Defects In Homology-directed Repair Research Articles

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP–AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

SummaryPARP inhibitors (PARPi) cause synthetic lethality in BRCA-deficient tumors. Whether specific vulnerabilities to PARPi exist beyond BRCA mutations and related defects in homology-directed repair (HDR) is not well understood. Here, we identify the ubiquitin E3 ligase TRIP12 as negative regulator of PARPi sensitivity. We show that TRIP12 controls steady-state PARP1 levels and limits PARPi-induced cytotoxic PARP1 trapping. Upon loss of TRIP12, elevated PARPi-induced PARP1 trapping causes increased DNA replication stress, DNA damage, cell cycle arrest, and cell death. Mechanistically, we demonstrate that TRIP12 binds PARP1 via a central PAR-binding WWE domain and, using its carboxy-terminal HECT domain, catalyzes polyubiquitylation of PARP1, triggering proteasomal degradation and preventing supra-physiological PARP1 accumulation. Further, in cohorts of breast and ovarian cancer patients, PARP1 abundance is negatively correlated with TRIP12 expression. We thus propose TRIP12 as regulator of PARP1 stability and PARPi-induced PARP trapping, with potential implications for PARPi sensitivity and resistance.

Abstract 2773: A DNA-damaging lupus autoantibody synergizes with PARP inhibitors against DNA repair-deficient tumor cells

Abstract The lupus anti-DNA autoantibody 3E10 is a compelling candidate for development as a targeted therapy for DNA repair-deficient malignancies. 3E10 has previously been shown to localize to tumors due to its attraction to DNA released by dying cancer cells, penetrate into cell nuclei, inhibit DNA repair, and kill cancer cells with defects in homology-directed repair (HDR) of DNA double-strand breaks. A more potent derivative of 3E10 with increased affinity for DNA has been developed (referred to here as 3E10EN), and identification of optimal combination therapies with 3E10EN is needed to facilitate planning for upcoming clinical trials. In the present study, we found that 3E10EN increases the activity of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) in HDR-deficient cells and hypothesized that combination treatment with 3E10EN and PARP inhibitors (PARPi) would yield synergistic effects on HDR-deficient cancer cell survival. PARP content and activity in HDR-deficient and proficient cells prior to and following treatment with 3E10EN was evaluated. 3E10EN did not impact PARP protein content but yielded a significant increase in pADPr signal in HDR-deficient cells, which suggests a compensatory increase in PARP activity in response to DNA damage accumulation in HDR-deficient cells. Combinations of 3E10EN and the PARPi olaparib were tested on a panel of HDR-deficient cells, and a matched pair of BRCA2-deficient and proficient DLD1 cells. Olaparib inhibited the increase in pADPr caused by 3E10EN, and colony formation assays analyzed by the Chou-Talalay method confirmed that 3E10EN and olaparib synergized against HDR-deficient cancer cells. Conversely, HDR-proficient cells were resistant to 3E10EN and olaparib combination treatment. The original 3E10 is a murine antibody isolated from a lupus mouse model, and in preparation for its further development as a new drug we have recently designed Deoxymab 1 (DX1), a humanized version of 3E10EN. DX1 exhibits improved activity relative to the 3E10EN prototype, and when tested on a panel of HDR-deficient and proficient cells, DX1 and olaparib exhibited synergistic effects similar to that observed with the 3E10EN prototype. In conclusion, we have found that both the prototype 3E10EN and humanized DX1 synergize with PARPi against HDR-deficient tumor cells. These findings provide the rationale for further studies to determine the potential for this approach to be translated into a clinically relevant therapeutic strategy. Citation Format: Zahra Rattray, Jaymin M. Patel, Philip W. Noble, Valentina Dubljevic, Deanne L. Greenwood, James A. Campbell, James E. Hansen. A DNA-damaging lupus autoantibody synergizes with PARP inhibitors against DNA repair-deficient tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2773.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

Human somatic cells deficient for RAD52 are impaired for viral integration and compromised for most aspects of homology-directed repair

Homology-directed repair (HDR) maintains genomic integrity by eliminating lesions such as DNA double-strand breaks (DSBs), interstrand crosslinks (ICLs) and stalled replication forks and thus a deficiency in HDR is associated with genomic instability and cancer predisposition. The mechanism of HDR is best understood and most rigorously characterized in yeast. The inactivation of the fungal radiation sensitive 52 (RAD52) gene, which has both recombination mediator and single-strand annealing (SSA) activities in vitro, leads to severe HDR defects in vivo. Confusingly, however, the inactivation of murine and chicken RAD52 genes resulted in mouse and chicken cells, respectively, that were largely aphenotypic. To clarify this issue, we have generated RAD52 knockout human cell lines. Human RAD52-null cells retain a significant level of SSA activity demonstrating perforce that additional SSA-like activities must exist in human cells. Moreover, we confirmed that the SSA activity associated with RAD52 is involved in, but not absolutely required for, most HDR subpathways. Specifically, a deficiency in RAD52 impaired the repair of DNA DSBs and intriguingly decreased the random integration of recombinant adeno-associated virus (rAAV). Finally, an analysis of pan-cancer genome data from The Cancer Genome Atlas (TCGA) revealed an association between aberrant levels of RAD52 expression and poor overall survival in multiple cancers. In toto, our work demonstrates that RAD52 contributes to the maintenance of genome stability and tumor suppression in human cells.

