The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance

Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.


Supplementary Figure 2. Camizestrant induces proteasome-mediated degradation of ERα
A) MCF7 or CAMA-1 cells were treated with DMSO or the indicated concentration of fulvestrant, camizestrant (AZD9833), or AZD9496 for 48 hours.ERα expression was determined by western blot.Equal protein concentrations of lysates were spiked with the same concentration of medium L-arginine (prepared lysate from MCF7 cells fully labeled with13 C6 L-arginine) and immunoprecipitated overnight at 4°C on Nunc Maxisorp plates pre-coated with protein A/G and antiestrogen receptor (ER) antibody (HC-20, Santa Cruz), followed by tryptic protein digestion.
The ER proteotypic peptide selected for relative quantification was the ligand-binding domain peptide LLFAPNLLLDR (aa402 to aa412).Analysis of this peptide was achieved by nanoflow liquid chromatography using an ultra-performance liquid chromatography M-Class System (Waters) and a Xevo TQS triple quadrupole mass spectrometer (Waters) operating in multiple-reaction monitoring mode.The resulting chromatographic peaks were integrated in TargetLynx (Waters).
Degradation half-life was measured using the one-phase exponential decay equation in GraphPad Prism (Y = Span.e−K.X + Plateau), where X is time and Y is response, which starts out as Span + Plateau and decreases to Plateau with a rate constant K.

Viability assays
Seven-day cell viability assays for combination treatments with camizestrant and palbociclib or abemaciclib in MCF7 parental and palbociclib-resistant cell lines (PC1, PC6, PC8) were measured using Cell Titer Glo (CTG, Promega).Cells were seeded in 60 μL medium 1 day before treatment.
Assay plates were dosed the next day (Day 0) and read on Day 7.An untreated plate was read on Day 0. These results were obtained with the addition of 30 μL of CTG and read for luminescence after incubation for 30 minutes at ambient temperature.Data were normalized to the original Day 0 seeding and the maximum Day 7 growth (DMSO only).Combination analysis resulting in the "matrix plots" heat maps was performed using GeneData Screener.

Juvenile rat uterine model
Female Wistar Han ® rats (Envigo, Netherlands) -16 days old at delivery, 21 days old at the start of the experimentswere used for this study.Animals were acclimatized to the laboratory conditions for at least 5 days prior to the start of experiments.Pups were housed with their dam in polysulfone type Sealsafe plus GR900 cages (Tecniplast, Lyon, France) on a bed of wood chips (Souralit, Girona, Spain) with free access to food (Rodent Maintenance Diet A04 from Safe) and water (0.2 μm filtered water).Species-appropriate environmental enrichment (Tunnel, Plexx, Uden, Netherlands) was added to the cages.The animal house was maintained under artificial lighting between 07:00-19:00 in a controlled ambient temperature of 22±2°C, and relative humidity was maintained at 55±10%.Pups were separated from their dam 1 day before the experiment start.
All test compounds were formulated 3 days before the start of the treatment period.Fulvestrant was formulated at 1 mg/mL in peanut oil; tamoxifen at 2 mg/mL in Tween 80 (1%); and camizestrant at 10 mg/mL by dissolution in 40% of the final volume of tetraethylene glycol, then addition of 50% of the final volume of Captisol ® (12.5% volume/volume [v/v]), followed by pH adjustment to 3-7.5 by addition of 250 μL of 1 M hydrochloric acid.The solution was then made up to the final volume with Captisol (12.5% v/v).
Before the experiment, animals were assigned to the different treatment groups (five animals per group, per cage) according to body weight, ensuring no significant difference between mean group body weights.Animals from the same litter were assigned to different groups.
Test compounds and respective vehicles were administered orally (PO) or subcutaneously once daily for 3 days, with a 24-hour interval (±1 hour).Fulvestrant was dosed subcutaneously at 5 mg/kg at a volume of 5 mL/kg.Tamoxifen was dosed at 10 mg/kg PO at a volume of 5 mL/kg.Camizestrant was dosed at 50 mg/kg PO at a volume of 5 mL/kg.
Animals were euthanized 24 hours after the last dose, and the uterus excised and removed from the cervix, with the horns still connected (sampling just above the cervix), and all visible excess fat was removed.A small incision was made around half-way along each horn, the horns were blotted on filter paper to remove fluids, and their weights recorded.
Individual uterus weights were entered into an Excel ® spreadsheet; all data entry was cross-checked by two people.Individual uterus weights were then normalized to rat body weight by dividing uterus weight by body weight.
Tissues sections were analyzed with HALO image analysis software (Indica Labs v3.4).To determine the percentage of epithelial cells, the tissue was segmented using a DenseNet classifier into two classes: uterus and epithelium.Percentage of individual animals' epithelial area was calculated.

