Antibodies and chemicals

The following antibodies were purchased from Cell Signaling Technology and used at the indicated dilution for Western blot analysis: phospho-4EBP1 (T37/46; #2855, 1:1000), 4EBP1 (#9644, 1:1000), phospho-S6 (S240/244; #5364, 1:20000), S6 (#2317, 1:1000), phospho-AKT (S473, #4060, 1:1000), AKT-pan (#4691, 1:5000), Cl-PARP (#9541, 1:1000), MCL1 (#94296, 1:2000), β-Actin (#3700, 1:100,000). These antibodies were obtained from Santa Cruz Biotechnology: Vinculin (sc-55465, 1:1000), Bcl-2 (sc-7382, 1:1000), V5 (sc-271944, 1:1000), and α-Tubulin (sc-8035, 1:1000). Bcl-XL (BD-556361, 1:1000) and eIF4E (BD-610269, 1:5000) antibodies were obtained from BD Biosciences. Horseradish peroxidase (HRP) linked secondary antibodies were from Cell Signaling Technology (#7076 anti-mouse or #7074 anti-rabbit, 1:4000). 4-Hydroxy-tamoxifen was purchased from SIGMA-ALDRICH INC (St. Louis, MO). Everolimus and INK128 were obtained from Revolution Medicine (South San Francisco, CA). Fulvestrant (#HY-13636/CS-1267) was purchased from MedChemExpress. MCL1-inhibitor S63845 was from MedChemExpress (#S63845).

The synthesis and characterization of RMC-6272 and RMC-5552 have been described elsewhere [38]. Both as bi-steric inhibitors in which the ether chemical handle was exchanged for a C40 carbamate to enable synthetic tractability and incorporated the XL388-derived (RMC-6272) and MLN0128-derived (RMC-5552) active-site inhibitors, respectively. Based on cellular assays using MDA-MB-468 cells, RMC-6272 and RMC-5552 display very potent inhibition of p4EBP1 (IC50: 0.44 nM and 0.48 M, respectively) and pS6K (IC50: 0.14 nM for both). Both compounds demonstrate selectivity for mTORC1 over mTORC2 (calculated as pAKT IC50/p4EBP1 IC50), with approximately 27-fold and 40-fold selectivity for RMC-6272 and RMC-5552, respectively.

Cell lines and tissue culture

BT20, BT474, BT549, HCC1428, HCC38, LY2, MCF-7, MDA-MB-231, T47D and ZR75B breast cancer cell lines were purchased from the American Type Culture Collection (ATCC). BT483, MDA-MB-157, MDA-MB-175VII, MDA-MB-361, MDA-MB-415, MDA-MB-436, MDA-MB-468, SKBR3, ZR-75-1 and ZR-75-30 breast cancer cells were purchased from the UCSF Cell Culture Facility (UCSF CCF). Tamoxifen-resistant (TAMR) MCF-7 (10 μM tamoxifen) cells were obtained from Dr. Pamela Munster (UCSF, San Francisco, CA). Palbociclib-resistant (PalboR) MCF-7 (1 μM palbociclib), ZR-75-1 (500 nM palbociclib), and T47D (100 nM palbociclib) cell lines were generated in the lab by gradually increase the concentration of palbociclib in the media. All resistant lines were grown and maintained in the presence indicated doses of the drug. Cell lines were confirmed by STR validation to ensure the authenticity of cell lines. No presence of Mycoplasma was found according to MycoAlertTM Mycoplasma Detection Kit from Lonza Group. Cell lines were grown according to the protocols in Supplementary Table 1.

Viral transduction and stable cell lines

For stable expression of eIF4E, the pLenti-MCV-Tet-eIF4E-flag plasmid was a gift from Dr. Davide Ruggero’s laboratory. Briefly, HEK293T cells were transfected with pLenti-MCV-Tet-eIF4E-flag together with packaging plasmid VSVG, Gag/pol and Rev. Virus particles were collected after 48 h transfection, filtered through 0.45-mm pore size Millipore filters. Filtered lentiviral supernatant was added to MCF-7 and ZR-75-1 cell lines with polybrene (8 µg/mL). After 72 h incubation, cells were selected in puromycin (2 mg/mL) for 72 h. To induce the expression of eIF4E-flag, cells were treated with doxycycline at indicated concentration.

For MCL1 overexpression, pLX304-MCL1-V5 or pLX304-Luciferase-V5 (a gift from Kevin Janes; Addgene plasmid # 98580) as a control were used to transfect HEK293T cells together with packaging plasmid VSVG, Gag/pol and Rev. Viruses were collected 48–72 h post transfection and concentrated using Lenti-X concentrator (Takara #631231) according to manufacturer’s instructions. MCF-7 cells were infected with viruses with polybrene (8 μg/mL) and blasticidin (10–20 mg/mL) was added 72 h after infection until non-infected cells all died.

Western blot

Cells were rinsed with PBS and lysed using the Laemmli sample buffer (50 mM Tris pH 6.8, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol, 5% BME). Lysates were boiled for 20 min, resolved using NuPAGE 4–12% SDS–PAGE gels (Life Technologies) and transferred to PVDF membranes (Millipore). Membranes were blocked using the Superblock T20 (TBS) Blocking Buffer (#37536 from Thermo Fisher Scientific), probed with primary antibodies overnight at 4 °C, and horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature for 1 h. The immune complexes were detected by SuperSignalTM West Dura Substrate (#34075 from Thermo Fisher Scientific).

