Defects In Double-strand Break Repair Research Articles

M6A demethylase OsALKBH5 is required for double-strand break formation and repair by affecting mRNA stability in rice meiosis.

N6-methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo-cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P-bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double-strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5-mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post-transcriptional regulation of meiotic progression in rice.

Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.

In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.

Tumour break load is a biologically relevant feature of genomic instability with prognostic value in colorectal cancer

BackgroundClinically implemented prognostic biomarkers are lacking for the 80% of colorectal cancers (CRCs) that exhibit chromosomal instability (CIN). CIN is characterised by chromosome segregation errors and double-strand break repair defects that lead to somatic copy number aberrations (SCNAs) and chromosomal rearrangement-associated structural variants (SVs), respectively. We hypothesise that the number of SVs is a distinct feature of genomic instability and defined a new measure to quantify SVs: the tumour break load (TBL). The present study aimed to characterise the biological impact and clinical relevance of TBL in CRC. MethodsDisease-free survival and SCNA data were obtained from The Cancer Genome Atlas and two independent CRC studies. TBL was defined as the sum of SCNA-associated SVs. RNA gene expression data of microsatellite stable (MSS) CRC samples were used to train an RNA-based TBL classifier. Dichotomised DNA-based TBL data were used for survival analysis. ResultsTBL shows large variation in CRC with poor correlation to tumour mutational burden and fraction of genome altered. TBL impact on tumour biology was illustrated by the high accuracy of classifying cancers in TBL-high and TBL-low (area under the receiver operating characteristic curve [AUC]: 0.88; p < 0.01). High TBL was associated with disease recurrence in 85 stages II–III MSS CRCs from The Cancer Genome Atlas (hazard ratio [HR]: 6.1; p = 0.007) and in two independent validation series of 57 untreated stages II–III (HR: 4.1; p = 0.012) and 74 untreated stage II MSS CRCs (HR: 2.4; p = 0.01). ConclusionTBL is a prognostic biomarker in patients with non-metastatic MSS CRC with great potential to be implemented in routine molecular diagnostics.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Diagnostic and Therapeutic Challenges for Infants with Radiosensitive (RS)-SCID Identified by Newborn Screening (NBS)

NBS for SCID has been universally implemented in the U.S. Patients with DNA double-strand break repair defects RS-SCID constitute a rare sub-group, who are susceptible to developing malignancies and non-lymphoid comorbidities. Here, we describe three patients and discuss challenges in clinical management of RS-SCID defects.

Patient derived ex vivo tissue slice cultures demonstrate a profound DNA double-strand break repair defect in HPV-positive oropharyngeal head and neck cancer

BackgroundHPV-positive head and neck squamous cell carcinoma of the oropharynx (OPSCC) are more sensitive towards radiation than HPV-negative OPSCC. Two main theories exist regarding the underlying mechanism. Stronger lymphocyte infiltration points to an enhanced immunogenicity, whereas data from HPV-positive HNSCC cell lines suggest an enhanced cellular radiosensitivity based on a defect in DNA double-strand break (DSB) repair. The critical limitation of the latter theory is that the evidence was largely derived from a small number of established HPV-positive HNSCC cell lines. Methods and materialsFresh patient-derived OPSCC samples were cut in 400 µm sections and cultured on cell culture inserts. Slice cultures were irradiated, in part combined with ATM inhibition, and fixed and frozen after 2 and 24 h. DSBs were analyzed by quantification of 53BP1 foci in nuclei co-stained with the SCC marker p63 via immunofluorescence microscopy. ResultsEx vivo OPSCC tumor slice cultures maintained stable oxygenation and proliferation characteristics for at least 3 days. Areas of p63-positivity in immunofluorescence microscopy matched histologically confirmed tumor cell areas in serial sections, indicating the suitability of p63 as a tumor cell marker. p63-positive nuclei in HPV-positive OPSCC tissues (n = 14) showed profoundly elevated numbers of residual radiation-induced DSBs as compared to those from HPV-negative OPSCC (n = 12) (3 Gy: on average 4.9 vs. 1.2 foci per nucleus; p < 0.0001). Within the HPV-positive subgroup, samples derived from patients with a smoking history of less than 10 pack years demonstrated higher residual DSBs as compared to those derived from patients with 10 or more pack years (3 Gy: on average 6.5 vs. 3.2 foci per nucleus; p = 0.0105). Additional ATM inhibition resulted in a substantial increase in residual foci in all 4 HPV-negative samples tested but strikingly only in 2 out of 11 HPV-positive samples. ConclusionsIn summary, our data provide robust, cell line-independent experimental evidence for an intrinsic DSB repair deficiency in HPV-positive OPSCC, strongly suggesting a meaningful contribution to the enhanced clinical radiosensitivity. The reduced effectiveness of ATM inhibition indicates a defect in the ATM-orchestrated DNA damage response. Lower numbers of residual 53BP1 nuclear foci in the ex vivo assay may identify HPV-positive patients with effective DSB repair who should potentially be excluded from de-intensification approaches.

