The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells

Abstract

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.

Keywords: BRCA1; CST complex; CTC1; DNA end resection; PARP inhibitor; STN1; TEN1; breast cancer; drug resistance; genetically engineered mouse model.

Graphical abstract

Figure 1

Multiple Independent CRISPR/SpCas9 Loss-of-Function Screens Identify CTC1 as a Driver of PARPi Resistance in BRCA1-Deficient Cells (A) Schematic overview of the screening approach utilized across the different screens. Each screen was performed on a different cell line and screened with a different library, which is indicated per screen. (B) SpCas9-expressing KB1P-G3 cells were screened with a DNA damage response (DDR)-focused library at 100× coverage. Cells were plated for clonogenic growth in the presence of olaparib (75 nM) or AZD2461 (250 nM) for 14 days, and sgRNA abundance in treated populations was compared with the starting population using MAGeCK software. Gene-based p values were log-transformed and plotted based on the positive rank (enrichment). Each dot represents a unique gene. (C) Brca1^−/−;Trp53^−/− mouse embryonic stem cells (mESCs) were screened with a genome-wide library in two independent transductions at 75× coverage. After 10 days of culture in the presence of olaparib (15 nM), treated populations were compared with the untreated population using MAGeCK software. Gene based p values were log-transformed and plotted based on the positive rank (enrichment). Each dot represents a unique gene. (D) A derivative of the BRCA1 mutant SUM149PT human triple-negative breast tumor cell line carrying a doxycycline-inducible SpCas9 expression construct was lentivirally infected with a genome-wide guide RNA library at more than 1,000× coverage. Cells were cultured in the presence of doxycycline plus 100 nM talazoparib for 2 weeks. The sgRNA abundance in treated populations was compared with the starting population using drugZ. Gene-based Z scores were log-transformed and plotted based on the positive z-rank (enrichment). Each dot represents an individual gene. (E) The top 20 genes in the KB1P-G3 screen and the top 200 genes in the mESC and SUM149PT screens were selected and plotted in a Venn diagram to identify consistent outliers.

Figure 2

Depletion of CTC1 Suppresses the Synthetic Lethal Interaction between BRCA1 Deficiency and PARP Inhibition (A) Schematic overview of the Ctc1 gene, in which putative OB fold domains and sgRNA target locations are indicated (adapted from Miyake et al., 2009). (B–D) KB1P-G3 cells were transfected with pX330puro vectors containing the indicated sgRNAs, and the target region was PCR-amplified to verify allele modification using TIDE software. (E) The indicated Ctc1-mutated KB1P-G3 cell lines were plated for clonogenic growth upon olaparib (75 nM) or AZD2461 (250 nM) treatment. Three independent experiments were performed, and each condition was plated in triplicate. One representative well per condition is shown for each independent experiment. (F) Quantification of crystal violet staining in (E). Data were plotted relative to the growth of untreated sgNT cells and are presented as mean ± SD (n = 3 independent experiments). Significance was calculated by two-way ANOVA with Dunnett’s multiple comparisons test (^∗∗∗∗adjusted p = 0.0001). (G) Relative cell proliferation was determined by IncuCyte Zoom Live – Cell Analysis System measurements. Each data point represents the average of three independent experiments, and in each experiment, six replicate wells were measured and averaged. Data represent mean ± SD (n = 3). Doubling times (exponential growth equation) were calculated using GraphPad software, and significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (H and I) SpCas9-expressing KB1P-G3 cells were transduced with doxycycline-inducible pLenti-sgRNA-tetR-T2A-PuroR vectors containing the indicated sgRNAs. Polyclonal populations were plated for clonogenic growth with or without AZD2461 (250 nM) (H). Cells were passaged every 10 days for a total of three times. At the endpoint, wells were fixed and stained with crystal violet, and allele distributions were determined from each condition using TIDE software (I). (J) KB1P-G3 cells were transfected with pX330puro vectors containing sgRNAs targeting Stn1 and Ten1 and cultured in the presence or absence of 75 nM olaparib as in (E). Data were analyzed as in (F) and reflect at least two independent experiments.

Figure 3

Loss of CST Complex Members Induces PARPi Resistance in BRCA1-Deficient mESCs and SUM149PT Breast Cancer Cells (A) Ctc1, Stn1, and Ten1 were targeted in R26^CreERT2;Brca1^SCo/Δ mESCs using pLentiCRISPRv2 vectors. Following transduction and selection, the Brca1-Sco allele was recombined by activation of CreERT2 via addition of 4-OHT, after which cells were plated out for clonogenic growth. Cells were fixed and stained with crystal violet. (B) Brca1 alleles from surviving populations were PCR-amplified using specific primers to detect Brca1^Sco (Sco) and recombined Brca1^ΔSco (DelSco) alleles. (C) BRCA1-mutant SUM149PT cells were transfected with the EditR CRISPR system, and the indicated CRISPR RNA (crRNA) and then continuously cultured in the presence of 50 nM talazoparib over a 14-day period, at which point cell viability was estimated by use of CellTiter-GLo reagent. Median effects from three independent experiments are shown. Error bars represent SEM. ^∗p = 0.0415 and 0.0201, respectively; ^∗∗p = 0.0013 and 0.0011, respectively; ^∗∗∗∗p = < 0.0001; unpaired two-tailed Student’s t test.