Cell lines and culture
All AZ cell lines were obtained from ATCC.Cells were tested and authenticated by short-tandem The CRISPR knock-in Y537S ESR1m MCF7 cell line was generated as described elsewhere.(2) Cells were used within 20 passages between thawing and use in the described experiments.
A terbium-labeled anti-GST antibody (PV3551, Invitrogen) was used to indirectly label the receptor by binding to its GST tag, and competitive binding was detected by a test compound's ability to displace a tracer fluorescent ligand (Fluormone ES2; P2645, Invitrogen) from the ERα-LBD (GST) resulting in a loss of TR-FRET signal between the terbium-anti-GST antibody and the tracer.The assay was performed with all reagents added using the Beckman Coulter BioRAPTR FRD microfluidic workstation.

Formulation of the SERDs and inhibitors
Camizestrant was formulated in triethylene glycol (TEG; 40% of final dosing volume) and 12.5% Captisol ® in water (60% of final volume).The vehicle control was formulated of 40% TEG and 60% Captisol at 12.5%.Fulvestrant was formulated at 50 mg/mL in peanut oil and dosed one or three times weekly as a 0.
For the camizestrant dose-response study in the CTC174 PDX model (hormone-independent), established tumors were removed from two donor mice and dissected into 3x3x3 mm pieces in RPMI 1640 media.Female NOD SCID gamma (NSG) mice (surgically ovariectomized at Jackson Laboratories, Bar Harbor, ME, USA) were anesthetized using 2% isoflurane.A small skin incision was made 0.5 cm dorsally to the third nipple, and a tumor fragment was inserted between the skin layer and the third mammary fat pad.The skin was closed with surgical glue (VetBond, 3M).TV was calculated as TV (mm3) = width2 x length x 0.52.
For the combination studies in the CTC174 PDX model (hormone-independent), established tumors were removed from donor mice and dissected into 3x3x3 mm pieces in RPMI 1640 media.Female NSG mice (CRL UK) were anesthetized using 2% isoflurane.A small skin incision was made 0.5 cm dorsally to the third nipple, and a tumor fragment was inserted between the skin layer and the third mammary fat pad.The skin was closed with surgical glue (VetBond, 3M).TV was calculated using the formula: TV (mm3) = (3.142x max (length:width) x min (length:width) x min (length:width))/6,000, or (mm3) = width2 x length x 0.52 for the CTC174 study.
For studies in the HBXF079-LTED PDX model (hormone-independent), established tumors were removed from donor mice and re-implanted between the skin layer and the third mammary fat pad in immunocompromised female NSG mice.Tumor volume was calculated as TV (mm3) = width2 x length x 0.52.The vehicle control or camizestrant at 10 mg/kg were dosed PO four times daily for 42 days at 0.1 mL per 10 g of mouse (n=8/9 per group).Tumors were harvested and snap frozen in liquid nitrogen 24 hours after the last dose.
For the CTG-1211 and CTG-2432 models (Champions Oncology) stock mice were produced in athymic nudes (plus estrogen supplementation).Established tumors were removed from donor mice and re-implanted unilaterally on the left flank in immunocompromised athymic nude mice.TV was calculated as TV (mm3) = width² x length x 0.52.
For the HBCx131, HBCx180 and HBCx169 models (Institute Curie), 140 mice (8 week-old female Swiss nude mice, Charles River Laboratories) were implanted for each efficacy assay.All implants came from the same tumor for each PDX.Animals grafted with HBCx131 and HBCx180 were continuously supplemented with 17β estradiol (Sigma-Aldrich) in their drinking water (2 µg/ml until start of treatment, 0.5 µg/ml after start of treatment).TV was calculated using the formula: TV = a x b2/2, where a is the largest diameter and b is the smallest diameter.
For the BB6RC160, HBCx3, HBCx19, HBCx22, HBCx34, and T272 models (XenTech), 5-11-weekold female athymic HsdCpb:NMRI-Foxn1nu mice (Envigo) were implanted for each efficacy assay (three mice per group).TV was calculated using the formula: TV (mm3) = [length x width2]/2.All implants came from the 3 to 24 donor tumors for each PDX, with no supplementation, except for the BB6RC160 model that received estrogen diluted in drinking water (β-oestradiol, 8.5 mg/l), from the date of tumor implant to the end of the study.
The resulting organoids were digested to perform cell viability assays.Organoids were pelleted at 500 rpm for 5 minutes in 15 mL tubes.After aspirating the supernatant, pellets were treated with TryPLE (ThermoFisher Scientific A1217701) for 5 minutes, 3 mL culture media was added, and tubes were centrifuged at 500 rpm for 5 minutes.Single cells were then seeded onto 96-well plates at 0.04-0.1x10 6cells/mL in culture media in suspension, and analyzed after drug treatment for 144 hours.Cells were treated with 10% alamarBlue (ThermoFisher DAL1025) and then incubated for 6 hours at 37°C.Two to three biological replicates of experiments were performed for six lines disassociated from six different CTC174 xenografted mice in three to four technical replicates.