Proliferation and apoptosis assays

Cells were seeded with their specified medium in 384-well plates (for slow growing cells: 2000 cells/well; for the normal growing cells: 1000 cell/well; for fast growing cells: 500 cells/well) overnight, and then treated with drugs for 72 h. Cell proliferation and cell death was measured by staining with Hoechst (Life Technologies #H3570) nuclear dye at 5 μg/ml and YOPRO-1 (Life Technologies #Y3603) at 500 nM, respectively, and analyzed using a Thermo CellInsight High Content microscope. The raw measurements from each treated well were normalized to the median of vehicle-treated control wells from the corresponding plate.

Clonogenic assay

Cells were seeded in 12-well microplates overnight at various density according to cell growth rate (for slow growing cells: ~10,000 cells/well; for the normal growing cells: ~6000 cells/well; for fast growing cells: ~3000 cells/well). For PalboR ZR-75-1 cells, due to decreased attachments of the cells to the plates, plates were treated with 5 μg/ml fibronectin (Sigma #F4759) prior to cell seeding. Cells then were treated with drugs at a series of concentrations or DMSO as a control, and media were replaced every 3–4 days. Cells were exposed to drugs or DMSO for about two weeks in total until the DMSO control wells reach confluence.

At harvest, cells were washed with PBS, fixed with cold methanol for 10 min and stained with 0.5% crystal violet for at least 1 h. Plates were rinsed with water and left for drying at room temperature. Pictures of stained colonies were taken using an EPSON Perfection V600 scanner on the following day. For quantification, stains were extracted using extraction buffer (0.1% SDS in 50% ethanol) and absorbance measured by a BioTek plate reader at 562 nm.

Ribosome profiling

Ribosome profiling was performed based on previous protocol [39]. To process the sequencing data, the ribosome profiling reads were first trimmed using cutadapt (v2.3) to remove the linker sequence AGATCGGAAGAGCAC. The fastx_barcode_splitter script from the Fastx toolkit was then used to split the samples based on their barcodes. Since the reads contain unique molecular identifiers (UMIs), they were collapsed to retain only unique reads. The UMIs were then removed from the beginning and end of each read (2 and 5 Ns, respectively) and appended to the name of each read using umitools (v0.3.3). Bowtie2 (v2.3.5) was then used to remove reads that map to ribosomal RNAs and tRNAs, and the remainder of reads were then aligned to mRNAs (we used the isoform with the longest coding sequence for each gene as the representative). Subsequent to alignment, umitools was used to deduplicate reads. The RNA-seq reads were directly aligned to the same mRNA reference using bowtie2. The translation efficiencies, i.e. the ratio between the RPF and RNA read counts per gene, were compared using Ribolog [40].

Mouse xenograft studies

Animal studies were conducted in compliance with the regulations of UCSF Institutional Animal Care and Use Committee (IACUC). MCF-7 TAMR cells were orthotopically implanted into the mammary fat pads of female NSG mice. Each mouse was injected with 5 million cells in the cleared mammary fat pads bearing slow release estrogen pellets and allowed to reach a tumor volume of around 150 mm3. The xenografts were then randomized into 4 cohort groups (n = 8) and treated with vehicle, fulvestrant, RMC-6272, or the combination of fulvestrant and RMC-6272. Fulvestrant was prepared in peanut oil at 25 mg/ml and subcutaneously injected 5 mg/mouse weekly, and RMC-6272 was prepared in 5:5:90 (v/w/v) Transcutol/Solutol HS 15/water and intraperitoneally injected 8 mg/kg weekly. Vehicle was a combination treatment of diluents delivered subcutaneously or intraperitoneally. Tumor volumes were measured every 3–4 days by caliper measurements.

MCF-7 PalboR model was developed by WuXi AppTec Co., Ltd (Suzhou, China). The MCF-7 PalboR tumors were derived in vivo from mice bearing MCF-7 tumors treated with palbociclib daily until resistant growth observed, and resistant tumors were serially implanted into next round of mice continuously treated with palbociclib. The resistant tumors (from passage 4) were implanted into 2–4 donor mice with palbociclib treatment, and when reached 500–1000 mm3, the tumors were removed and cut into fragments of ~30 mm3, which were then implanted into study mice (Balb/c nude female mice 6–8 weeks old) supplemented with subcutaneous injection of estrogen solution (0.04 mg/mouse) twice weekly since the day of implantation. Mice were randomized into treatment groups with mean tumor volume of ~200 mm3 and enrolled into study.

PDX models CTG-1211, CTG-1260, and CTG-2891 were done by Champions Oncology, Inc. (Rockville, MD). Athymic Nude-Foxn1nu female mice 6–8 weeks old were implanted subcutaneously with fragments from PDX tumors. When tumors reached ~150–300 mm3, mice were randomized into treatment groups and enrolled into studies.

Statistical analyses

Data are expressed as averages, with the standard error of mean (±SEM) indicated. Statistical analyses were performed in Graphpad Prism 7 software using unpaired t test with Welch’s correction unless otherwise stated in figure legends. Results were considered statistically significant for P < 0.05.