Robust homology-directed repair within mouse mammary tissue is not specifically affected by Brca2 mutation.

The mammary gland undergoes significant proliferative stages after birth, but little is known about how the developmental changes impact DNA double-strand break (DSB) repair. Mutations in multiple genes involved in homology-directed repair (HDR), considered a particularly accurate pathway for repairing DSBs, are linked to breast cancer susceptibility, including BRCA2. Using reporter mice that express an inducible endonuclease, we find that HDR is particularly robust in mammary tissue during puberty and pregnancy, accounting for 34–40% of detected repair events, more than in other tissues examined. Brca2 hypomorphic mutation leads to HDR defects in mammary epithelium during puberty and pregnancy, including in different epithelial lineages. Notably, a similar dependence on Brca2 is observed in other proliferative tissues, including small intestine epithelium. Our results suggest that the greater reliance on HDR in the proliferating mammary gland, rather than a specific dependence on BRCA2, may increase its susceptibility to tumorigenesis incurred by BRCA2 mutation.

Abstract 3020: Homology-directed repair in mouse mammary tissue

Abstract Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Defects in homology-directed repair (HDR)_considered the most accurate of the three major pathways for repairing double-strand breaks (DSBs) in DNA in mammalian cells_can lead to genomic instability and are associated with tumor predisposition. Mutations in multiple genes involved in HDR are linked to breast cancer susceptibility including BRCA1 and BRCA2. The mammary gland is highly influenced by its hormonal environment and undergoes significant changes during pregnancy, lactation and involution, but many questions remain about how the DNA damage response in mammary tissue, and specifically DNA DSB repair, is impacted by these developmental changes. A widely used reporter for studying DSB repair by HDR is DR-GFP in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats, resulting in a functional GFP gene. We have recently generated DR-GFP containing mice that express I-SceI under the control of a tetracycline-inducible promoter. Induction of I-SceI by doxycycline administration ex vivo results in substantially increased HDR in primary cultures of mammary epithelial cells compared to transient transfection experiments. To gain insight into the effects of breast cancer predisposing mutations on HDR in mammary epithelial cells, the I-SceI/DR-GFP mice were crossed to mice carrying hypomorphic mutations in Brca1 (Brca1tr/tr) or Brca2 (Brca2ex27/ex27). An approximately 3-fold reduction in HDR was observed in primary mammary epithelial cells from virgin Brca1tr/tr I-SceI DR-GFP mice compared to littermate controls, while an 8-fold reduction was observed in primary mammary epithelial cells from virgin Brca2ex27/ex27 mice. Primary ear fibroblasts from these animals showed similarly reduced HDR levels. In order to understand how DSB repair processes in the mammary gland are affected by developmental changes we are assessing HDR at different stages of mammary gland development in vivo. We observe a significant increase in HDR relative to overall repair in mammary tissue from mice at mid-pregnancy compared to virgin littermates. We are now attempting to determine if there is a relationship between repair proficiency, lineage specificity of mammary epithelial cells and fate after involution in mammary tissue from normal and BRCA mutant mice. Citation Format: Elizabeth Kass, Mary Ellen Moynahan, Maria Jasin. Homology-directed repair in mouse mammary tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2015-3020

Dysfunctional DNA Double-Strand Break Repair Is Present in a Subset of Primary t-AML/t-MDS Myeloblasts

