Error-free DNA Repair Research Articles

Abstract 4686: Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer

Abstract Breast cancer (BC), the most prevalent malignancy in women, ranks fourth in cancer mortality as of 2022. Mutations in the BRCA genes significantly elevate BC risk and are associated with markedly reduced survival compared to non-carriers. These genes are crucial in homologous recombination, a mechanism essential for error-free DNA repair. Despite advancements, treatment options for BRCA mutation-driven cancers remain limited. Synthetic lethality (SL) has emerged as a promising strategy, with poly(ADP-ribose) polymerase inhibitors (PARPi) being the first developed drugs based on this principle. However, PARPi's effectiveness is limited, as nearly 40% of BRCA-mutated BCs are unresponsive, and resistance often develops in initial responders. Therefore, novel mutation-specific targeted therapies are urgently needed. In this study, we employed the novel computational method 'Mining Synthetic Lethals (MiSL)' to analyze TCGA pan-cancer primary tumor data, identifying ART3 as a potential SL partner of the BRCA1 germline mutation. Our approach focused on the intersection of genetic interactions and cancer vulnerabilities, providing a unique insight into SL relationships. siRNA-mediated silencing of ART3 in BRCA1-mutated cells consistently led to decreased cell viability, apoptosis induction, and G2/M cell cycle arrest. Following stable transfection with an inducible ART3 shRNA construct, we confirmed significant reduction in cell viability in BRCA1-deficient cells through colony formation assays. RNA sequencing of ART3 knockdown cells revealed transcriptional changes in genes involved in cell cycle progression, apoptosis, and growth and proliferation pathways compared with control cells. Immunofluorescent staining revealed increased DNA damage foci with decreased DNA repair in ART3 knockdown BRCA1-deficient cells. In conclusion, our study identifies ART3 as a novel SL partner of the BRCA1 mutation in BC. This discovery paves the way for developing new therapeutic strategies for BRCA1-mutated breast cancers and novel chemoprevention approaches for individuals carrying germline BRCA1 mutations. Future research will focus on further elucidating the molecular mechanisms underlying this SL interaction and exploring its therapeutic potential in clinical settings. Citation Format: Hannah Neiger, Xinyu Pei, Anh Nguyen, Wenjia Wang, Emily L. Siegler, James Zhou, Kristie Lau, Alexandra Tan, Yihui Shi. Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4686.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase(AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection.

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.

