Alternative Non-homologous End-joining Pathway Research Articles

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

Nonhomologous end joining as key to CRISPR/Cas-mediated plant chromosome engineering.

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process.

Somatic Mutations in Oncogenes Are in Chronic Myeloid Leukemia Acquired De Novo via Deregulated Base-Excision Repair and Alternative Non-Homologous End Joining.

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.

The repair of topoisomerase 2 cleavage complexes in Arabidopsis.

DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

Targeting the MALAT1/PARP1/LIG3 Complex Induces DNA Damage and Apoptosis in Multiple Myeloma Mouse Models

Molecular pathogenesis studies of multiple myeloma (MM) have focused on coding genes rather than long non-coding RNAs (lncRNAs). However, evidence indicates that lncRNAs are involved in the initiation and progression of almost all kinds of cancers, including MM. MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is one of the most highly expressed conserved nuclear lncRNAs. Here, we demonstrated that MALAT1 plays important roles in MM DNA repair and cell death. We found that bone marrow plasma cells from patients with MGUS and MM express elevated levels of MALAT1 relative to those from healthy individuals. Our functional studies suggested that in MM, MALAT1 is key in regulating the DNA repair alternative non-homologous end-joining (A-NHEJ) and apoptosis pathways. Using RNA Antisense Purification-Mass Spectrum (RAP-MS), we identified MALAT1 binding proteins in MM, including PARP1 and LIG3, two key components of the A-NHEJ pathway protein complex. Degradation of the MALAT1 RNA by RNAse H using antisense gapmer DNA oligos in MM cells induced poly-ADP-ribosylation of nuclear proteins, defected the DNA repair pathway, and further induced apoptotic pathways. Anti-MALAT1 therapy combined with PARP1 inhibitor or proteasome inhibitor in MM cells showed a synergistic effect in vitro. Furthermore, using a novel single wall carbon nanotube (SWCN) conjugated with anti-MALAT1 oligos, we successfully knocked down MALAT1 RNA in cultured MM cell lines and in two xenograft mouse models. Most importantly, anti-MALAT1 therapy also could induce DNA damage and cell apoptosis in vivo, indicating that MALAT1 could serve as a potential novel therapeutic target for MM treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting MALAT1 induces DNA damage and sensitize non-small cell lung cancer cells to cisplatin by repressing BRCA1.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA which has been identified to be involved in alternative non-homologous end joining (A-NHEJ) pathways by binding with PARP1 and LIG3 in myeloma cells. This study aims to explore the roles of MALAT1 in DNA repair processes in non-small cell lung cancer (NSCLC). The interactions between MALAT1 and proteins were identified by co-immunoprecipitation and RNA pulldown. The interactions between MALAT1 and microRNAs (miRNA) were predicted by bioinformatics tools and confirmed by luciferase assay and RNA pulldown. The DNA damages were quantified by comet assay. The cell viability was examined by MTT assay and the cell apoptosis was determined by flow cytometry. MALAT1 is identified to be involved in A-NHEJ pathway in NSCLC cells. However, in LIG3-null cells where A-NHEJ pathway is inactivated, targeting MALAT1 still increases DNA damages, suggesting that MALAT1 participates in other DNA repair pathways. Subsequently, MALAT1 is identified to bind with miR-146a and miR-216b, which directly target the 3'UTR of BRCA1. MALAT1 is confirmed to functions as a competing endogenous RNA (ceRNA) absorbing miR-146a and miR-216b, upregulating BRCA1 expression and protecting Homologous Recombination (HR) pathway in NSCLC cells. Finally, overexpression MALAT1 protects NSCLC cells from the cytotoxic effect of cisplatin. While, targeting MALAT1 in NSCLC cells induces DNA damages by repressing HR pathway and sensitizes NSCLC cells to cisplatin which had the potential for NSCLC treatment. MALAT1 is involved in HR pathway by protecting BRCA1 and targeting MALAT1 induces DNA damages in NSCLC.