In vitro gene expression analysis in cell lines and PDX models
For Figure 4B  Individual animal percentage inhibitions were calculated using the following equation: ℎ = ([]/ℎ   )) × 100%

B)
MCF7 and CAMA-1 cells cultured in the presence of heavy L-arginine were switched to media containing light L-arginine plus vehicle (0.1% DMSO) or 100 nM camizestrant (shown on graph as AZ'6724) or fulvestrant at start (T 0 ).At the indicated time point, cells were collected and the proportion of ERα containing heavy L-arginine was assessed by mass spectrometry.Individual data points from three independent experiments are presented with a one-phase decay line of best fit.C) MCF7 cells were pre-incubated for 1 hour with 10 µM of MG132.Vehicle or 100 nM camizestrant (AZD9833) or fulvestrant was then added to the MG132-containing media and incubated with cells for a further 5 hours.The level of ERα was assessed by western blotting, with GAPDH acting as a control.D) MCF7 cells were incubated with vehicle (DMSO) or the indicated concentration of fulvestrant or camizestrant (shown in figure as AZ'6724) for 3 or 24 hours.Lysates were fractionated into cytoplasmic and nuclear fractions and assessed by western blot.ER, estrogen receptor; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase Supplementary Figure 3. Binding and activity of camizestrant in clinically relevant ERα mutations A) ER degradation in a panel of ESR1wt cell lines and B) ER and PR MCF7 ESR1m Y537S treated at 100 nM camizestrant, fulvestrant, and elacestrant (RAD1901) for 48 hr, measured by western blot.C) In MCF7 cells expressing Y537S, camizestrant (AZD9833) demonstrated anti-tumor activity in a dose-dependent manner, with maximal anti-tumor activity at 10 mg/kg.D) Efficacy correlated with ER degradation measured by western blot from tumors taken at the end of the efficacy dosing period.Statistical analyses were performed by one-tailed, unequal variance t-test versus log (change in tumor volume) compared with vehicle control at the final day of treatment.ns, not significant; *p<0.05;**p<0.01;***p<0.001;**** p<0.0001ER, estrogen receptor; ESR1(m), (mutated) estrogen receptor 1 gene; PR, progesterone receptor Supplementary Figure 4. Camizestrant causes dose-dependent cytostasis or regression in PDX models CTC174 tumors and plasma were collected at a variety of times after the last dose of camizestrant, at a variety of doses for at least 3 days.Residual ERα was determined by western blot, normalized to vehicle control, and the concentration of camizestrant in the plasma, corrected for plasma binding, was determined by liquid chromatography/mass spectrometry.A) Points represent observed data from individual animals, and the line describes a concentration:response relationship derived from these data.B) Points represent mean residual ERα (± standard deviation) from groups of tumors treated with the indicated concentration of camizestrant and collected at the indicated time after the last dose.Residual ERα was determined by western blot and immunohistochemistry and compared with vehicle control tumors.ERα immunohistochemistry was scored by H-score.Data represent individual tumors; line and error bars are geometric mean ± 95% confidence interval.C) Modeled mean residual ERα levels over a 28-day dosing period for camizestrant 0.8, 3, 10, 20, and 40 mg/kg four times daily were calculated, plotted, and compared with the observed 28-day tumor growth inhibition for that dose.Points represent each camizestrant dose in ascending order.The dotted line represents 13% residual ERα, the point at which additional ERα degradation appears to have no further anti-tumor effect.ER, estrogen receptor; PDX, patient-derived xenograft.Supplementary Figure 5. Camizestrant is a pure ER antagonist A) Ishikawa cells were incubated with camizestrant or fulvestrant for 48 hours and lysates analyzed by western blot.ER or PR protein levels are expressed relative to the AZD9496 100 nM level.B) Juvenile female rats were treated with the indicated dose of compound for 3 days.Twenty-four hours after the last dose, the uterus was removed.Uterine horns were processed for hematoxylin and eosin staining.The morphology of the endometrial epithelium tissue was assessed by a pathologist with representative images shown.C, D) The uterine horn was weighed after removal and the epithelial area was calculated via computational pathology.Data points represent individual animals.Lines represent geometric mean ± 95% CI. * Tamoxifen vehicle, ** SERDs vehicle.