Human Germline HLTF E259K Mutation Identified in Familial MDS Patients Accumulates DNA Damage through Impaired PCNA Polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been discovered, the genetic landscape and the molecular pathogenesis still remain unclear. To explore novel causal genetic alterations and their pathogenetic significance, we performed whole-exome sequencing analysis using four samples from a Japanese familial MDS pedigree, two affected and two unaffected as controls. In total, 16889 non-synonymous germline substitutions were obtained. Among them, twelve candidate single-nucleotide variations (ANK1, C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, RYK, TTN) were elucidated. Of the candidates, we elucidated HLTF, encoding a member of the SWItch/Sucrose Non-Fermenting (SWI/SNF) family, as a putative associated gene. HLTF works as an E3-ubiquitin ligase and binds with Methyl Methanesulfonate Sensitive 2 (MMS2)/Ubiquitin Conjugating Enzyme E2N (UBC13) complex. This complex is known to promote the Lys-63 linked polyubiquitination of proliferating cell nuclear antigen (PCNA), followed by an error-free postreplication DNA repair by arranging template switching (Mailand et al. Nat Rev Mol Cell Biol 2013). The obtained germline mutation of HLTF was p.Glu259Lys (E259K), heterozygous mutation.To study the functional role of HLTF, we firstly evaluated the effects of Hltf silencing on hematopoiesis by in vitro colony-formation assay with Hltf-knocked down mouse primary bone marrow cells, and found that these cells have enhanced in vitro colony-formation capacity. In agreement with the acquisition of an immature phenotype, the cells expressing c-kit, a hematopoietic stem/ progenitor cell marker, were increased in Hltf shRNA-expressing colonies after the first round. We then focused on DNA damage in HLTF knockdown HEL cells according to the function of HLTF post-replication DNA repair. We examined the phosphorylation of gamma H2AX foci by immunofluorescent staining and found that the number of foci was increased in HLTF knockdown cells compared with control cells, suggesting that DNA damage was accumulated in HLTF knockdown cells.To investigate the effects of exogenous expression of HLTF E259K mutant protein, we evaluated in vitro apoptosis status with HEL cells transduced with empty vector (EV), HLTF wild type (WT), and HLTF E259K mutant (MT), respectively. In apoptosis assay, annexin V positive cells were significantly increased in MT-overexpressed HEL cells compared with WT-overexpressed HEL cells. In addition, we found that the number of gamma H2AX foci was increased in HLTF MT-overexpressed HEL cells, suggesting that DNA double-strand breaks were accumulated in MT-overexpressed cells. To explore the mechanism of accumulated DNA double-strand breaks by mutant HLTF, we evaluated the binding ability of HLTF mutant protein with PCNA, and MMS2/UBC13, all of which play a critical role in the DNA double-strand break repair process. We found that binding of HLTF MT to MMS2 or UBC13 was significantly attenuated compared with HLTF WT although binding to PCNA remained unchanged, suggesting HLTF MT cannot efficiently form a complex that induces polyubiquitination of PCNA. In line with this, PCNA polyubiquitination was impaired in HLTF MT-overexpressed cells compared with HLTF WT-overexpressed cells.Finally, we searched somatic HLTF mutation in sporadic MDS bone marrow samples. Whole HLTF coding regions of 40 samples were sequenced. As a result, a novel HLTF missense heterozygous mutation (p.Thr50Ala, T50A) was identified in one sample (1/40 = 2.5%). These data suggested the possibility that HLTF mutation might contribute to the pathogenesis not only in familial MDS but also in sporadic MDS.In conclusion, our results indicated that HLTF E259K germ line mutation accumulates DNA damage through impaired PCNA polyubiquitination. The aberrant function of HLTF mutant might play a role in the pathogenesis of familial MDS. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Suzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria. Harada:NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding; Celgene: Research Funding. Kurokawa:Bristol-Myers Squibb: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau.

Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications.

Human bocavirus 1 (HBoV1) was discovered in human nasopharyngeal specimens in 2005. It is an autonomous human parvovirus and causes acute respiratory tract infections in young children. HBoV1 infects well differentiated or polarized human airway epithelial cells in vitro. Unique among all parvoviruses, HBoV1 expresses 6 non-structural proteins, NS1, NS1-70, NS2, NS3, NS4, and NP1, and a viral non-coding RNA (BocaSR), and three structural proteins VP1, VP2, and VP3. The BocaSR is the first identified RNA polymerase III (Pol III) transcribed viral non-coding RNA in small DNA viruses. It plays an important role in regulation of viral gene expression and a direct role in viral DNA replication in the nucleus. HBoV1 genome replication in the polarized/non-dividing airway epithelial cells depends on the DNA damage and DNA repair pathways and involves error-free Y-family DNA repair DNA polymerase (Pol) η and Pol κ. Importantly, HBoV1 is a helper virus for the replication of dependoparvovirus, adeno-associated virus (AAV), in polarized human airway epithelial cells, and HBoV1 gene products support wild-type AAV replication and recombinant AAV (rAAV) production in human embryonic kidney (HEK) 293 cells. More importantly, the HBoV1 capsid is able to pseudopackage an rAAV2 or rHBoV1 genome, producing the rAAV2/HBoV1 or rHBoV1 vector. The HBoV1 capsid based rAAV vector has a high tropism for human airway epithelia. A deeper understanding in HBoV1 replication and gene expression will help find a better way to produce the rAAV vector and to increase the efficacy of gene delivery using the rAAV2/HBoV1 or rHBoV1 vector, in particular, to human airways. This review summarizes the recent advances in gene expression and replication of HBoV1, as well as the use of HBoV1 as a parvoviral vector for gene delivery.

Breast Cancer Predisposition Genes and Synthetic Lethality.

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.

Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis

Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H.pylori CagA for gastric carcinogenesis.

