 blocks estrogen-dependent recruitment of ER to the TFF1 promoter. MCF7 cells were treated with ligand (E2: 5 × 10⁻¹⁰ M; ER antagonists: 10^–6 M) as indicated for 45 min. Cells were fixed with formaldehyde and chromatin was immunoprecipitated with anti-ER antibody. Real-time PCR was used to assess the relative amount of ER bound to the TFF1 gene promoter. Error bars indicate the SD of triplicate samples

Fig. 3

G1T48 inhibits ER-positive breast cancer cell growth. a ER-positive MCF7, b ER-positive BT474 , c ER-positive ZR-75–1 were treated for 7 days with 10^–10 M E2 in addition to ER antagonists (10^–11–10^–5 M). d ER-negative MDA-MB-231 cells were treated for 7 days with ER antagonists (10^–11–10^–5 M). Cellular proliferation was assessed by measuring DNA content (Hoechst stain) and is presented as fold induction over vehicle control. Error bars indicate the SD of triplicate samples

Fig. 4

G1T48 inhibits ER signaling in models of endocrine therapy resistance in vitro a SKBR3 breast cancer cells were transfected with an estrogen-responsive reporter gene together with ER (wtER, ER-Y537S, or ER- D538G) expression plasmids prior to 18 h of treatment with 17β-estradiol (1.0 nM) and ER antagonists (10^–11–10^–5 M). Firefly and renilla luciferase activity were then assessed using dual luciferase reagent. Error bars indicate the SD of triplicate samples. b G1T48 inhibits cell growth mediated by endocrine refractory ER mutants. MCF7 cells expressing ER variants ER-D538G and ER-Y537S were treated for 7 days with doxycycline plus increasing dose of antiestrogens (10^–12–10^–5 M). Cellular proliferation was assessed by measuring DNA content (Hoechst stain) and is presented as relative fluorescence units. Error bars indicate the SD of triplicate samples. c G1T48 inhibits growth factor-mediated breast cancer cell growth. MCF7 cells were treated for 7 days with insulin (20 nM) plus increasing dose of anti-estrogens (10^–12–10^–7 M). Cellular proliferation was assessed by measuring DNA content (Hoechst stain) and is presented as relative fluorescence units. Error bars indicate the SD of triplicate samples

Fig. 5

Combination strategy of G1T48 and the CDK4/6 inhibitor lerociclib inhibit estrogen-dependent and tamoxifen-resistant (TamR) breast cancer xenograft models in vivo a Ovariectomized estrogen-treated female nu/nu mice bearing MCF7 xenograft tumors were randomized to treatment with vehicle, lerociclib (50 mg/kg) or G1T48 (30 or 100 mg/kg), alone or together, p.o. daily for 28 days. 2-way ANOVA comparison of average tumor volumes throughout treatment, followed by Bonferroni multiple comparison test, indicated significant tumor growth inhibition by all treatments, as well as increased response to the combination of G1T48 (30 mg/kg) and lerociclib (50 mg/kg). Error bars represent SEM. b–d Ovariectomized tamoxifen-treated female nu/nu mice bearing TamR xenograft were randomized to treatment with vehicle, palbociclib (100 mg/kg), lerociclib (50 or 100 mg/kg) (b), fulvestrant (200 mg/kg), or G1T48 (30 or 100 mg/kg) (c), with lerociclib and G1T48 being tested alone and in combination (d), p.o. daily for 28 days. 2-way ANOVA comparison of average tumor volumes throughout treatment, followed by Bonferroni multiple comparison test, indicated significant tumor growth inhibition by all treatments, as well as increased response to the combination of G1T48 (30 mg/kg) and lerociclib. Error bars represent SEM

Fig. 6

Combination strategy of G1T48 and the CDK4/6 inhibitor lerociclib in vivo in an estrogen-deprived xenograft model. Ovariectomized vehicle-treated female nu/nu mice bearing LTED xenograft were randomized to treatment with vehicle, lerociclib (50 or 100 mg/kg) or palbociclib (100 mg/kg) (a) or G1T48 (5 or 100 mg/kg) or fulvestrant (25 mg/kg) (b), alone or together (c), p.o. daily for 28 days. 2-way ANOVA comparison of average tumor volumes throughout treatment, followed by Bonferroni multiple comparison test, indicated significant tumor growth inhibition by all treatments, as well as increased response to the combination of G1T48 (30 mg/kg) and lerociclib. Error bars represent SEM

Fig. 7

Evaluation of the combined efficacy of lerociclib and G1T48 in a Patient-Derived Xenograft Model harboring the ER-Y537S Mutation. a Female nu/nu mice were engrafted with a START Patient-Derived Xenograft Model (START-PDX) model, designated ST2177, harboring an ER-Y537S mutation. Mice were randomized to vehicle, fulvestrant (5 mg/animal), G1T48 (30 or 100 mg/kg), lerociclib (50 mg/kg), or the combination of G1T48 and lerociclib and treated for 60 days. b, c Kaplan–Meier analysis is presented as time for tumors to reach endpoint (2.5 times original tumor volume). *Kaplan–Meier analysis followed by a Mantel–Cox test for significance demonstrated significantly greater tumor growth delay for these comparisons using an adjusted Bonferroni threshold of p < 0.012. Error bars represent SEM