Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) Breast datasets were accessed through cBioPortal (3,11–14). Area under the curve (AUC) values for cell lines were retrieved from the CTD2 Data Portal (15,16). TP53 mutational data was extracted from the Cancer Cell Line Encyclopedia (CCLE) portal (17).
Cell Lines and Culture Methods
Cal51 cells were obtained from DSMZ. Sum149PT cells were a generous gift from Naoto Ueno (MD Anderson, Houston, TX). All other cell lines were obtained from ATCC. Information for all cell lines and media compositions is found in Supplemental Table 2. For all Dox-inducible cell lines, regular FBS was replaced with Tet-system approved FBS (Clontech) in media. TP53 DNA-resequencing of cell lines was performed with the help of the MD Anderson Advanced Technology Genomics (ATGC) core. All cell lines tested negative for mycoplasma, as assayed by PCR. Identities of cell lines were confirmed with short tandem repeat (STR) DNA fingerprinting, as previously described (18).
Generation of Dox-Inducible sgGPX4 Cell Lines
MDA-MB-468 iCas9 clones were generated previously by infection with Dox-inducible Lenti-Cas9 (Tet-on pCW-Cas9, Addgene plasmid #50661) (19). sgGPX4 and control constructs in the pCRISPR backbone were purchased from GeneCopoeia (HCC299543-SG01 and CCPCTR01, respectively). Cells were seeded in 60mm plates, then transfected with 2ug plasmid DNA and 6uL XtremeGENE 9 (Roche) in Opti-MEM (Invitrogen). Cells were selected with hygromycin for 7 days, after which all non-transfected cells had died. Resulting cells were screened for GPX4 knockout using western blot, and Cas9 expression was induced with doxycycline.
Generation of Dox-Inducible TP53-Mutant Cell Lines
TP53 cDNA was inserted into the pCR8/GW/TOPO vector (Invitrogen), then point mutagenized using the QuikChange Lightning II kit (Agilent) following the manufacturer’s protocol. Primers were designed using QuikChange Primer Design Software (Agilent), and underlined amino acids represent the nucleic acid changed from wild type.
Resulting constructs were Gateway cloned into the pInducer20 vector, using LR Cloanse II (Invitrogen). Insertion of mutant TP53 into pInducer20 was confirmed with restriction digest mapping and DNA sequencing. Lentiviral vectors were prepared with co-transfection of viral helper plasmids pCMV-VSV-G and pCMV-dr8.2 dvpr (Addgene plasmids #8454 and #8455, respectively) and transfected into HEK293T cells using X-tremeGENE (Roche). MDA-MB-436 cells were stably infected with viral media supplemented with polybrene for 48 hours, then selected with G418.
The drug library was curated from three separate libraries: the BROAD “Informer Set” of drugs as published by Seashore-Ludlow et. al. (16), the Texas A&M Institute for Biosciences and Technology’s Custom Clinical Library, and the Powel Brown Library, a collection of drugs targeting signaling pathways and nuclear receptors which are of interest to the Brown lab, totaling 453 unique drugs with known mechanisms of action. Drug annotations were manually curated and cataloged from databases or vendor sources and visualized using Protein Analysis Through Evolutionary Relationships (PANTHER) classification (20).
Method for High-Throughput in Vitro Drug Screening using an ATP Luminescence Assay
Briefly, cells were seeded in 384-well plates (Greiner), incubated at 37oC for 24 hours, and treated with 10uM, 1uM, or 0.1 uM of each drug in duplicate with the aid of an Echo 550 and Access Workstation (Labctye) for 72 hours. As a surrogate for viability, ATP levels of treated cells were determined with CellTiterGlo (Promega) using an Infinite M1000pro plate reader (Tecan). All cell lines were screened twice with separate cell line passages, and screening fitness was determined with the screening window coefficient Z’.