Biallelic variants in YRDC cause a developmental disorder with progeroid features

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient’s dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair.

During meiosis, homologous chromosomes pair and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, double-strand breaks (DSBs) initiate recombination within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have been identified. We identified Zcwpw1, containing H3K4me3 and H3K36me3 recognition domains, as having highly correlated expression with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognises CpG dinucleotides. Male Zcwpw1 knockout mice show completely normal DSB positioning, but persistent DMC1 foci, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest ZCWPW1 recognition of PRDM9-bound sites at DSB hotspots is critical for synapsis, and hence fertility.

Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Abstract 2823: DNA methyltransferase inhibitors sensitize NSCLC cells to PARP inhibitors by induction of a double strand break repair defect

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the US. Poly (ADP-ribose) polymerase inhibitors (PARPi) show efficacy as radiosensitizers in NSCLC, but arguably have gained greater importance in cancer therapeutics by exhibiting synthetic lethality (SL) in tumors with defects in the homologous recombination (HR) pathway of double strand break repair (DSBR). Mechanistically, PARPi cytotoxicity is partially mediated through trapping of PARP1 at sites of DNA damage, with next generation PARPis such as talazoparib (Tal) being potent PARP trappers. Our group has previously demonstrated that combining Tal with a DNA methyltransferase inhibitor (DNMTi) enhances PARPi cytotoxicity in acute myeloid leukaemia and breast cancer models. DNMTis are cytosine analogs that become incorporated into replicating DNA, where they covalently bind their target enzyme DNMT1. When combined with PARPi, DNMTis enhance PARP trapping at damage sites, which collapse into cytotoxic double strand breaks (DSB) when encountered by a replication fork. In the current study, we demonstrate synergistic cytotoxicity of Tal in combination with the DNMTi 5-azacytidine (Aza) in NSCLC cell lines by combination index analyses and colony forming assays. Further potentiation of Tal+Aza cytotoxicity is observed following induction of DNA damage via IR. These results were substantiated in an A549 xenograft model (n=8 per group) where Tal+Aza+IR significantly reduced tumor volume and increased survival compared to vehicle (p&amp;lt;0.001) or Tal+Aza without IR (p&amp;lt;0.05). We and others have previously reported that low dose DNMTi results in stable transcriptional changes in the cancer epigenome, including DNA damage response pathways. Here, we show by microarray analysis that NSCLC cell lines (A549, H23, H1299, H460) treated with Aza exhibit up- and downregulation of multiple genes in the DNA repair reactome, including decreased expression of the key HR-related gene FANCD2, and the non-homologous end joining (NHEJ) gene Ku80. These results were validated by mRNA and protein quantification in Aza-treated NSCLC cell lines and xenograft tumor samples. In keeping with a FANCD2-associated HR defect, Aza exposure is associated with reduced accumulation of RAD51 foci following IR, and reduced HR activity in an extrachromosomal HR assay. Additionally, increased sensitivity to IR post Aza treatment can be explained by decreased expression of NHEJ factor Ku80. These results indicate that Aza treatment in NSCLC induces a DSBR deficiency that can be targeted for SL by PARPi and can further sensitize to IR. Given these agents are currently in use in the clinic, this combination offers a promising therapeutic strategy that could be rapidly translated into clinical practice. Citation Format: Rachel Abbotts, Michael Topper, Daniel Fontaine, Christopher Biondi, Eun Yong Choi, Rena Lapidus, Stephen Baylin, Feyruz Rassool. DNA methyltransferase inhibitors sensitize NSCLC cells to PARP inhibitors by induction of a double strand break repair defect [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 2823.

