Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps

Abstract

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

Keywords: 53BP1; BRCA1; DNA damage response; DNA ligase III; PARP1, PARP inhibitor; drug resistance; replication fork; ssDNA gaps; vulnerabilities.

Figure 1.. Depletion of LIG3 increases sensitivity to PARPi in BRCA1-deficient cells, independent of 53BP1 loss

(A) Outline of shRNA dropout screens. Screens were done at olaparib concentrations of 25 and 50 nM for ES-B1P.R and ORG-KB1P.R, respectively. (B) Plot of log₂ ratio (fold change [treated versus untreated]) versus abundance (mean of normalized [norm] counts) of shRNAs extracted from ES-B1P.R mESCs and ORG-KB1P.R organoids treated with olaparib or left untreated for 3 weeks. To eliminate artifacts of significant cell death without PARPi, the analysis considered fold change between untreated and treated conditions and removed genes that were already depleted at T0 (day of seeding). Analyzed using MAGeCK. (C) Comparison of the screening outcome between indicated cell lines. p value by MAGeCK. (D–F) Quantification of long-term clonogenic assays with ORG-KB1P.R (D), ORG-KB1P.S (E), and ORG-KP organoids (F) treated with olaparib or left untreated. (G) Schematic representation of the Brca1 selectable conditional allele in R26^creERT2;Brca1^SCo/−;Trp53bp1^−/−;Trp53^−/− (ES-P.R) mESCs. Incubation of these cells with 4-hydroxytamoxifen (4OHT) induces CreERT2 recombinase, resulting in R26^creERT2;Brca1^−/−;Trp53bp1^−/−;Trp53^−/− (ES-B1P.R) cells lacking BRCA1. (H) Quantification of long-term clonogenic assay in ES-P.R and ES-B1P.R cells treated with olaparib. See also Figure S2. (I) Quantification of long-term clonogenic assays in RPE1-P, RPE1-B1P.S and RPE1-B1P.R cells treated with olaparib. See also Figure S2. Data are represented as mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s., not significant (two-tailed t test).

Figure 2.. Resistance to PARPi in 53BP1-deficient KB1P cells is mediated by nuclear LIG3

(A) Generation of nuclear LIG3 mutants in KB1P.R cells. Lig3 contains two ATG translation initiation sites flanking a mitochondrial targeting sequence (MLS). Translation initiated at the upstream ATG site produces a mitochondrial protein, whereas translation from the downstream ATG site produces the nuclear form. Ablation of the downstream ATG allows cells to retain mitochondrial, but not nuclear LIG3 function. CRISPR-Cas9 was used to introduce in-frame ATG > CTC mutation in the nuclear ATG through delivery of a homology repair template. (B) Western blot analysis of LIG3 in whole-cell lysates of KB1P.R, KB1P.R(LIG3^mut/wt) B1, KB1P.R(LIG3^mut/mut) A3, and KB1P.R(LIG3^mut/mut) F5 cells. (C) LG3 immunofluorescence and MitoTracker staining in nuclear LIG3 mutant cells. Scale bar, 9 μm. (D) Quantification of long-term clonogenic assays with KB1P.S, KB1P.R cells, and nuclear LIG3 mutant clones B1, A3, and F5, treated with olaparib or untreated. (E) Western blot analysis of total and nuclear LIG3 in KB1P.R and nuclear LIG3 mutant KB1P.R^(ΔnucLIG3) cells. Expression of LIG3 constructs was induced with doxycycline (Dox) for 2 days prior to analysis. (F) Quantification of long-term clonogenic assay with KB1P.R and nuclear LIG3 mutant KB1P.R^(ΔnucLIG3) cells, treated with olaparib or untreated. Expression of LIG3 constructs was induced with doxycycline (Dox) starting 2 days before the assay and maintained for the duration of the assay. Data are represented as mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s., not significant (two-tailed t test).

Figure 3.. LIG3 is required at replication forks in BRCA1-deficient cells treated with PARPi

(A) Experimental setup, representative images, and quantification of LIG3-EdU proximity ligation assay (PLA) foci in KB1P.S+hB1 and KB1P.S cells incubated for 10 min with 20 μM EdU, in the absence or presence of olaparib. Scale bar, 5 μm. (B) Experimental setup, representative images, and quantification of LIG3-EdU PLA foci in KP and KB1P.S cells incubated for 10 min with 20 μM EdU, in the absence or presence of PDDX-001. Scale bar, 5 μm. (C) Experimental setup of DNA fiber assay. Cells were pre-incubated with 0.5 μM olaparib for 80 min, followed by sequential labeling with CldU (red) and IdU (green) in the presence of olaparib for 20 min each. RF progression was quantified by measuring tract lengths of CldU and IdU in micrometers. (D) Quantification of RF speed in CldU tracks, following the indicated treatments, in KB1P.S+hB1 and KB1P.S cells after siRNA-mediated depletion of LIG3. See also Figure S6. (E) Quantification of RF speed in CldU tracks, following the indicated treatments, in nuclear LIG3 mutant KB1P.R^(ΔnucLIG3) cells. See also Figure S6. (F) Representative images of symmetric and asymmetric RFs. (G) Quantification of RF symmetry following the indicated treatments in KB1P.R cells. The box represents the 10th to 90th percentile range. (H) Quantification of RF symmetry following the indicated treatments in KB1P.R and KB1P.R^(ΔnucLIG3) cells. The box represents the 10th to 90th percentile range. Data are represented as mean. ****p < 0.0001; n.s., not significant (Mann-Whitney U test).