In Vitro Drug Screen Data Processing
Data organization and analysis was performed using Pipeline Pilot (Biovia, 2018 Server edition) integrated with R. Raw cell counts were first normalized as the growth rate index (GR), purposed by Hafner et al., using the following equation:
wherein Drug D3 is the value of drug treatment values, Control D0 is the cell count values from a separate plate fixed on the day of drug addition, and Control D3 is the on-plate negative control (21). The normalized growth rate versus concentration was then fit using a cascade of 6 different models to provide an optimal fit to a broad range of curve shapes. The first model tested attempted to fit normalized data to a hill slope (4-parameter logistic regression) using iteratively reweighted least squared method found in the robust R package (https://cran.r-project.org/web/packages/robust/robust.pdf). Failure to converge, defined by a threshold of 0.00001, results in the data being passed to a series of constrained logistic regression or linear models. The constrained models used are: a 3PL which either fixes the max and fits the minimum, slope, and EC50 or fixes the minimum and fits the max, slope, and EC50; a 2PL that fixes the top and bottom to either the 2nd and 98th percentile or min and max of the dataset, respectively, and fits the slope and EC50; or a linear model if all other models fail. After curves were fit, the area under the curve (AUC) was calculated using numerical integration and subsequently normalized to the maximum theoretical value, which results in values between 0 (Full active) and 1 (in-active).
A cut point of AUC ≤ 0.5 in at least one p53-mutant cell line was required for the drug to progress into the counter screen. For the counter screen, a difference of > 0.1 between average p53 wild-type AUC and average p53-mutant AUC was considered a candidate in vitro drug.
Method for in Silico Drug Screening
Cancer Therapeutics Response Portal (CTRPv2.0) Informer Set AUC values were retrieved from the CTD2 Data Portal and integrated with TP53 mutational data extracted from the CCLE, representing 486 drugs. AUC values were normalized to the maximum theoretical value. A minimum cut point of AUC ≤ 0.5 in at least one p53-mutant cell lines was required for progression into the counter screen. Average AUC values of drugs that passed cut point were determined for p53 wild-type and p53-mutant breast cancer cell lines. A difference of > 0.1 between average p53 wild-type AUC and average p53-mutant AUC was considered a candidate in silico drug.
Cell Proliferation Assays of Breast Cancer Cell Lines
Cells were plated in 96-well plates (Nunc) incubated at 37oC overnight, then treated with drugs at indicated concentrations for 72 hours. Plates were fixed with paraformaldehyde, stained with DAPI, and imaged with an ImageXpress Pico (Molecular Devices). Nuclei were segmented and counted by defining a threshold value of pixel intensity over background and object size, using the cell scoring algorithm of the CellReporterXpress Software (Molecular Devices). Results were either reported as fold growth or Hafner’s growth rate. Resulting values were fit into logistic regression models using Prism 9.2 (GraphPad), from which IC50 and AUC values were extracted.
DRAQ7 Cell Death Assay of MDA-MB-468 Cells
MDA-MB-468 cells were plated in 96-well plates (Nunc), incubated at 37oC overnight, and treated with indicated drugs for 24 hours. Cells were co-stained live with DRAQ7 (300nM) and Hoechst 33342 (10uM) for 20 minutes and imaged at 4X with an ImageXpress Pico (Molecular Devices), using DAPI and Cy5 filter cubes. Live and dead nuclei were segmented and counted by comparing pixel intensity to background and object size, using the cell scoring algorithm of CellReporterXpress Software (Molecular Devices).
Annexin V / PI Flow Cytometry
Cell lines were treated with DMSO, ML-162 (500nM, 24 hours), or staurosporine (1uM, 3 hours), then stained with Annexin-V and PI (Invitrogen) and prepared for flow cytometry analysis. Briefly, cells were washed with cold PBS, resuspended in Annexin V binding buffer, and incubated with FITC-conjugated Annexin V and PI for 15 minutes. Cells were analyzed for Annexin V and PI intensity with a Gallios 561 (Beckman Coulter) with the help of the MD Anderson Flow Cytometry and Cellular Imaging Core.