Abstract 1352: Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors

Abstract Chromothripsis and chromoanasynthesis are two forms of genomic instability leading to complex genomic rearrangements that affect one or very few chromosomes. These one-off catastrophic events play a role in numerous tumor entities as well as in some congenital diseases. The availability of murine models recapitulating both phenomena would substantially facilitate the investigation of the mechanistic aspects underlying catastrophic genomic events. Homologous recombination repair (HR) and canonical Non-Homologous-End-Joining (cNHEJ) represent the two major processes for DNA double-strand break repair in mammalian cells. Conditional inactivation of key factors of either of these two pathways, such as Brca2 for HR and Xrcc4 or Lig4 for cNHEJ in nestin-expressing or Emx1-expressing murine neural progenitor cells leads to medulloblastomas and gliomas in a p53-deficient background. We showed by whole-genome sequencing that these tumors frequently display chromothripsis or chromoanasynthesis (33 to 73% of the analyzed tumors, n= 27) and that catastrophic rearrangements drive tumor development. FISH analysis identified a link between chromoanasynthesis and increased numerical and structural aberrations and with the presence of marker chromosomes. In addition, amplifications of c-Myc and n-Myc likely facilitate catastrophic events. Detailed analysis of the microhomologies at the breakpoint junctions on the chromosomes affected by complex genomic rearrangements identified cNHEJ and alternative end-joining as likely repair processes involved in chromothripsis and chromoanasynthesis. Treatment of cells derived from the mouse tumors with inhibitors of HR and/or alternative end-joining (e.g. RAD51 and PARP inhibitors, respectively) in combination with DNA damage revealed the dependence of these tumor cells on specific repair processes and showed that these DNA repair deficiencies can be utilized for synthetic lethality approaches. Comparison of the mouse tumors with whole-genome sequencing data from human medulloblastomas (n=68) and gliomas (n=32) identified an association between chromothripsis and deficiencies in repair processes, by analyzing copy-number level aberrations affecting repair factors and mutational signatures of DNA double-strand break repair defects. This link between DNA repair deficiency and chromothripsis was further confirmed in additional tumor entities such as breast cancer (n=356) and melanoma (n=69). In analogy to the clinical use of PARP inhibitors in the context of BRCA-deficient breast cancer, our findings point towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements. Citation Format: Manasi Ratnaparkhe, John Wong, Pei-Chi Wei, Mario Hlevnjak, Paul Northcott, David T. Jones, Marcel Kool, Anna Jauch, Agata Pastorczak, Andrey Korshunov, Rajiv Kumar, Susanna M. Downing, Stefan M. Pfister, Marc Zapatka, Peter J. McKinnon, Frederick W. Alt, Peter Lichter, Aurelie Ernst. Inactivation of factors of DNA double-strand break repair by homologous recombination or non-homologous end-joining leads to frequent catastrophic genomic events in murine and human tumors [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 1352.

MOF influences meiotic expansion of H2AX phosphorylation and spermatogenesis in mice.

Three waves of H2AX phosphorylation (γH2AX) have been observed in male meiotic prophase I: the first is ATM-dependent and occurs at leptonema, while the second and third are ATR-dependent, occuring at zygonema and pachynema, respectively. The third wave of H2AX phosphorylation marks and silences unsynapsed chromosomes. Little is known about H2AX phosphorylation expands to chromatin-wide regions in spermatocytes. Here, we report that histone acetyltransferase (HAT) MOF is involved in all three waves of H2AX phosphorylation expansion. Germ cell-specific deletion of Mof in spermatocytes by Stra8-Cre (Mof cKO) caused global loss of H4K16ac. In leptotene and zygotene spermatocytes of cKO mice, the γH2AX signals were observed only along the chromosomal axes, and chromatin-wide H2AX phosphorylation was lost. In almost 40% of early-mid pachytene spermatocytes from Mof cKO mice, γH2AX and MDC1 were detected along the unsynapsed axes of the sex chromosomes, but failed to expand, which consequently caused meiotic sex chromosome inactivation (MSCI) failure. Furthermore, though RAD51 was proficiently recruited to double-strand break (DSB) sites, defects in DSB repair and crossover formation were observed in Mof cKO spermatocytes, indicating that MOF facilitates meiotic DSB repair after RAD51 recruitment. We propose that MOF regulates male meiosis and is involved in the expansion of all three waves of H2AX phosphorylation from the leptotene to pachytene stages, initiated by ATM and ATR, respectively.