Effects of germline and somatic events in candidate BRCA-like genes on breast-tumor signatures.

Mutations in BRCA1 and BRCA2 cause deficiencies in homologous recombination repair (HR), resulting in repair of DNA double-strand breaks by the alternative non-homologous end-joining pathway, which is more error prone. HR deficiency of breast tumors is important because it is associated with better responses to platinum salt therapies and PARP inhibitors. Among other consequences of HR deficiency are characteristic somatic-mutation signatures and gene-expression patterns. The term "BRCA-like" (or "BRCAness") describes tumors that harbor an HR defect but have no detectable germline mutation in BRCA1 or BRCA2. A better understanding of the genes and molecular events associated with tumors being BRCA-like could provide mechanistic insights and guide development of targeted treatments. Using data from The Cancer Genome Atlas (TCGA) for 1101 breast-cancer patients, we identified individuals with a germline mutation, somatic mutation, homozygous deletion, and/or hypermethylation event in BRCA1, BRCA2, and 59 other cancer-predisposition genes. Based on the assumption that BRCA-like events would have similar downstream effects on tumor biology as BRCA1/BRCA2 germline mutations, we quantified these effects based on somatic-mutation signatures and gene-expression profiles. We reduced the dimensionality of the somatic-mutation signatures and expression data and used a statistical resampling approach to quantify similarities among patients who had a BRCA1/BRCA2 germline mutation, another type of aberration in BRCA1 or BRCA2, or any type of aberration in one of the other genes. Somatic-mutation signatures of tumors having a non-germline aberration in BRCA1/BRCA2 (n = 80) were generally similar to each other and to tumors from BRCA1/BRCA2 germline carriers (n = 44). Additionally, somatic-mutation signatures of tumors with germline or somatic events in ATR (n = 16) and BARD1 (n = 8) showed high similarity to tumors from BRCA1/BRCA2 carriers. Other genes (CDKN2A, CTNNA1, PALB2, PALLD, PRSS1, SDHC) also showed high similarity but only for a small number of events or for a single event type. Tumors with germline mutations or hypermethylation of BRCA1 had relatively similar gene-expression profiles and overlapped considerably with the Basal-like subtype; but the transcriptional effects of the other events lacked consistency. Our findings confirm previously known relationships between molecular signatures and germline or somatic events in BRCA1/BRCA2. Our methodology represents an objective way to identify genes that have similar downstream effects on molecular signatures when mutated, deleted, or hypermethylated.

The Impact of Single- and Double-Strand DNA Breaks in Human Spermatozoa on Assisted Reproduction.

Several cellular insults can result in sperm DNA fragmentation either on one or both DNA strands. Oxidative damage, premature interruption of the apoptotic process and defects in DNA compaction during spermatogenesis are the main mechanisms that cause DNA breaks in sperm. The two-tailed Comet assay is the only technique that can differentiate single- (SSBs) from double- (DSBs) strand DNA breaks. Increased levels of the phosphorylated isoform of the H2AX histone are directly correlated with DSBs and proposed as a molecular biomarker of DSBs. We have carried out a narrative review on the etiologies associated with SSBs and DSBs in sperm DNA, their association with reproductive outcomes and the mechanisms involved in their repair. Evidence suggests a stronger negative impact of DSBs on reproductive outcomes (fertilization, implantation, miscarriage, pregnancy, and live birth rates) than SSBs, which can be partially overcome by using intracytoplasmic sperm injection (ICSI). In sperm, SSBs are irreversible, whereas DSBs can be repaired by homologous recombination, non-homologous end joining (NHEJ) and alternative NHEJ pathways. Although few studies have been published, further research is warranted to provide a better understanding of the differential effects of sperm SSBs and DSBs on reproductive outcomes as well as the prognostic relevance of DNA breaks discrimination in clinical practice.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