Western blots were performed as described previously (22). Primary antibodies were: GPX4 (Abcam, ab125066, 1:1000), TP53 (Santa Cruz, sc-126, 1:1000), Caspase 3 (Cell Signaling Technology, 14220, 1:1000), Caspase 7 (Cell Signaling Technology, 12872, 1:1000), Caspase 9 (Cell Signaling Technology, 9508, 1:1000), Actin (Sigma, SAB4301137, 1:4000), and Vinculin (Sigma, 05-386, 1:4000).
Brightfield Cell Imaging
Cells were seeded in 6-well plates in their respective media. Treatments and incubations were performed as indicated, following which cells were imaged live at 20X with an Eclipse Ti (Nikon) using NIS Elements Software v3.2 (Nikon).
Small Molecule Death Inhibition Assay
Cells were plated in 96-well plates (Nunc) and pre-treated with DMSO control or 10uM inhibitors Z-VAD-fmk, Necrostain-1, or Ferrostatin-1 (Cayman Chemical) for 24 hours, following which cells were treated with ML-162 with or without inhibitors for 72 hours. Plates were fixed, stained with DAPI, and imaged at 4x with an ImageXpress Pico (Molecular Devices). Nuclei were segmented and counted by comparing pixel intensity to background and object size, using the cell scoring algorithm of CellReporterXpress Software (Molecular Devices). Resulting nuclei counts were normalized with Hafner’s growth rate metric.
C11-BODIPY581/591 Fluorescent Imaging
Cells were seeded into 96-well optically clear black well plates (Nunc) and grown for 24 hours at 37oC with or without FS-1. Cells were treated with 100nM ML-162, with or without FS-1 for 6 hours, then with 5uM C11-BODIPY581/591 (Molecular Probes) in HBBS for 30 minutes at 37oC. Images were captured at 40X using an ImageXpress Pico (Molecular Devices) with FITC and TRITC filter cubes. Fluorescence intensity was calculated using CellReporterXpress Software (Molecular Devices).
Experiments using nude mice (Jackson Laboratory) were performed with M.D. Anderson Institutional Animal Care and Use Committee (IACUC)-approved protocols. MDA-MB-468 or MDA-MB-231 cells were injected into the mammary fat pads of nude mice (5x106 or 7.5x105 cells, respectively, per animal in 100μl PBS, ). When tumors developed and reached approximately 50-100 mm3, mice were randomized into groups to receive ML-162 (50mg/kg) or vehicle (DMSO), injected intratumorally 5 day per week. Tumor sizes were measured at indicated time points with digital calipers, and tumor volume was calculated with the formula: Volume = (width2 x length)/2. Individual tumor growth rates were calculated with log-transformed linear regression.
H&E and Immunohistochemistry
Tumor samples were fixed using 4% paraformaldehyde and embedded in paraffin. Sections of tissue were mounted on slides and processed for immunohistochemical (IHC) staining, as previously described (23). For IHC staining, tissues were incubated with primary antibodies overnight at 4oC: Lab Vision anti-Ki67 (Thermo Scientific, prediluted), anti-cleaved caspase 3 (Thermo Scientific, prediluted), or anti-4-hydroxynonenal (R&D Systems, 1:1000).
Data Analysis and Statistical Considerations
Z’ was calculated as in Zhang et al. (24). A Z’ of > 0.5 was required for each cell line replicate to be added to its respective primary or counter drug screen. Fold DRAQ7+ was calculated as the ratio between drug and DMSO control treated cell DRAQ7 percent positivities. Fold growth of cells was compared using a mixed-effects model with a Geisser-Greenhouse correction. Slopes of tumor growth were calculated with log10-transformed linear regression, then compared with Student’s t-test. Kaplan-Meier curves were compared with log-rank analysis. All other experimental significance was determined with Student’s t-test. For all experiments, a p-value of < 0.05 was considered statistically significant.