Abstract PS19-03: Dna repair imbalance and immune response in breast cancer mortality disparities

Abstract Background: Black breast cancer patients have p53 loss in 60% of their tumors, compared to 35% p53 loss in white breast cancer patients. The tumor suppressor p53 has pleiotropic effects on DNA repair, as it regulates both error prone and error-free DNA repair pathways. These effects on DNA repair represent molecular vulnerabilities that influence chemotherapy response, both directly and indirectly through the activation of immune responses. While studies have begun to elucidate DNA repair imbalance and immune response in human cancer, little is known about how these pathways differ by race. Methods: To study DNA repair and immune response in breast cancer, we performed gene expression analysis on FFPE samples from the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled black and younger women. We curated a list of DNA repair genes representing regulators of error prone and error free DNA repair. Pathways included Nucleotide Excision Repair (NER), Fanconi Anemia (FA), Mismatch repair (MMR), Base Excision Repair (BER), Homologous Recombination (HR), Translesion Synthesis (TLS), Alternative End Joining (AEJ), Checkpoint, and APOBEC. In addition, we developed a 50-gene immune panel representing 12 individual immune cell types (B cells, T cells, Treg cells, T help cells, T follicular helper cells, CD8 T cells, NK cells, Eosinophils, Neutrophils, M1 & M2 Macrophages) and both adaptive and innate arms of the immune system. A total of 1464 patients (53% black, 53% under 50) were included in the current analysis. We used consensus clustering to identify groups of patients based on DNA repair gene expression and used linear regression to estimate the relative frequency differences between these classes and demographic and clinical characteristics. Results: We found that breast cancers grouped into four clusters based on DNA repair gene expression. One cluster, ‘Repair High’, represented 32% of the tumors, and had high expression of NER, NHEJ, HR, and FA genes, suggesting a broad DNA repair response. Another group, ‘HR/FA High’ represented 23% of the tumors and was enriched for high expression of HR and FA genes. An “APOBEC High” group consisted of 32% of the tumors and was enriched for high expression of APOBEC family genes (APOBEC3D, APOBEC1, APOBEC3A, APOBEC3H, APOBEC3B). Finally, 13% of tumors, had a ‘Heterogeneous Repair’ pattern of high expression of HR, NHEJ, and FA genes, but lower expression of NER genes. The HR/FA and Heterogeneous Repair groups were enriched for TP53 mutant-like tumors (93% vs. 5% and 61% vs. 38% Mutant vs. Wildtype respectively). In addition, the Heterogeneous Repair group was enriched for Hormone Receptor positive samples ([RFD] 8.2% (0.613, 15.3), 77% vs. 23% in positive vs. negative respectively), while the HR/FA High group was significantly enriched for TNBC ([RFD] HR/FA: 51.2% (45.1, 57.1), 75% vs. 25% TNBC vs. non-TNBC respectively). The Repair High group was the only group enriched for non-black race ([RFD]: 10.4% (4.0, 16.7), 42% vs. 58% in blacks vs. non-blacks respectively). Finally, DNA repair classes were associated with immune scores, with the APOBEC High tumors having a significantly higher Eosinophil score (p = 0.021) and Neutrophil score (p = 0.007) compared to the other four groups. Conclusion: DNA repair expression is highly variable across breast tumors and may depend upon TP53 status, tumor subtype, and race. Differential immune marker expression by DNA repair group suggests some DNA repair groups may have differential response to immune-targeted therapies. DNA repair, immune response, and race are inter-related in breast cancer and unraveling and ultimately targeting breast cancer disparities may require coordinated evaluation of these pathways. Citation Format: Andrea Walens, Alina M Hamilton, Markia A Smith, Xiaohua Gao, Erin L Kirk, Stephen D Hursting, Katherine A Hoadley, Cyrus Vaziri, Melissa A Troester. Dna repair imbalance and immune response in breast cancer mortality disparities [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-03.

More than Meets the ISG15: Emerging Roles in the DNA Damage Response and Beyond.

Maintenance of genome stability is a crucial priority for any organism. To meet this priority, robust signalling networks exist to facilitate error-free DNA replication and repair. These signalling cascades are subject to various regulatory post-translational modifications that range from simple additions of chemical moieties to the conjugation of ubiquitin-like proteins (UBLs). Interferon Stimulated Gene 15 (ISG15) is one such UBL. While classically thought of as a component of antiviral immunity, ISG15 has recently emerged as a regulator of genome stability, with key roles in the DNA damage response (DDR) to modulate p53 signalling and error-free DNA replication. Additional proteomic analyses and cancer-focused studies hint at wider-reaching, uncharacterised functions for ISG15 in genome stability. We review these recent discoveries and highlight future perspectives to increase our understanding of this multifaceted UBL in health and disease.

SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.

Abstract 2567: TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability

Abstract TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in “trunk” clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing. Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution. Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed. Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Thomas Webber, Nnennaya Kanu, Charles Swanton. TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2567.

Increased error-free DNA repair gene expression through reprogramming in human iPS cells.

IntroductionMany studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied.MethodsWe analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming.ResultsThrough reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information.ConclusionsHigh PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.

Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

Abstract 3307: BRCA1 promoter methylation analyzed in 1032 primary breast cancers predicts favorable response to chemotherapy independently of p53 mutations

Abstract Individuals with germline mutations in the breast cancer susceptibility gene, BRCA1, are at increased risk of developing breast- and ovarian cancer. BRCA1 is involved in DNA double strand break repair (DSB) by homologous recombination (HR), an error-free DNA repair pathway that uses an intact sister chromatid for the repair. BRCA1 mutated tumors are therefore HR deficient and sensitive to drugs that stall DNA replication forks, resulting in DSBs. Tumor cells lacking the protein are characterized by genomic instability and a pattern of specific genome-wide mutation signature as a consequence of dependence on alternative error prone repair pathways. BRCA1 can also be inactivated epigenetically by CpG promoter hypermethylation. These BRCA1 epigenetically silenced tumors are more likely to be of the basal-like subtype and are strongly associated with this pattern of mutational signature characteristic for individuals with germline mutations in BRCA1. This suggests that the tumorigenesis in the sporadic cancer cases is similar to tumors where germline mutations are present, highlighting the importance of epigenetic silencing of BRCA1. The aim of the study was to examine the clinical significance of epigenetic silencing of BRCA1 in primary breast cancer, focusing on the response to chemotherapy. DNA methylation analysis of the BRCA1 promoter was performed by pyrosequencing tumor samples from 1032 patients with primary breast cancer of which 966 were sporadic, 61 were BRCA2 germline mutation carriers and 5 were BRCA1 mutation carriers. The p53 mutational status was also studied in the same primary tumors by DNA sequencing. We analysed the methylation status of BRCA1 in tumors derived from 1032 patients and found BRCA1 to be promoter hypermethylated in 29 sporadic tumors (29 out of 966, 3.0 %) whereas none of the tumors derived from BRCA mutation carriers were BRCA1 promoter hypermethylated. Patients with BRCA1 promoter hypermethylation receiving chemotherapeutic drug treatment show significantly better breast cancer specific survival (P=0.0192). The trend is opposite in patients not receiving chemotherapeutic treatment, were patients with BRCA1 hypermethylated tumors show worse survival (P=0.074). The impact of BRCA1 methylation with respect to survival in chemotherapy treated patients was found to be independent of p53 mutational status, ER status, tumorsize, lymph node status, year- and age at diagnosis (HR = 0.108; 95% CI = 0.015-0.794; P = 0.0288). BRCA1 promoter hypermethylation predicts improved disease outcome in anti-cancer drug treated patients. Therefore, BRCA1 promoter methylation status in primary breast cancer is an important factor to be considered when deciding treatment options. Citation Format: Olafur A. Stefansson, Holmfridur Hilmarsdottir, Kristrun Olafsdottir, Laufey Tryggvadottir, Asgerdur Sverrisdottir, Jon G. Jonasson, Jorunn E. Eyfjord, Stefan Sigurdsson. BRCA1 promoter methylation analyzed in 1032 primary breast cancers predicts favorable response to chemotherapy independently of p53 mutations [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 3307.