Figure 4.. Loss of LIG3 in BRCA1-deficient cells results in an increase in PARPi-mediated ssDNA regions

(A) Experimental setup to quantify amount of ssDNA gaps per nucleus by quantitative image-based cytometry (QIBC) analysis of mean intensity of native BrdU per nucleus. Cells were incubated with BrdU for 48 h followed by 2 h treatment with 0.5 μM olaparib or left untreated. Scale bar, 50 μm. (B–D) QIBC analysis of ssDNA in KB1P.S+hB1 and KB1P.S cells (B), KB1P.S and KB1P.R cells (C), and KB1P.S and KB1P.R^(ΔnucLIG3) cells (D). See also Figure S7.

Figure 5.. Increase in ssDNA gaps results in increased genomic instability in LIG3-deficient cells

(A) QIBC analysis of ssDNA gaps in KB1P.S and nuclear LIG3 mutant KB1P.R^(ΔnucLIG3) cells. Cells were treated with 25 μM mirin for 48 h prior to treatment with olaparib or transfected with siRNA targeting CHD4. See also Figure S7. (B and C) Representative electron micrographs of normal RF (B) and RF with internal ssDNA gaps behind the fork (C). Scale bar for large panels: 250 nm = 1,214 bp; scale bar for small panels: 50 nm = 242 bp. P, parental strand; D, daughter strand. (D) Quantification of internal ssDNA gaps behind RFs observed in KB1P.S, KB1P.R, and KB1P.R^(ΔnucLIG3) cells upon treatment with 0.5 μM olaparib for 2 h. KB1P.S and KB1P.R^(ΔnucLIG3) cells were additionally treated with 25 μM mirin for 48 h prior to treatment with olaparib or transfected with siRNA targeting CHD4. Data were acquired using electron microscopy. Data are represented as mean ± SD. ****p < 0.0001; n.s., not significant (two-way ANOVA). (E) Quantification of RF symmetry in KB1P.R and KB1P.R^(ΔnucLIG3) cells following indicated treatments. KB1P.R^(ΔnucLIG3) cells were additionally treated with 25 μM mirin for 48 h prior to treatment with olaparib, or transfected with siRNA targeting CHD4. Data are represented as mean, and the box represents the 10th to 90th percentile range. ****p < 0.0001; n.s., not significant (Mann-Whitney U test). (F) Quantification of chromosomal aberrations in KB1P.S, KB1P.R, and KB1P.R^(ΔnucLIG3) cells following 2 h treatment with 0.5 μM olaparib and recovery for 6 h. KB1P.S and KB1P.R^(ΔnucLIG3) cells were additionally treated with 25 μM mirin for 48 h prior to treatment with olaparib or transfected with siRNA targeting CHD4. Data are represented as mean ± SD. ***p < 0.001; n.s., not significant (two-tailed t test). (G) Quantification of the different types of chromosomal aberrations identified in (F).

Figure 6.. LIG3 depletion increases in vivo efficacy of PARPi and is overexpressed in a fraction of human tumors

(A) Outline of in vivo experimental set up. Organoids were modified in vitro and transplanted into the mammary fat pad of syngeneic, wild-type FVB/NRj mice. Upon tumor outgrowth, mice were treated with olaparib or vehicle for 28 consecutive days. (B and C) Kaplan-Meier survival curves of mice transplanted with KB1P.S (B) or KB1P.R organoid lines (C), after in vitro shRNA-mediated depletion of LIG3. ***p < 0.001 and ****p < 0.0001 (log rank [Mantel-Cox]). (D and E) Summary and representative images of immunohistochemistry (IHC) analysis of LIG3 expression in triple-negative breast cancers (D) and ovarian serous carcinomas (E). Scale bar, 100 μM.

Figure 7.. Proposed model

BRCA1-deficient cells can bypass chromatin-trapped PARP1 lesions via two different mechanisms of gap suppression: one dependent on loss of 53BP1 and another that is LIG3 dependent. 53BP1-mediated ssDNA gap induction may result from loss of HR-mediated gap repair and/or defective Okazaki fragment processing. LIG3-mediated gap suppression might require repriming activities mediated by Polα, PRIMPOL or another unknown primase, resulting in small gaps that depend on LIG3 to be filled. Upon loss of LIG3, recruitment of MRE11 by CHD4 leads to unscheduled processing of the small gaps into longer stretches of post-replicative ssDNA, resulting in RF stalling and increased genomic instability. PARPi-sensitive BRCA1-deficient cells exhibit post-replicative PARPi-induced ssDNA gaps, which are mediated by 53BP1. Accumulation of PARPi-induced post-replicative ssDNA gaps mediated by 53BP1 and by loss of LIG3 underlies PARPi hyper-sensitivity of BRCA1/LIG3 double-deficient cells. PARPi-resistant BRCA1/53BP1 double-deficient cells lack 53BP1-mediated gap formation, and PARPi-induced ssDNA gaps only occur upon loss of LIG3, resulting in accumulation of longer stretches of post-replicative ssDNA, RF stalling, genomic instability, and PARPi sensitivity.