Cell lines and cell culture
MDA-MB-436 and HCC1937 were purchased from ATCC. MDA-MB-436 and Ovcar8 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. HCC1937 cells were cultured in RPMI 1640 supplemented 10% FBS and 1% penicillin/streptomycin. All cell lines were analyzed and authenticated by morphologic inspection and biochemical examination of the BRCA1 mutation pathway as well as short tandem repeat profiling analysis. Mycoplasma testing was also performed to exclude the possibility of mycoplasma contamination of all cell lines.
Chemical Compounds
Olaparib (Catalog# A4154), cisplatin (Catalog# A8321), carboplatin (Catalog# A2171), and 5-fluorouracil (Catalog# A4071) were all purchased from APExBIO company. UPF 1096 (Catalog# S8038), NMS-P118(Catalog# S8363), stenoparib (E7449) (Catalog# S8419), niraparib (Catalog# S2741), rucaparib (Catalog# S4948), and veliparib (ABT-888) (Catalog# S1004) were all purchased from Selleckchem. Cisplatin (Catalog# A10221) was purchased from AdooQ Bioscience. ART-558(Catalog# HY-141520), DNA-PK inhibitors PIK-75 hydrochloride (Catalog# HY-13281), Nedisertib (Catalog# HY-101570) and AZD-7648 (Catalog# HY-111783), RAD51 Inhibitor B02 (Catalog# HY-101462), and olaparib (for in vivo experiment: Catalog# HY-10162) were all purchased from MCE (MedChem Express). ART-812 was synthetized by Dr. Wayne Childers at Temple University School of Pharmacy. All compounds were dissolved, aliquoted, and stored following the manufacturer's instructions.
Pooled Genome-wide CRISPR/Cas9 Screen
The GeCKO CRISPR library was purchased from Addgene (#1000000048), amplified, and packaged as lentivirus based on the instructions on Addgene website. The CRISPR screen was performed as described previously8. In brief, MDA-MB-436 cells were transduced with lentivirus carrying GeCKO library, and puromycin selection was performed for 3 days. Then we treated transduced MDA-MB-436 cells with olaparib for 14 days, the medium was changed with adding fresh olaparib every three days during 14 days screen, and the surviving cells were harvested. The genomic DNA was extracted, and PCR was carried out before deep sequencing of sgRNA sequence in the surviving cells’ genome. All deep sequencing data are available at GEO (series accession number GSE205221). For data analysis, we calculated the enrichment score as: The enrichment score= (sgRNA number from the reads)/ (sgRNA number in the library) X log2 (average abundance). The sgRNAs used for validations were synthesized and constructed as described8. Primer sequences are shown in Supplementary Table S1.
T7EN1 assays and DNA sequencing
The T7EN1 assay was performed as described previously8. To identify the ZNF251 mutations, the purified PCR product was cloned into the pCR2.1-TOPO TA vector (TOPO TA cloning kit; Life Technologies) and sequenced by Sanger sequencing. The primers used for Sanger sequencing were listed in Supplementary Table S1.
Generation of mutant single clones
500 transduced MDA-MB-436 cells were mixed with 1 ml of methylcellulose (MethoCult H4034 Optimum, Stem Cell Technologies) in a 6-well cell culture plate and cultured at 37°C in a 5% CO2 incubator. Two weeks later, single colonies were picked and cultured in a 96-well plate with the complete medium supplemented with 2% penicillin/ streptomycin. The cells were passaged every two or three days, and 1/3 of cells were collected for genomic DNA extraction. Then ZNF251 target region was PCR amplified and sequenced.
Cell viability assay
1×104 cells were cultured with 100µl of complete medium in a 96-well plate and treated as indicated. Cell viability was measured at different time points as described with the trypan blue exclusion viability test. The final viable cell number was calculated based on the growth standard curve. All the key viability experiments were confirmed by the MTS assay (Promega, Catalog# G3582) and CCK-8 assay (APExBIO company, Catalog# K1018).
