The oral selective oestrogen receptor degrader (SERD) AZD9496 is comparable to fulvestrant in antagonising ER and circumventing endocrine resistance

Abstract

Background: The oestrogen receptor (ER) is an important therapeutic target in ER-positive (ER+) breast cancer. The selective ER degrader (SERD), fulvestrant, is effective in patients with metastatic breast cancer, but its intramuscular route of administration and low bioavailability are major clinical limitations.

Methods: Here, we studied the pharmacology of a new oral SERD, AZD9496, in a panel of in vitro and in vivo endocrine-sensitive and -resistant breast cancer models.

Results: In endocrine-sensitive models, AZD9496 inhibited cell growth and blocked ER activity in the presence or absence of oestrogen. In vivo, in the presence of oestrogen, short-term AZD9496 treatment, like fulvestrant, resulted in tumour growth inhibition and reduced expression of ER-dependent genes. AZD9496 inhibited cell growth in oestrogen deprivation-resistant and tamoxifen-resistant cell lines and xenograft models that retain ER expression. AZD9496 effectively reduced ER levels and ER-induced transcription. Expression analysis of short-term treated tumours showed that AZD9496 potently inhibited classic oestrogen-induced gene transcription, while simultaneously increasing expression of genes negatively regulated by ER, including genes potentially involved in escape pathways of endocrine resistance.

Conclusions: These data suggest that AZD9496 is a potent anti-oestrogen that antagonises and degrades ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models.

Fig. 1

AZD9496 is comparable to fulvestrant in endocrine-sensitive ER +  cells. a Cell growth assay of T47D, MCF7, ZR75-1, 600MPE, and MDA-MB-415 parental cells treated for 6 days with different endocrine treatments. b Immunoblot for ER expression and signalling (PR) in MCF7 cells in the presence of 5% cs-FBS (ED-medium, ED), ED+E2, or 10% FBS and two different concentrations 10^–6 M (–6) and 10^–7 M (–7) of fulvestrant or AZD9496. c mRNA levels of progesterone receptor (PGR) in MCF7 cells were assessed using real-time quantitative PCR (RT qPCR). The mRNA expression was normalised to the actin housekeeping gene, and expression levels are presented as –ΔΔCT compared with E2 control. d ERE-luciferase activity assay in MCF7 cells treated by ED, ED+E2, or 10% FBS for 24 h. SEM are shown (n = 3); **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

AZD9496, like fulvestrant, inhibits tumour growth and ER signalling in vivo in the presence of E2. Mice bearing MCF7 xenografts in the presence of exogenous E2 were randomised to continued E2 or switched to ED, all plus vehicle (Veh), fulvestrant (Ful), or AZD9496 (9496), and monitored for 8 days. a Tumour growth curve of MCF7 cells in E2 or ED condition in the presence of vehicle, fulvestrant, or AZD9496 (n = 4–5). Graphs show the model-predicted values and 95% confidence intervals at each time point. For display purposes, predicted values have been ‘normalised’ by dividing predicted values and 95% confidence limits by the day 0 treatment group predicted value. Measurements of individual tumour sizes from each mouse were normalised in the same manner and plotted. b Representative ER IHC and quantification of ER protein level by IHC using H-score. c Immunoblot of ER downstream target gene PR and β-actin control in available E2 or ED-treated tumours. d Log2 fold change of the average gene expression in tumours from each treatment group. Heat map of genes differentially expressed in the E2 treated groups. SEM are shown; *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3

AZD9496 effect in endocrine-resistant ER +  cells. a Immunoblot for ER and downstream ER targets PR and BCL2 in MCF7 and T47D parental (P) and endocrine-resistant models (EDR, TamR, and FulR). b, c Effect of different endocrine therapies, all in ED-medium, ED, tamoxifen 10^–7 M (Tam), fulvestrant 10^–7 M (Ful), and AZD9496 10^–7 M (9496), on the growth of resistant cell models: MCF7 EDR, TamR, and FulR (b); T47D EDR, TamR, and FulR (c). Cell viability is expressed as relative percentage compared to its own control (hatched bars; EDR to ED, TamR to Tam, and FulR to Ful) at day 6 of treatment. SEM are shown (n = 3); *p < 0.05; ***p < 0.001; ****p < 0.0001

Fig. 4

AZD9496 is comparable to fulvestrant in delaying tumour growth and reducing ER level in endocrine-resistant tumours in vivo. Effect of AZD9496 and fulvestrant on in vivo transplantable model of MCF7 TamR (a–d, orange boxes) and MCF7 EDR (e, f, blue boxes). a Kaplan–Meier curves showing the tumour tripling time of MCF7 TamR tumours. Tumour volume is assessed in the presence of tamoxifen control (Tam), stop tamoxifen treatment and switch to drug vehicle (Veh), fulvestrant (Ful), or 3 different doses of AZD9496 (9496), 0.5, 5, and 50 mg/kg (for each group n = 12). Data are reported as change of the average of tumours in the same group. b ER protein levels by western blot and IHC (highlighted square in the western blot for the same sample) of representative short-term (10 days) treated tumours with quantification of ER protein level by IHC using H-score. c ER protein levels in representative long-term treated TamR tumours and quantification of ER protein level of the long-term treated tumours by IHC using H-score. d Heat map of significant differentially expressed genes in at least one of the treated groups compared to the tamoxifen control group. e Kaplan–Meier curves of transplantable MCF7 EDR tumours in the presence of vehicle (Veh; n = 7), fulvestrant (Ful; n = 6), or AZD9496 (9496; n = 6) for tumour tripling time from baseline followed up for about 90 days (±1–2 weeks). f Quantification of ER protein level by IHC using H-score. SEMs are shown; **p < 0.01; ****p = < 0.0001, ^#p < 0.01 compared to Tam and Veh, respectively. Cuzick test on (b and c) for Veh, 0.5, 5, and 50 mg/kg AZD9496 (not shown) p < 0.0001