Fulvestrant

To select subgenomic RNAs (sgRNAs) (Additional file 1: Table S1) for CRISPR-Cas9 genome-editing of T47D cells [12 (link)–15 (link)], we utilized a web tool (http://crispr.mit.edu) entering the sequence flanking Y537S and D538G mutations. The oligos were cloned into PX458 (www.addgene.com), also coding for Cas9, tracrRNA, green fluorescent protein (GFP), and the resulting plasmid was transfected along with the respective double-stranded 70 bp oligos into T47D cells. GFP+ cells were sorted by fluorescence-activated cell sorting (FACS), and the mutation was confirmed by Sanger sequencing (Additional file 2: Figure S1) and digital droplet PCR (ddPCR) using previously described methods [16 (link)] (Fig. 1a). We obtained two clones for Y537S, three clones for D538G, and three clones for ESR1 wild-type (WT), which were kept as individual clones, and pooled for experimental studies as indicated.Fig. 1

Generation and characterization of ESR1 mutant, genome-edited MCF7 and T47D cell line models. a ESR1 mutation allele frequency in DNA and RNA was determined by digital droplet PCR. b T47D and MCF7 wild-type (WT) or mutant clones were pooled and treated with vehicle, 1 nM estradiol (E2) or 1 μM of fulvestrant (Ful) for 24 h, and lysates were immunoblotted as indicated. The blot is representative of three independent experiments. ER estrogen receptor. c T47D and MCF7 clones were pooled after hormone deprivation, transfected with ERE-TK, and relative light units (RLU) were determined (one-way analysis of variance (Anova), **p < 0.01). The experiment was repeated three times and the figure shows one representative experiment with two biological replicates. d Hormone-deprived T47D and MCF7 cells were treated with vehicle, 1 nM E2, 1 μM fulvestrant or 1 nM E2 with 1 μM fulvestrant for 12 h, and RNA was isolated, and RT-qPCR was performed (one-way Anova for comparison of basal level, Student’s t test for comparison of fulvestrant response in the presence of E2, *p < 0.05, **p < 0.01)

Gene targeting of ESR1 in MCF7 cells was carried out using recombinant adeno-associated virus (AAV) technology as previously described [17 (link)]. Briefly, ESR1 was targeted using one AAV vector for both the ESR1 Y537S and D538G mutations. AAV vectors were generated by ligating WT homology arms into an AAV plasmid backbone (Agilent, La Jolla, CA, USA), and site-directed mutagenesis was utilized to generate the Y537S and D538G mutations within the targeting construct. Virus was prepared by co-transfecting HEK-293 T cells with pHelper, pRC (Agilent) and the respective ESR1 mutation carrying rAAV targeting plasmid: 10⁶ cells were infected, neomycin-resistant clones were isolated using a modified PCR screening strategy [18 (link)], and the cells were then exposed to Cre-expressing recombinant adenovirus to remove the neomycin cassette. Clones were confirmed by Sanger sequencing (Additional file 2: Figure S1), and ddPCR (Fig. 1a). Single-stranded cDNA was generated using the First Strand cDNA Synthesis Kit (Amersham Biosciences). Two clones and a targeted WT control for the ESR1 exon 10 locus were isolated for each ESR1 mutation. Primer sequences for PCR amplification, mutagenesis, targeting, and sequencing are shown in the Additional file 1: Table S2.

A total of 5,000 cells/well of the parental or resistant cell lines, cultured with their individual treatments, were plated in 96-well plates 24 hours before beginning respective additional treatments, which consisted of 10 μg/ml trastuzumab, 1 μM lapatinib, the combination of trastuzumab with lapatinib, or 10^-7 M fulvestrant. Cell growth was assessed at different time points (zero, three, six, and nine days). Cell cultures were fixed with 4% glutaraldehyde and stained with 0.05% methylene blue. The dye was subsequently extracted with 3% HCl and absorbance measured at 655 nm. Growth fold change was determined by ((O.D. 655 nm at six days/O.D. 655 nm at zero days) Treatment)/((O.D. 655 nm at six days/O.D. 655 nm at zero days) Control). Growth curve and growth fold change experiments were executed in quadruplicate.

This protocol was approved by the Mayo Clinic institutional review board and each site institutional review board within the participating Translational Breast Cancer Research Consortium (TBCRC) institutions (Supplement 1). Participants provided written informed consent. Postmenopausal women with ER⁺/ERBB2⁻ MBC or ER⁻/ERBB2⁻ MBC with a history of primary ER⁺/ERBB2⁻ disease were preregistered. The study defined ER⁺ disease (by clinical assay) as 10% or more cells positive to limit inclusion of basal-like ER⁻/ERBB2⁻ MBC that did not acquire ET resistance due to ERα downregulation. Additional key preregistration criteria included 2 or fewer prior MBC chemotherapy regimens, prior treatment with fulvestrant for ER⁺ MBC, measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, willingness to limit daily alcohol intake, and no visceral crisis. Unlimited prior treatment with ET was allowed.
During the preregistration period, a biopsy specimen of a metastatic site was obtained for central ERα testing (for stratification). Key registration criteria included adequate blood cell counts and chemistry results, an Eastern Cooperative Oncology Group (ECOG) performance score of 0 to 1, no systemic therapy 21 days or fewer before registration, no need for treatment with a chronic proton pump inhibitor or H2 antagonist, and no visceral crisis.

Alisertib was administered as 50 mg, oral, twice daily on days 1 to 3, 8 to 10, and 15 to 17 of a 28-day cycle. Fulvestrant was administered as 500 mg, intramuscularly on days 1 and 15 of a 28-day cycle (cycle 1) and then day 1 of all subsequent cycles.
Arm 1 patients experiencing progression of disease (PD) could crossover to arm 2 if (1) ER was 10%or greater positive in preregistration or PD tumor tissue; (2) recovery from toxic effects to grade 0 to 1; (3) adequate blood cell counts and chemistry results; and (4) treatment initiated within 35 days of PD.