Motor Neuron Disease-Associated Loss of Nuclear TDP-43 Is Linked to DNA Double-Strand Break Repair Defects

Loss of nuclear TDP-43 has been linked to amyotrophic lateral sclerosis (ALS), which also features increased genome damage in affected neurons. Although TDP-43 binds to DNA, its role in the DNA damage response (DDR) has not been investigated. Here, we report that nuclear TDP-43 is a critical component of the DNA double-strand break (DSB) repair machinery in motor neurons. TDP-43 is rapidly recruited at DSB sites where it stably interact with non-homologous end joining (NHEJ)- mediated DSB repair and DDR factors in spinal motor neurons derived from human neural stem cells. Total or nucleus-specific loss of TDP-43 in motor neurons and C. elegans markedly increased genomic DSBs by impairing NHEJ and sensitized cells to DSB stress. TDP-43 pathology strongly correlated with DSB accumulation and DDR activation in the spinal cord of postmortem ALS patients. Our findings link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease.

Micronuclei Frequency in Tumors Is a Predictive Biomarker for Genetic Instability and Sensitivity to the DNA Repair Inhibitor AsiDNA.

Therapeutic strategies targeting DNA repair pathway defects have been widely explored, but often only benefit small numbers of patients. Here we characterized potential predictive biomarkers for treatment with AsiDNA, a novel first-in-class DNA repair inhibitor. We evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double-strand breaks (DSB). For each cell line, we monitored constitutive PARP activation, spontaneous DNA damage by alkaline comet assay, basal micronuclei levels, the number of large-scale chromosomal rearrangements (LST), and the status of several DNA repair pathways by transcriptome and genome analysis. Sensitivity to AsiDNA was associated with a high spontaneous frequency of cells with micronuclei and LST and specific alterations in DNA repair pathways that essentially monitor DSB repair defects. A high basal level of micronuclei as a predictive biomarker for AsiDNA treatment was validated in 43 tumor cell lines from various tissues and 15 models of cell- and patient-derived xenografts. Micronuclei quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to AsiDNA treatment. That micronuclei frequency can be measured in biopsies and does not reveal the same genetic instability as conventional genome assays opens new perspectives for refining the classification of tumors with genetic instability. Cancer Res; 77(16); 4207-16. ©2017 AACR.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis.

DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma.

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV- HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10-14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC.

Functions of the MRE11 complex in the development and maintenance of oocytes.

The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11ATLD1 allele. Mre11ATLD1/ATLD1 females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1−/− and Trp13Gt/Gt mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11ATLD1/ATLD1. Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11ATLD1/ATLD1 follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-015-0535-8) contains supplementary material, which is available to authorized users.

