Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.     

Synthetic Lethal Interactions between EGFR and PARP Inhibition in Human Triple Negative Breast Cancer Cells

Few therapeutic options exist for the highly aggressive triple negative breast cancers (TNBCs). In this study, we report that a contextual synthetic lethality can be achieved both in vitro and in vivo with combined EGFR and PARP inhibition with lapatinib and ABT-888, respectively. The mechanism involves a transient DNA double strand break repair deficit induced by lapatinib and subsequent activation of the intrinsic pathway of apoptosis. Further dissection of the mechanism reveals that EGFR and BRCA1 can be found in the same protein complex, which is reduced by lapatinib. Interestingly, lapatinib also increases cytosolic BRCA1 and EGFR, away from their nuclear DNA repair substrates. Taken together, these results reveal a novel regulation of homologous recombination repair involving EGFR and BRCA1 interaction and alteration of subcellular localization. Additionally, a contextual synthetic lethality may exist between combined EGFR and PARP inhibitors.     

The interplay between BRCA1 and 53BP1 influences death,aging, senescence and cancer

In proliferating cells DNA double strand breaks (DSBs) are a common occurrence during DNA replication. DSB repair using homologous recombination is essential for the error-free repair of such breaks and proliferating cells require some level of HR activity for their viability. The BRCA1 tumour suppressor has an important role in this process and is believed to channel the DSBs into the HR pathway. The related 53BP1 gene is known to positively regulate repair of DSBs outside of S phase, but via the NHEJ pathway. Two new studies suggest a new role for 53BP1 as an inhibitor of HR [1], [2]. These genetic studies establish that 53BP1, but not other components of the NHEJ machinery, can inhibit the early resection step of HR. In cells defective for BRCA1, which is required for efficient HR, the balance between promoting and inhibiting HR is thrown towards inhibition. Simultaneous loss of 53BP1 can rescue the HR defect of BRCA1-defective cells and restore cellular viability. Here, I provide an overview of these studies and discuss their implications for tumourigenesis.     

Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.     

The BRCA1 ubiquitin ligase and homologous recombination repair

Impairment of homologous recombination (HR), a vital process employed during repair of DNA double strand breaks and stalled DNA replication, provides a valuable opportunity for the cell to become transformed. Once transformed, the impairment turns to be a target for therapy as exemplified by the synthetic lethal strategy such as poly (ADP-ribose) polymerase (PARP) inhibitor for BRCA1/2-defective breast and ovarian cancer. Hence, improving mechanistic understanding of HR has emerged as an urgent issue to address due to the high clinical demand. Ubiquitin modification plays a central role in HR and more than a few E3 ubiquitin ligases have been implicated in the process. However, the significance of the activity of one such key E3 ligase, BRCA1, has not yet been clarified and remains as a major obstacle in the field. Here, we review recent advances in our understanding of BRCA1 function in HR and discuss possible roles of the activity.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

Minding the gap: the underground functions of BRCA1 and BRCA2 at stalled replication forks

The hereditary breast and ovarian cancer predisposition genes, BRCA1 and BRCA2, participate in the repair of DNA double strand breaks by homologous recombination. Circumstantial evidence implicates these genes in recombinational responses to DNA polymerase stalling during the S phase of the cell cycle. These responses play a key role in preventing genomic instability and cancer. Here, we review the current literature implicating the BRCA pathway in HR at stalled replication forks and explore the hypothesis that BRCA1 and BRCA2 participate in the recombinational resolution of single stranded DNA lesions termed "daughter strand gaps", generated during replication across a damaged DNA template.     

DNA helicases FANCM and DDX11 are determinants of PARP inhibitor sensitivity

The encouraging response rates of BRCA1- and BRCA2-mutated cancers toward PARP inhibitors make it worthwhile to identify other potential determinants of PARP inhibitor responsiveness. Since the Fanconi anemia (FA) pathway coordinates several DNA repair pathways, including homologous recombination in which BRCA1 and BRCA2 play important roles, we investigated whether this pathway harbors other predictors of PARP inhibitor sensitivity. Lymphoblastoid cell lines derived from individuals with FA or clinically related syndromes, such as Warsaw breakage syndrome, were tested for PARP inhibitor sensitivity. Remarkably, we found a strong variability in PARP inhibitor sensitivity among different FANCD1/BRCA2-deficient lymphoblasts, suggesting that PARP inhibitor response depends on the type of FANCD1/BRCA2 mutation. We identified the DNA helicases FANCM and DDX11 as determinants of PARP inhibitor response. These results may extend the utility of PARP inhibition as effective anticancer treatment.     

Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: BRCA2: One Small Step for DNA Repair,One Giant Protein Purified

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer.     

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.     

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions.     

Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

The CHD1 gene, encoding the chromo‐domain helicase DNA‐binding protein‐1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double‐strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR‐mediated DNA repair but not non‐homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.     

BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.     

BRCA2 Function and the Central Nervous System

Defective responses to DNA double strand breaks (DSBs) in the nervous system can lead to neurodegeneration or tumorigenesis. A key player in the repair of DNA DSBs is the tumor suppressor BRCA2, an essential component of the homologous recombination repair pathway and the Fanconi Anemia complex. We recently demonstrated that BRCA2 was required for normal neurogenesis and prevention of medulloblastoma brain tumors. Here, we discuss how this study contributes both to our understanding of BRCA2 functions in vivo, and the tissue-specific requirements for DNA repair and damage-signaling pathways.     

Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers

Purpose

This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs) and to poly(ADP-ribose) polymerase (PARP) inhibitors.

Methods

Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.

Results

Thirty five (22.2%) of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7%) of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%), and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%). In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined) were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined) were identified in the hereditary non-triple-negative group.

Conclusions

Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.     

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G₁ phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G₁ to homologous recombination in S/G₂.     

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.     

BRCA2 is required for homology-directed repair of chromosomal breaks

The BRCA2 tumor suppressor has been implicated in the maintenance of chromosomal stability through a function in DNA repair. In this report, we examine human and mouse cell lines containing different BRCA2 mutations for their ability to repair chromosomal breaks by homologous recombination. Using the I-SceI endonuclease to introduce a double-strand break at a specific chromosomal locus, we find that BRCA2 mutant cell lines are recombination deficient, such that homology-directed repair is reduced 6- to >100-fold, depending on the cell line. Thus, BRCA2 is essential for efficient homology-directed repair, presumably in conjunction with the Rad51 recombinase. We propose that impaired homology-directed repair caused by BRCA2 deficiency leads to chromosomal instability and, possibly, tumorigenesis, through lack of repair or misrepair of DNA damage.