BRCA1 Mutation-Specific Responses to 53BP1 Loss-Induced Homologous Recombination and PARP Inhibitor Resistance

Abstract

BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brca1^ΔC/ΔC mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1^ΔC/ΔC;53bp1^-/- mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance.

Keywords: 53BP1; BRCA1; PARP inhibitors; homologous recombination; resistance.

Figure 1.. Effects of 53BP1 Depletion on BRCA1 Protein Activity

(A) Cartoon showing BRCA1 constructs expressed in MDA-MB-436 cells: BRCA1 full-length, BRCA1-Δ11q, BRCA1-ΔBRCT, and BRCA1-ΔCC+BRCT. Retained peptide domains and protein interactions are indicated. Red indicates the RING domain location, orange indicates exon-11-encoded amino acids, black indicates coiled-coil domain amino acids, green shows BRCT domain locations, and blue shows non-assigned-domain amino acids. (B) Western blot analyses of MDA-MB-436 cells engineered to express mCherry, BRCA1 full-length (FL) protein, BRCA1-Δ11q (Δ11q), BRCA1-ΔBRCT (ΔBRCT), and BRCA1-ΔCC+BRCT (ΔCC+BRCT) truncated proteins as well as non-target (NT) or 53BP1 shRNA (53). Arrows indicate truncated BRCA1 proteins, and asterisks indicate nonspecific bands. (C) Cells from (B)were assessed for RPA32 IRIF formation using immunofluorescence assays. Forcom parison, FL-BRCA1-expressing cellsare included assolid black bars in graphs showing data for mCherry-expressing cell lines. At the indicated time points post-γ-irradiation (IR) (10Gy), cells were fixed and those with at least 10 foci per nucleus counted positive for IRIF . For each protein ,a minimum of 100 nuclei per time point and cell line were counted per experiment. Mean and SEM percentages of cells containing RPA32 foci are shown from three independent experiments. Solid lines represent NT shRNA, and dashed lines represent 53BP1 shRNA. Data were analyzed using two-way ANOVA. Significant differences between NT and 53BP1 shRNA at each time point are denoted (***p < 0.001; **p < 0.01; *p < 0.05). See Figure S1C for representative images. (D) Cells were assessed as in (C) for mean and SEM percentages of cells containing at least 5 BRCA1 foci per nucleus from three independent experiments. See Figure S2 for representative images. (E) Cells were assessed as in (C) for mean and SEM percentages of cells containing at least 5 RAD51 foci per nucleus from three independent experiments. See Figure S2 for representative images. (F) Cells were incubated with rucaparib and assessed for colony formation. Mean and SEM colony formation was calculated as a percentage of DMSO-vehicle-treated cells from three independent experiments. Full-length BRCA1 colony formation data (black line) is shown as a comparator in graphs with mCherry expressing cells. NT and 53BP1 shRNA-treated cells are shown with solid and dashed lines, respectively.

Figure 2.. Generation and Assessment of Brca1^ΔC/ΔC;53bp1^−/− Mice

(A) Brca1 coiled-coil genomic region targeted using CRISPR/Cas9. Red arrow indicates single guide RNA (sgRNA)-targeting location and the resulting c.4080–4083 4-bp deletion mutation. The Brca1 coiled-coil wild-type peptide region is shown with the critical L1363 residue (equivalent to human L1407) highlighted green. Amino acid changes resulting from the 1361fs28X mutation are red. Bottom : cartoon of predicted truncated protein product. (B) Representative photograph of 6-week-old littermates with indicated genotypes. See also Table 1. (C) Weights of individual mice with the indicated genotypes at 6 weeks of age. **p < 0.01, ***p < 0.001, NS, not significant (one-way ANOVA with multiple comparisons). (D) Kaplan-Meier survival curves of mice with the indicated genotypes. (E) Representative photograph of Brca1^ΔC/ΔC;53bp1^−/− mouse with thymic lymphoma. H&E of thymus, liver, bone marrow, and kidney showing tumor infiltrates (*). Scale bar, 20 μm. (F) Primary MEFs (before passage 4) with the indicated genotypes were assessed for Brca1 protein expression by western blotting. Arrows indicate the expected gel migrations of Brca1 w ild-type (WT) (top arrow), a potential Brca1^ΔC protein (m iddle arrow), and the Brca1^Δ11 protein (lower arrow). Asterisk indicates a nonspecific band.