Abstract 4739: Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer

Abstract Colorectal cancer (CRC) is the second most common fatal cancer in the U.S., with ∼ 30% cases familial (FCRC). Genetic predispositions have only been defined for 25% of FCRC (including Lynch syndrome, Familial Adenomatous Polyposis (FAP), and MutYH polyposis). For the genetically undefined group (uFCRC), understanding of disease pathogenesis is limited, and the absence of defined defects in relevant pathways that could support molecular diagnosis results in under- and over-screening. We hypothesized that uFCRC would arise distinctly from moderately penetrant germline defects in repair of DNA double-strand breaks (DSBs). Such defects in DSB repair would lead to a moderate level of constitutional genomic instability (CGI) that could be assayed from patient peripheral blood lymphocytes (PBLs). We further hypothesized that such DNA lesions would often be heterozygous, associated with haploinsufficiency or dominant-negative effects, and would impair protein interactions important for DNA repair. We combined exome sequencing with biological assays to screen a rich resource of uFCRC clinical samples from the Fox Chase Cancer Center Risk Assessment Program. Exome sequencing of PBLs from patients (N = 25) identified a mean of 1.4 rare variants/patient that were 1) strongly predicted to damage protein function using multiple functional predictors (SIFT, Polyphen-2, Provean, and MutationAssessor), 2) involved in pathways that suppress DSBs, and 3) have not been previously implicated in CRC. Further, many of the variant genes were commonly mutated in CRCs and other cancers in TCGA database. Comparison of PBLs from patients and age- and sex- matched controls revealed increased DSB levels (basal and with DNA damaging agents) in 18/25 patients vs. 1/25 normal controls (P = 0.001, AUC = 0.85). Next, knockdown of variant genes in HCT116 CRC cells increased DSB formation with or without damaging agents. As a proof-of-concept, we studied one patient in detail who had candidate disease-causing variants in 2 DNA repair genes, ERCC6 and WRN. Both variants were dysfunctional in biochemical tests. We established patient-derived EBV-lines and observed, at baseline and with several DNA damaging agents, elevated levels of γH2AX foci and larger nuclear comets, indicative of DSBs. This phenotype could be rescued by expression of wild type WRN or ERCC6 and conversely induced by knockdown in CRC cells. Further, the patient derived lines exhibited reduced expression of WRN and ERCC6, implicating haploinsufficiency of these genes in the phenotype observed. Our data support the hypothesis that defects in DSB repair genes cause moderate-level CGI that commonly presents as uFCRC. Our studies identify novel genetic subsets of FCRC, and could potentially predict familial risk and improve screening and diagnosis. Citation Format: sanjeevani arora, Hong Yan, Iltaeg Cho, Hua-Ying Fan, Biao Luo, xiaowu gai, dale bodian, joe vockley, yan zhou, elizabeth handorf, mark andrake, emmanuelle nicolas, Ilya Serebriiskii, tim yen, michael hall, greg enders, erica golemis. Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer. [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 4739. doi:10.1158/1538-7445.AM2015-4739

Effect of a poly(ADP‐ribose) polymerase‐1 inhibitor against esophageal squamous cell carcinoma cell lines

Effective molecular target drugs that improve therapeutic efficacy with fewer adverse effects for esophageal cancer are highly anticipated. Poly(ADP-ribose) polymerase (PARP) inhibitors have been proposed as low-toxicity agents to treat double strand break (DSB)-repair defective tumors. Several findings imply the potential relevance of DSB repair defects in the tumorigenesis of esophageal squamous cell carcinoma (ESCC). We evaluated the effect of a PARP Inhibitor (AZD2281) on the TE-series ESCC cell lines. Of these eight cell lines, the clonogenic survival of one (TE-6) was reduced by AZD2281 to the level of DSB repair-defective Capan-1 and HCC1937 cells. AZD2281-induced DNA damage was implied by increases in γ-H2AX and cell cycle arrest at G2/M phase. The impairment of DSB repair in TE-6 cells was suggested by a sustained increase in γ-H2AX levels and the tail moment calculated from a neutral comet assay after X-ray irradiation. Because the formation of nuclear DSB repair protein foci was impaired in TE-6 cells, whole-exome sequencing of these cells was performed to explore the gene mutations that might be responsible. A novel mutation in RNF8, an E3 ligase targeting γ-H2AX was identified. Consistent with this, polyubiquitination of γ-H2AX after irradiation was impaired in TE-6 cells. Thus, AZD2281 induced growth retardation of the DSB repair-impaired TE-6 cells. Interestingly, a strong correlation between basal expression levels of γ-H2AX and sensitivity to AZD2281was observed in the TE-series cells (R2 = 0.5345). Because the assessment of basal DSB status could serve as a biomarker for selecting PARP inhibitor-tractable tumors, further investigation is warranted.