Uracil DNA Glycosylase of Gammaherpesvirus alters somatic hypermutation through error-free DNA repair

Abstract The gammaherpesviruses (gHV) which include human Epstein Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are associated with B cell malignancy including Burkitt lymphoma. All herpesvirus encode a viral homolog of the mammalian Uracil DNA-glycosylase (UNG) gene. In B cells, mammalian UNG plays a critical role in the antibody diversification processes of class switch recombination (CSR) and somatic hypermutation (SHM). UNG processes U:G mismatches created by activation-induced cytidine deaminase (AID) in an error-prone fashion to accomplish CSR and SHM. During primary infection, gHV expands in the germinal center B cells which actively express AID and undergo CSR and SHM. However, the function of viral UNG (vUNG) on viral pathogenesis and host genome integrity during the mutagenic germinal center reaction is an open question. Using a MHV68 mouse infection model we find that germinal center cells are the major viral targets during acute infection. Analysis of infected cells demonstrates significant overlap of vUNG and AID expression. Using a cell based hypermutation reporter assay, we find that host UNG supports error-prone repair and mutagenesis while vUNG results in only high-fidelity error-free repair of AID lesions. In vitro biochemical characterization finds several differences between vUNG and host UNG, including the ability of vUNG to bind abasic sites. These results demonstrate a differential ability of UNGs to support error-prone versus error-free repair. The finding that vUNG is expressed in germinal center cells suggest a novel mechanism whereby gammaherpesvirus can affect antibody diversification and B lymphocyte function.

Abstract B04: RAD51 expression as a biomarker of homologous recombination deficiency in ovarian cancer

Abstract RAD51 is a critical component of the homologous recombination pathway, forming a nucleoprotein filament that enables strand exchange and templated error-free DNA repair. The tumor suppressors BRCA1 and BRCA2 interact with RAD51 to control its activity on DNA. Defects in homologous recombination in tumors are clinically relevant, with evidence of synthetic lethality of such cancers to poly ADP ribose polymerase (PARP) inhibitors. Mutations in RAD51 are uncommon in cancer, but aberrant over-expression of RAD51 has been reported as a mechanism to overcome a recombination defect in in-vitro models. However there are no large scale studies on RAD51 expression in clinically annotated data sets. Here we report a series of experiments to study RAD51 protein expression in ovarian cancer, a tumor type where recombination defects are prominent and being evaluated in several clinical trials of PARP inhibition. Analysis of DNA repair protein expression in formalin fixed clinical tissue is challenging, and our experiments highlight recent advances in quantitative microscopy that are generalizable to other clinical scenarios as well. We first evaluated several commercially available monoclonal antibodies to RAD51 using siRNA depleted cell blocks, to identify an appropriate reagent and staining conditions for formalin fixed paraffin embedded (FFPE) material. We then analyzed RAD51 expression in a collection of 600 ovarian cancer samples obtained through the British Columbia Cancer Agency (BCCA) OvCare consortium, using a combination of multiplexed immunofluorescence staining and automated spectral microscopy to quantify staining on a per-cell basis. We find that RAD51 expression displays a Gaussian distribution in this cohort of high grade epithelial cancers, with no obvious correlation to BRCA mutation status. RAD51 sub-nuclear foci, which are a commonly used measure of homologous recombination in-vitro, did not prove to be accurately quantifiable in FFPE material. We then stained for RAD51 in a collection of approximately 250 ovarian cancer samples obtained from the SCOTROC4 trial. This clinical trial enrolled women with ovarian cancer into two treatment arms with fixed dose and escalated doses of carboplatin. Platinum sensitivity has been shown to correlate with sensitivity to PARP inhibitors, due to the requirement of homologous recombination for repair of platinum adducts. Therefore this trial offers a unique opportunity to study the predictive significance of biomarkers of homologous recombination in-vivo. In our analysis of this data set, we find that samples in the lowest quartile of RAD51 expression displayed a significantly improved survival after platinum chemotherapy, consistent with decreased HR in these cases. These are the first data on RAD51 in a clinical trial dataset of platinum sensitivity. Our study highlights methods and technical challenges for the quantitative analysis of DNA repair proteins in human clinical trial specimen, and describes a potential biomarker for synthetic lethal approaches targeting homologous recombination deficiency. Citation Format: Michal M. Hoppe, David SP Tan, Diana GZ Lim, Anthony Karnezis, David Huntsman, Jennifer Steel, Xinxue Liu, James Paul, Liz-Anne Lewsley, Nadeem Siddiqui, Robert Brown, Anand D. Jeyasekharan. RAD51 expression as a biomarker of homologous recombination deficiency in ovarian cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B04.

The Shu complex is a conserved regulator of homologous recombination.