Figure 3.. Assessment of HR and PARPi Sensitivity

(A) MEFs were subject to 10-Gy IR and assessed for Rpa32 and Rad51 foci formation as described in Figure 1. The number of foci-positive cells (mean and SEM) from three independent experiments is shown (*p < 0.05, ***p < 0.001, compared to WT, unpaired t test). Representative images of MEFs with the indicated genotypes are shown. Scale bar, 10 μm. (B) Cell lines were electroporated with plasmids expressing Rosa26-targeting sgRNA and Cas9 cDNA, plasmid enhanced green fluorescent protein (pEGFP), and a homology donor sequence. The number of gene conversion events from three independent repeats is shown. ***p < 0.001 (unpaired t test). See Figures S3D–S3G for additional controls and information. (C) Metaphase spreads were prepared from the indicated genotypes of MEFs treated with 500 nM rucaparib for 24 hr. The mean number of breaks and radial chromosomes per 10 metaphases are shown. The number of metaphases assessed were 29 fo r Brca1^+/+;53bp1^+/+, 46 for Brca1^Δ11/Δ11,53bp1^+/+, 29 for Brca1^Δ11/Δ11 ;53bp1^−/−, and 56 for Brca1^ΔC/ΔC;53bp1^−/− MEFs. Scale bars, 10 μm. (D) MEFs were treated with rucaparib for 7 days and stained with Sulforhodamine B (SRB). Solubilized stains were quantified, and cell growth (mean and SEM) is expressed as percentage of vehicle-treated cells from three independent experiment. (E) Mice were treated with vehicle or 200 mg/kg rucaparib twice daily for 5 continuous days, and survival was measured by Kaplan-Meier analyses. (F) Peripheral blood from mice described in (E) was assessed at the end of vehicle (V) or rucaparib (R) treatment for neutrophil and lym phocyte cell numbers. **p < 0.01 compared with vehicle (unpaired t test).

Figure 4.. 53BP1 Depletion in BRCA1 Mutant Cancer Cell Lines

(A) Cartoon showing the FL BRCA1 protein and the relative location of mutations (*) in BRCA1 mutant cancer cell lines. The predicted effects of mutations are indicated. L56BRC1, SUM149PT, and UWB1.289 harbor exon 11 mutations and are capable of expressing the BRCA1-Δ11q splice isoform (shown below). (B) Western blot analyses of the indicated proteins in MDA-MB-231 (231), SUM149PT (149), UWB1.289 (UWB), L56BRC1 (L56), MDA-MB-436 (436), HCC1395 (1395), and SUM1315MO2 (1315) cells. Full-length and BRCA1-Δ11q proteins are indicated by arrows. (C) Western blot analyses of 53BP1 protein expression after the indicated cell lines were subject to NT or 53BP1 shRNA sequence 1 or 2. (D) Cell lines were treated with increasing concentrations of rucaparib and colony formation assessed. BRCA1 WT MDA-MB-231 cells were also assessed for rucaparib sensitivity as a comparator. Values represent mean and SEM. LC50 values for 3 independent experiments are shown. ***p < 0.001; **p < 0.01; *p < 0.05 compared with NT shRNA for each cell line (unpaired t test). See Figure S5A for fold change values. (E) Cells expressing NT or 53BP1 shRNA#2 were assessed for RPA32 IRIF by immunofluorescence as described in Figure 1. The number of foci-positive cells (mean and SEM) for 3 independent experiments is shown, **p < 0.01; *p < 0.05 compared to NT (unpaired t test). See Figure S5C for representative images. (F) Cells were assessed for RAD51 IRIF as described in Figure 1. MDA-MB-231 BRCA1 WT cells were included as a positive control for RAD51 foci. To account for potential cell cycle or growth differences between cell lines, RAD51-foci-positive cells were normalized to geminin-positive cells. The number of foci-positive cells (mean and SEM) that were also geminin staining positive for 3 independent experiments is shown. **p < 0.01; *p < 0.05 compared to NT (unpaired t test). See Figure S5D for representative images.