Abstract 2415The chromosomal aberrations found in treatment-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/t-MDS) cells suggest that disease initiation and progression may result from the inappropriate response to double-strand DNA breaks (DSBs) induced by prior exposure to radiation or chemotherapy. We hypothesized that dysregulation of DSB repair by homology-directed repair (HDR) or nonhomologous end joining (NHEJ) in t-AML/t-MDS may result from acquired mutations in HDR/NHEJ pathway genes. To test this possibility, we used next-generation sequencing technology to identify somatic genetic variants in 21 canonical HDR and 9 NHEJ DNA repair genes, as well as a subset of 7 DNA damage response genes using tumor DNA and paired normal DNA obtained from 25 t-AML/t-MDS patients. We identified 6 patients with somatic changes in 3 of these genes (RAD51L3, EME1, TP53). As dysfunctional DSB repair from epigenetic or post-translational modifications in DSB repair pathway genes or abnormalities in other DNA repair pathway genes would be missed using this approach, in parallel we performed functional studies of DSB repair using primary bone marrow cells from 16 of these t-AML/t-MDS patients and CD34+ cells from 5 normal donors. We evaluated DSB by measuring phosphorylated histone H2AX (pH2AX), a well established marker for DSB, in myeloblasts (CD45 dim, low side scatter) and lymphocytes (a surrogate for normal cells) in these samples. Baseline measurements of primary cells, coupled with a time course to measure pH2AX induction and decay after 2 Gy of irradiation (IR) were used to assess the basal DSB burden and response to acute damage, respectively. pH2AX levels were measured by flow cytometry and the geometric mean of the fluorescence intensity was converted to mean equivalent soluble fluorophore (MESF) through the use of standard beads included in each experiment.We found that 4 of 16 t-AML/t-MDS patients had myeloblasts that displayed baseline and post-damage pH2AX levels similar to normal CD34+ controls, while 12/16 patients had abnormal pH2AX levels which fell into one of three major patterns. 1) The first subset had myeloblasts in which baseline pH2AX levels were elevated compared to normal donor CD34+ (average MESF 23,107 vs 11,490, respectively; p<=0.002) suggesting an increased basal DSB burden in these cells. Furthermore, the myeloblasts showed impaired pH2AX induction (measured at 30 min. post IR) compared to CD34+ controls (1.53 vs 2.97 fold increase in pH2AX over baseline, p<=0.002), suggesting a defect in detecting DSB. This phenotype was unique to patients harboring trisomy 8 and was tumor specific, as their lymphocytes displayed baseline and post-induction pH2AX levels similar to lymphocytes from normal controls. No somatic (tumor) sequencing variants were present in the interrogated genes, raising the possibility that trisomy 8 could be driving an abnormal DNA damage response. 2) A second subset of patients had impaired pH2AX induction compared to normal donor CD34+ cells (1.44 vs 2.97 fold increase in pH2AX over baseline, p<=0.01), again suggesting a defect in detecting DSBs. These patients also lacked somatic changes in HDR/NHEJ pathway genes. 3) The final subset of patients had delayed resolution of pH2AX levels compared to CD34+ controls post IR either at 4 hours (average MESF 39,260 vs 25,480, p<0.05) or delayed resolution over the entire 24 hour period compared to controls (p<0.001). These data are consistent with a DSB repair defect and similar to our data showing cells lacking BRCA2, a gene central to the HDR pathway, have elevated pH2AX levels at 4–24 hours post DSB induction compared to BRCA2 sufficient cells (p=0.01). One of these patients had an acquired mutation in the HDR gene RAD51L3. We are currently determining the sensitivity of primary t-AML/t-MDS cells with abnormalities in pH2AX levels to a combination of DSB inducing chemotherapy and PARP inhibition, which is synthetically lethal in the setting of HDR defects. We show cell lines lacking RAD51L3 are more sensitive to PARP inhibition compared to isogenic controls (surviving fraction (SF)50 5 nM vs 20,000 nM). In total, this study confirms that DNA repair genes are mutated in t-AML/t-MDS, suggests that dysfunctional DSB repair is present in t-AML/t-MDS myeloblasts, and provides a rationale to test whether the abnormal DNA damage response can be exploited therapeutically using a synthetic lethal approach in this disease. Disclosures:No relevant conflicts of interest to declare.

Abstract B56: Identification of genes involved in mammary tumor susceptibility and resistance in two strains of mice

Abstract Loss of p53 can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53+/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53+/−females, but are rare in C57BL/6-Trp53+/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) through mitotic recombination is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The Balb/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis is that BALB/c-Trp53+/− mice are more susceptible to mammary tumors due to impaired dsb repair leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53+/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J-Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsb longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs suggesting a decreased efficiency of homologous recombination. Using a GFP reporter assay, homology-directed repair was decreased by 36% in MEFs from BALB/c-Trp53+/− compared to C57BL/6-Trp53+/−. Palb2 is a candidate gene as it lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer. Although no coding SNPs were found within Palb2, preliminary results show the BALB/c-Trp53+/− cells have a 30% decrease in Palb2 RNA compared to C57BL/6-Trp53+/− cells as assayed by RT-qPCR. These results identify defects in homology-directed repair of dsb in BALB/c which are linked to susceptibility to mammary tumors in this strain. Experiments are in progress analyzing these features in congenic strains. The results will elucidate potential low penetrance modifiers that determine resistance to mammary tumors in both mice and humans which can be used as targets for therapy as well as breast cancer screening. Citation Information: Cancer Res 2009;69(23 Suppl):B56.

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

Variant XRCC3 implicated in cancer is functional in homology-directed repair of double-strand breaks

Polymorphisms in DNA repair genes, including double-strand break (DSB) repair genes, are postulated to confer increased cancer risk. A variant of the XRCC3 gene, which is involved in DSB repair, has been associated with increased risk of malignant skin melanoma and bladder cancer. We tested the hypothesis that this variant, Thr241Met, may affect cancer risk by disrupting a critical function of XRCC3, i.e., promoting homology-directed repair (HDR) of chromosomal DSBs. Using a quantitative fluorescence assay, we find that the variant XRCC3 protein is functionally active for HDR, complementing the HDR defects of an XRCC3 mutant cell line as well as the wild-type protein. We also examined cells expressing this variant for sensitivity to the interstrand cross-linking agent, mitomycin C (MMC), as HDR mutant cell lines, including the XRCC3 mutant, have been found to be hypersensitive to this DNA damaging agent. Cells expressing the variant protein were found to be no more sensitive than cells expressing the wild-type protein. These results suggest that the increased cancer risk associated with this variant may not be due to an intrinsic HDR defect.