Homologous recombination (HR) is an error-free DNA repair mechanism that maintains genome integrity by repairing double-strand breaks (DSBs). Defects in HR lead to genomic instability and are associated with cancer predisposition. A key step in HR is the formation of Rad51 nucleoprotein filaments which are responsible for the homology search and strand invasion steps that define HR. Recently, the budding yeast Shu complex has emerged as an important regulator of Rad51 along with the other Rad51 mediators including Rad52 and the Rad51 paralogs, Rad55-Rad57. The Shu complex is a heterotetramer consisting of two novel Rad51 paralogs, Psy3 and Csm2, along with Shu1 and a SWIM domain-containing protein, Shu2. Studies done primarily in yeast have provided evidence that the Shu complex regulates HR at several types of DNA DSBs (i.e. replication-associated and meiotic DSBs) and that its role in HR is highly conserved across eukaryotic lineages. This review highlights the main findings of these studies and discusses the proposed specific roles of the Shu complex in many aspects of recombination-mediated DNA repair.

More end resection is not merrier.

Unrestrained 53BP1 activity causes fusions of dysfunctional telomeres and embryonic lethality associated with misrepair of DNA double-strand breaks in BRCA1-deficient mice. However, the physiological role of 53BP1 remains unclear, because it presumably did not evolve to carry out these pathological functions. A new report proposes that 53BP1 activity prevents hyper-resection and thereby promotes error-free DNA repair while suppressing alternative mutagenic pathways.

Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3′ to 5′ direction and is essential for parasite growth

Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.

Abstract P4-07-07: Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models

Abstract Background: Homologous recombination deficiency (HRD) in cancer cells can occur due to mutations (germline or sporadic), methylation or other epigenetic causes. HRD leads to a defect in the conservative, error-free DNA repair mechanism and is associated with enhanced susceptibility to DNA targeting chemotherapy. Currently functional HRD assays are not broadly available for clinical use. Many of the HRD assays used in the experimental setting require fresh frozen tissue for optimal results, or require specialized expertise to interpret the results. We evaluated an immunohistochemical (IHC) assay using formalin fixed paraffin embedded (FFPE) tissue to measure protein expression of Rad51 and geminin, a cell proliferation marker, to assess HRD in breast cancer cell line models and clinical breast cancer samples. We hypothesize that Rad51, which is involved in the later stages of HR, can serve as a functional marker of HRD. Methods: The MCF-7 human breast cancer cell line was used as a model with intact HR. Western blotting of total cell lysates from cells grown in culture was performed to confirm HR response following treatment with DNA damaging chemotherapeutic agents, cisplatin and doxorubicin. Paclitaxel, a microtubule targeting agent, was used as a negative control. Mice with MCF-7 xenograft tumors were also treated with cisplatin, or doxorubicin at two dose levels (low and high) and various time points post treatment to assess the dose and time response to HR markers. Tumors from mice treated with paclitaxel were used as a negative control. Xenograft tumors were fixed and analyzed by IHC using an antibody specific for total Rad51 and geminin expression. DNA damage was also assessed in a portion of the tumor using a pulse gel electrophoresis assay. We also analyzed FFPE breast cancer clinical samples from patients with BRCA1 mutations for Rad51 and geminin expression. Results: In MCF-7 grown in vitro, total Rad51 was elevated as soon as 4 hours following exposure to doxorubicin and cisplatin, but not in response to paclitaxel treatment. In xenograft tumors, baseline Rad51 and geminin expression were relatively high illustrating proficient HR in an actively proliferating tumor model. Rad51 expression increased post treatment with cisplatin and doxorubicin as early as 6hrs and peaked at 16-24hrs. Geminin expression correlated well with expression of Rad51 at baseline and in time response to treatment. Pulse gel electrophoresis in paired tumor samples confirmed DNA damage was occurring compared to vehicle control treated tumors. However, this technique did not show a strong dose or time response. Five breast tumors from patients with known BRCA1 mutations were stained for Rad51 and geminin expression. High geminin expression and low Rad51 expression was noted in the majority of these tumors. Conclusions: An IHC assay using FFPE tissue to measure Rad51/geminin is a promising method to assess HRD in breast cancer. Further analytical and clinical validation of this approach is ongoing. Citation Format: Chalasani P, Nagy D, Livingston RB, Weterings E, Nagle R, Singh S, Barnes M, Grogan T, Ridder R, Baker AF, Kandavel S. Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-07.