Off-target effect examination
Off-target sites were predicted using an online search tool (http://crispr.mit.edu). 3bp mismatches compared with the target consensus sequence were allowed. The predicted off-target sequences were searched using UCSC browse, and 500bp flanking the sites were PCR-amplified in primary cells and single mutation clones. The PCR product was subjected to the T7EN1 assay to determine the mutation. The PCR product was then cloned into a TA vector and Sanger sequenced to identify mutations.
ZNF251 complementation experiment
Exponentially growing MDA-MB-436 ZNF251 WT and KD cells were seed in six-well plates (1 million cells/well) and transfected with pcDNA3.1 vector or human ZNF251 on pcDNA3.1 plasmid carrying a neomycin resistance (neo) gene. After transfection with 1µg and 2 µg plasmid respectively, the cells were selected with G418 (400 ug/ml) in the culture medium for 2 weeks to keep selecting neomycin resistant cells for generating stably transfected cell lines9,10. Then plated into 96-well plates at a density of 1× 104 cells per well in triplicates. Next day, the transfected cells were treated with DMSO or olaparib for 3 days and cell viability was measured. Expression of ZNF251 (ZNF251 mRNA) was measured by real-time PCR in control and human ZNF251 on pcDNA3.1 plasmid transfected MDA-MB-436 cells. It was performed with iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad cat#1725150). The expression level of ZNF251 was normalized to housekeeping GAPDH gene.
Immunoblot analysis
Nuclear and total cell lysates were obtained as described before11 and analyzed by SDS-PAGE using primary antibodies against: ATM (Santa Cruz Biotechnology #sc-135663), CtIP (Abcam #ab-70163), 53BP1 (Abcam #ab-175933), SLFN11 (Santa Cruz Biotechnology #sc-515071), BRCA1 (ThermoFisher Scientific #MA1-23164), BRCA2 (Santa Cruz Biotechnology #sc-28235), PALB2 (Proteintech #14340-1-AP), RAD51 (Abcam #ab-88572), RAD52 (Santa Cruz Biotechnology #sc-365341), RAD54 (Santa Cruz Biotechnology #sc-374598), DNA-PKcs (Bethyl #A300–518A), Ku70 (Santa Cruz Biotechnology #sc-17789), Ku80 (ThermoFisher Scientific #MA5–15873), DNA ligase 4 (ThermoFisher Scientific #PA5-40826), PARP1 (Santa Cruz Biotechnology #sc-74470), PARP2 (Santa Cruz Biotechnology #sc-393310), PARP3 (Santa Cruz Biotechnology #sc-390771), DNA ligase 3 (Santa Cruz Biotechnology #sc-135883), Polθ (MyBioSource #MBS9612322), lamin B (Abcam #ab-16048–100), and β-actin (Santa Cruz Biotechnology #sc-47778) and the following secondary antibodies conjugated to HRP (horseradish peroxidase): goat anti-rabbit (EMD Millipore #12–348) and goat anti-mouse (EMD Millipore #AP181P). ZNF251 western analysis was performed with ZNF251 antibody (Proteintech cat# 25601-1-AP) and GAPDH antibody (Cell signaling technology cat#2118). For quantification of western analysis, ImageJ software was used to measure the density of the protein bands.
DNA damage/repair assays
DSBs were detected by neutral comet assay as described before11 with modifications. Briefly, comet assays were performed using the Oxiselect Comet Assay Kit (Cell Biolabs #STA-355) according to the manufacturer's instructions. Images were acquired by an inverted Olympus IX70 fluorescence microscope using a FITC filter, and the percentage of tail DNA of individual cells was calculated using the OpenComet plugin of ImageJ. HR, D-NHEJ, and Alt-NHEJ were measured using DR-GFP (HR), EJ2-GFP (D-NHEJ), and EJ5-GFP (Alt-NHEJ) reporter cassettes as described before11. Briefly, the reporter plasmid was digested by I-SceI endonuclease, and the repaired GFP cells were counted by flow cytometer. The result was calculated by total restored GFP positive cells / total transfected M-cherry or BFP positive cells.