Figure 4

CTC1 Functions as a Resection Antagonist on Non-telomeric DSBs (A and B) CTC1 depletion induces RPA-coated ssDNA overhangs at sites of DNA damage in BRCA1-defient KB1P cells. (A) Representative images of RPA-negative and RPA-positive 53BP1-labeled alpha tracks in the indicated CRISPR/SpCas9-targeted KB1P-G3 cells (highlighted by the white arrowheads). Scale bars represent 5 μm. (B) RPA co-localization was quantified 1 hr after irradiation with an Americium-241 (²⁴¹Am) point source. The experiment was performed three times, and in each independent experiment, a minimum of 100 tracks were analyzed. Data are plotted as mean ± SEM. Significance was calculated by unpaired two-tailed Student’s t test (^∗∗p < 0.01). (C and G) CTC1 depletion restores formation of DNA damage-induced RAD51 foci in BRCA1-deficient cells. (C) Representative confocal images of CRISPR/SpCas9-expressing KB1P-G3 cells targeted with the indicated sgRNAs. Cells were stained 3 hr after 10 Gy of ionizing radiation (IR) for the indicated proteins. RAD51-positive cells are highlighted by the white arrowheads. The scale bar represents 5 μm. (D–G) Quantification of confocal images, plotted as a box and whiskers plot. The box represents the 25^th to 75^th percentiles, and the whiskers show the minimum to maximum values. The experiment was performed at least twice, and data are plotted as a percentage of yH2AX- (D), 53BP1- (E), or RIF1-positive cells (> 10 foci) (F) or RAD51-positive cells (> 5 foci) (G) per field. Statistics were performed by Kruskal-Wallis non-parametric test followed by Dunn’s multiple comparisons test. The indicated cell lines were compared with sgNT-treated cells (^∗∗∗∗p < 0.0001). See also Figure S3.

Figure 5

CTC1 Facilitates c-NHEJ at Telomeric and Non-telomeric DSBs (A–C) CTC1 depletion suppresses end-to-end fusions of uncapped telomeres. (A) Schematic overview of the telomere fusion assay. TRF2ts mouse embryonic fibroblasts (MEFs) of the indicated genotypes were cultured at the non-permissive temperature (39°C) for 24 hr before harvesting. (B) Representative images of metaphase spreads showing chromosomes unfused or fused at their telomeres (examples highlighted by white arrowheads). Chromosomes were stained with DAPI and a telomere-specific FISH probe (green). (C) Metaphases were detected and imaged automatically by Metafer. At least two independent experiments were performed, in each independent experiment, more than 2,000 chromosomes were counted manually. Genotypes were blinded during counting. Data are plotted as mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test (^∗p ≤ 0.05; ^∗∗p ≤ 0.01). (D and E) Heterozygous inactivation of Ctc1 impairs IgM-to-IgA class switch recombination (CSR) in CH12 B cells. (D) FACS analysis of CH12 clones of the indicated genotype 40 hr after induction of CSR by incubation with CD40Ab, IL-4, and TGF-β-1 (CIT). (E) Quantification of FACS data, representing mean ± SD of two independent experiments. Significance was calculated by unpaired Student’s t test (^∗∗p value ≤ 0.01).

Figure 6

Depletion of CTC1 Induces PARP Inhibitor Resistance In Vivo (A) mRNA expression levels of Ctc1 in matched treatment-naive and PARPi-resistant BRCA1-deficient mouse mammary tumors. The y axis indicates the log2 (counts per million) value. (B) Schematic overview of the generation of isogenic Ctc1-mutated and control tumors via ex vivo manipulation of tumor organoids. (C and D) Example TIDE plots of untreated mammary tumors derived from Ctc1-mutated KB1P4 tumor organoids of the indicated genotype. (E) Survival of mice orthotopically transplanted with modified KB1P4 tumor organoids. Mice were stratified into untreated (n = 3) or olaparib-treated (100 mg/kg intraperitoneally daily for 56 consecutive days, n = 7) groups when tumors reached a size of 50–100 mm3. Significance was calculated by log rank (Mantel-Cox) test (^∗∗p < 0.01).