Figure 5.. 53BP1 Gene KO Using CRISPR/Cas9

(A)Cells were treated with sg_G FP orsg_53BP1. Forsg_53BP1-treated cells,we established multiple clones per cell lineand assessed 53BP1 protein expression by western blotting. Selected clones with undetectable 53BP1 protein expression are shown for protein expression relative to the sg_GFP control cells. (B) Cells from (A) were treated with increasing concentrations of rucaparib and colony formation assessed. Mean and SEM colony formation is shown from 3 independent experiments. (C) Cells from (A) were assessed for RAD51 IRIF as described in Figure 1. The number of foci-positive cells (mean and SEM) from 3 independent experiments and representative images are shown. *p < 0.05, **p < 0.01 compared to GFP (unpaired t test). MDA-MB-436+BRCA1 FL proteins were also assessed as a positive control. Scale bar, 10 μm. (D) SUM149PT sg_53BP1 KO clone#12 cells were subjected to scram bled (Sc) or each of 3 different BRCA1-targeting siRNAs. Top: western blot showing BRCA1-Δ11q depletion. Bottom : mean and SEM colony formation of cells treated with 1 μM rucaparib normalized to vehicle-treated cells from 3 independent experiments. *p < 0.05, **p < 0.01 compared to Sc (unpaired t test). (E) Cells from (D) were assessed for RPA32 and RAD51 IRIF as described in Figure 1. The number of foci-positive cells (mean and SEM) from 3 independent experiments and representative images are shown. ***p < 0.001 compared to Sc (unpaired t test). Scale bar, 10 μm.

Figure 6.. Effect of BRCA1 and 53BP1 Mutational Status on Tumor PARPi Sensitivity

(A) Western blot analyses of MDA-MB-436 cells engineered to express mCherry (mCh), FL BRCA1 (FL), and BRCA1-ΔBRCT (ΔBRCT)that were WT for the coiled-coil region (WT) or harbored the L1407P(L-P) missense mutation and subjected to immunoprecipitation using hemagglutinin (HA) antibody and immunoblotted for the indicated proteins. (B) Western blot analyses of MDA-MB-436 cells expressing either BRCA1 FL or BRCA1-DBRCT (DBRCT) proteins, which are eithercoiled-coil WT or harbor the L1407P (L-P) mutation. Additionally, cells expressed NT or 53BP1 (53) shRNA. (C) Cells from (B) were assessed for RPA32 and RAD51 IRIF as described in Figure 1. Left: representative images and number of foci-positive cells (mean and SEM) are shown from 3 independent experiments. ***p < 0.001 compared with relative NT shRNA cells (unpaired t test). Scale bar, 10 μm. (D) Cells from (B) were assessed for colony formation in the presence of 100 nM rucaparib. Mean and SEM from 3 independent experiments normalized to colony formation of BRCA1 FL-expressing cells are shown. **p < 0.01 compared with relative NT shRNA cells (unpaired t test). (E) MDA-MB-436 tumor xenografts expressing or BRCA1-ΔBRCT proteins that are either coiled-coil WT or harbor the L1407P (L-P) mutation and treated with NT or 53BP1 shRNA targeting. BRCA1 and 53BP1 expression status in tumor xenografts was confirmed by IHC staining. MDA-MB-436 cells with mCherry and BRCA1 FL add-back tumors were included as controls for staining. Scale bar, 10 μm.