Mice and in vivo studies
6–8 weeks-old female NOD/SCID/IL-2Rγ (NSG) mice (Jackson Laboratories) were injected subcutaneously with 1x106 MBA-MD-436 cells in the flank. Mice were randomized to treatment groups when tumor sizes reached 50–60 mm3. For the first set of the experiments, all animals with wildtype or ZNF251 KD tumors of 50–60 mm3 were randomized into two groups (n = 4/group), which were intraperitoneally treated with vehicle or olaparib (10mg/kg) daily for four weeks, respectively. For the second set of experiments, all mice were randomly divided into four groups and intraperitoneally injected daily with either vehicle, olaparib (10mg/kg), DNA-PK inhibitor PIK-75 (10mg/kg), or olaparib (10mg/kg) plus PIK-75(10mg/kg) for four weeks. Since the start of the experiment, tumor volumes (V) were measured every three days based on the formula V = L×W2×0.5, where L represents the largest tumor diameter and W represents the perpendicular tumor diameter12. After four weeks, all mice were euthanized and tumors were dissected out, imaged, weighed, or used for further characterization. All experiments involving animals were approved by the Cooper University and the IPhase Pharma Services LLC Institutional Animal Care and Use (IACUC) Committee.
Fork protection assay/DNA fiber assay
At stalled forks, degradation of DNA fibers was assessed as follows. Exponentially growing MDA-MB 436 ZNF251 WT and KD cells were treated with 5 uM Olaparib and/or 8 uM Plk-75 for 48 hrs. Cells were sequentially pulse-labeled with 50 µM of 5-chloro-2′-deoxyuridine thymidine (CldU) (Sigma-Aldrich) and 250 µM of idoxuridine (IdU) (Sigma-Aldrich) for exactly 30 min each, washed once with 1× PBS, and treated with 4 mM HU for 4 hr. Cells were collected and resuspended in 1× PBS at a concentration of 500 cells/ul. 2.5 µl of cell suspension was diluted with 7.5 µl of lysis buffer (200 mM Tris-HCl pH 7.5, 50 mM EDTA, and 0.5% [w/v] SDS) on a glass slide and incubated for 8 min at RT. The slides were titled at 15–60°, air-dried, and fixed with 3:1 methanol/acetic acid for 10 min. Slides were denatured with 2.5 M HCl for 90 min, washed with 1× PBS, and blocked with 2% BSA (Carl Roth) in PBS for 40 min. The newly replicated CldU and IdU tracks were labeled for 1.5 hr with anti-BrdU antibodies recognizing CldU (1:300, Abcam) and IdU (1:100, BD Biosciences), followed by 1 hr incubation with secondary antibodies anti-mouse Alexa Fluor 594 (1:500, #A11062, Life Technologies) and anti-rat Alexa Fluor 488 (1:500, #A21470, Life Technologies). The incubations were performed in the dark in a humidified chamber. After 5 washes in PBST for 3 min, mount coverslip with 20 ul mounting media. DNA fibers were visualized using a Leica SP8 Confocal microscope at a 63X objective magnification, and images were analyzed using ImageJ software.
Bioinformatics analysis of ZNF251 expression in the cells sensitive and resistant to PARPi olaparib
To analyze the expression of ZNF251 expression in the cells sensitive and resistant to PARPi olaparib, we performed bioinformatics analysis. Datasets for the respective inhibitors were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo), a large public repository for high-throughput molecular abundance data — specifically, gene expression data13. Dataset GSE165914 was utilized for the analysis of ZNF251 expression in olaparib sensitive and resistant cells14. Statistical analysis was performed using the Graph Pad Prism 9.
Quantification and Statistical Analysis
All statistical analyses were performed by GraphPad Prism 8. Cell viability data were analyzed by two-way ANOVA tests. The neural comet assay data was analyzed by Mann-Whitney Rank Sum Test. The data of DNA repair assay and in vivo experiments were analyzed by unpaired t-test with Welch's correction.
Data availability
The data generated in this study are available within the article and its supplementary data files. All deep sequencing data of our CRISPR screen are available at GEO (series accession number GSE205221).