Estrogen response in luminal breast cancer

Estrogen receptor α (ERα) is the defining and driving transcription factor in luminal breast cancers and genes modulated by ERα can dictate cell growth and endocrine response. Nonetheless, genomic mechanisms which govern ERα-driven tumorigenesis, acquisition of chemoresistant phenotype and tumor metastases remain elusive. Recently, scientists have explored to shed light on ER regulated genomic events in primary breast cancer with divergent clinical outcome and in distant ERα+ metastases. Mapping of genome-wide ER binding events by chromatin immunoprecipitation followed by high-throughput sequencing revealed differential FoxA1-mediated ERα-chromatin binding programming that results in predictive gene signatures exclusive for ERα+ breast cancer clinical outcome, and is characterized by remarkable intensification of ERα binding signal in tumors that progress towards a poor prognosis [1,2]. Furthermore, ERα-chromatin interactions occur regardless of tumor endocrine therapy sensitivity. Nevertheless, there is differentially stronger ERα binding signal in tamoxifen resistant in comparison with tamoxifen sensitive lineages [1]. Although the mechanisms underlying ERα binding plasticity in breast cancer remain to be elucidated, the influence of specific stimuli, as those triggered by growth factors pathways [3,4], may result in differential ERα binding patterns that regulate gene expression programs, sensitivity to endocrine therapy and overall clinical outcome in ERα+ breast tumors. It is worthwhile to emphasize that the only DNA motif found enriched in the core of ER binding events is the estrogen response element (ERE) [1]. Of note, innovative model in ERα-driven tumorigenesis and cancer aggressiveness has emerged from the concept of the tag-team model of gene expression in luminal breast cancer, according to which amplified distant EREs (DEREs) coordinately and remotely modulates the transcription of distant genes through long-rage chromatin interactions [5]. The model hypothesis highlights DERE axes as hot spots for concomitant and aberrant genome amplification in luminal breast cancer; thus coordinately and persistently deregulating target transcriptome, including co-amplification of oncogenes and repression of tumor-suppressor loci for cancer development and endocrine therapy resistance. Using integrated next-generation sequencing approaches, two densely ERα-bound DERE regions, frequently amplified in ERα+ luminal breast cancers, were mapped on chromosomes 17q23 and 20q13 [5]; genomic amplification of which has been associated with endocrine therapy relapse and overall poor prognosis in breast cancer [6]. Moreover, integration of 3C dataset with published time-course study of gene expression revealed 95 loci remotely interacting with 20q13 DEREs, 38 genes with 17q23 DEREs, and 46 estrogen-responsive targets [5]. Interestingly, a strong body of evidences has demonstrated the novel role of the DEREs targeted genes THRAP1 and ZIM2 as tumor suppressors in luminal breast cancer. It is reasonable to speculate that the increased frequency of chromatin interactions might play a role to elicit epigenetic repression of DERE-regulated genes. The aforementioned phenomenon derives from genomic alterations induced by chronic estrogenic exposure of mammary cells that persist in breast progenitor cells [5], and progressively accumulate in malignant differentiating cells, in agreement with previous findings that points to the intensification of the ERα binding signal and ERE amplification during breast cancer worsening prognosis [1,2]. Precisely, these findings open new avenues to explore amplified DEREs in 17q23 and 20q13 as potential prognostic markers in luminal breast cancer, based on the rationale that the aberrant amplification of DEREs may enable functionality of residual estrogen/ERα regardless of the administration of selective ER blockers, such as tamoxifen [5]. This concept, at least partially, can explain the benefit of using aromatase inhibitors in post-menopausal women, which present the higher incidence of luminal breast cancer. The inhibition of the main source of estrogen production in these patients may prevent the E2/ERα-induced anomalous amplification of DEREs. On the other hand, the amplified DEREs axes and the tag-team model of gene expression modulation in luminal breast cancer currently challenges the investigators to determine the precise factors and mechanisms that trigger the malignant transformation of normal progenitor mammary cells. Amplified DNA regulatory elements may result from sustained amplification of DERE-DERE interactions that may intensify chromatin interaction within regions harboring clustered breakpoints, exquisitely prone to genomic rearrangements, as a result of genomic instability [5,7]. During neoplastic transformation, defects in double-strand break repair may destabilize these physical interactions, promoting insertions and self-duplication of 20q13 DERE clusters, for example, into regions of seven derivative chromosomes [5]. In contrast to this concept, it is puzzling that mammary cells sustain the benign phenotype in the majority of women regardless of the estrogenic milieu. At present, it is also important to evaluate the occurrence of aberrant DERE amplification and its peculiarities in controlling target genes expression in ovarian cancer, commonly lethal and tamoxifen resistant. Nonetheless, cumulative evidence hint the potential roles of the amplified distant DNA response elements axes and the tag-team model of gene expression modulation in hormone-driven carcinogenesis and cancer progression. More importantly, knowledge gained out of the study [5] should be instrumental for further understanding of cancer genomics and bringing of innovative interventions to control cancer.