Exploiting MYC-induced PARPness to target genomic instability in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of the alternative nonhomologous end joining pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by olaparib impaired MM cell viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1 inhibition were correlated with an increase of DNA double-strand breaks, activation of the DNA damage response and apoptosis. Importantly, by comparing a gene expression signature of PARP-inhibitor sensitivity to our plasma cell dyscrasia gene expression profiling, we identified a subset of MM patients who could benefit from PARP inhibitors. In particular, gene set enrichment analysis suggested that high MYC expression correlates with sensitivity to PARP inhibitors in MM. Indeed, we identified MYC as a promoter of PARP1-mediated repair in MM and, consistently, we demonstrated that cytotoxic effects induced by PARP inhibition are mostly detectable in MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 alternative non-homologous end joining repair, which therefore represents a druggable target in this still incurable disease.

Altered DNA ligase activity in human disease.

The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

Both the classical and alternative non-homologous end joining pathways contribute to the fusion of drastically shortened telomeres induced by TRF2 overexpression

ABSTRACTThe double-stranded telomeric binding protein TRF2 is expressed in many human cancers at elevated levels. Moreover, experimental overexpression of TRF2 in human cells causes replication stalling in telomeric tracts, which leads to drastic telomere shortening and fusion of deprotected chromosome ends. To understand which end joining pathway is involved in mediating these chromosome fusions, we overexpressed TRF2 in human HCT116 cell lines that were deficient for the DNA Ligase 4 (Lig4)-dependent classical non-homologous end joining (C-NHEJ) or the DNA Ligase 3 (Lig3)-dependent alternative non-homologous end joining (A-NHEJ) pathway. Surprisingly, abrogation of either Lig4 or nuclear Lig3 significantly reduced inter-chromosomal fusion of drastically shortened telomeres, suggesting that both the C-NHEJ and A-NHEJ pathways are involved in mediating this type of fusion. Fusion between deprotected sister chromatids, however, only required the Lig3-dependent A-NHEJ pathway. Interestingly, a previous study reported similar end joining pathway requirements for the fusion of critically shortened telomeres during a telomere attrition-based cellular crisis. We speculate that, as in cellular crisis, the same repair pathway(s) may drive clonal and genomic evolution in human cancers containing elevated TRF2 levels.

Lack of associations between LIG3 gene polymorphisms and neuroblastoma susceptibility in Chinese children.

Accumulating evidence suggests that dysregulation of the DNA non-homologous end-joining (NHEJ) repair system is a causative factor in many cancers, including high-risk neuroblastoma. A number of studies have shown that polymorphisms in the DNA ligase III (LIG3) gene, one of the key genes in the error-prone alternative NHEJ (a-NHEJ) pathway for DNA double-strand break (DSB) repair, are associated with a variety of cancers. Nevertheless, whether LIG3 polymorphisms contribute to neuroblastoma risk remains unknown. We investigated the correlation between neuroblastoma susceptibility and two LIG3 polymorphisms (rs1052536 C>T and rs4796030 A>C) among 469 neuroblastoma patients and 998 healthy controls from China. Our results failed to detect any relationship between the analyzed SNPs and neuroblastoma risk in either overall analysis or stratification analysis. These results suggest that rs1052536 C>T and rs4796030 A>C are unrelated to neuroblastoma susceptibility in the Chinese population. Further studies with larger sample sizes and multiple ethnicities are necessary to verify our results.

Enhancement of Radiation Effectiveness in Cervical Cancer Cells by Combining Ionizing Radiation with Hyperthermia and Molecular Targeting Agents.

Hyperthermia (HT) and molecular targeting agents can be used to enhance the effect of radiotherapy (RT). The purpose of this paper is to evaluate radiation sensitization by HT and different molecular targeting agents (Poly [ADP-ribose] polymerase 1 inhibitor, PARP1-i; DNA-dependent protein kinase catalytic subunit inhibitor, DNA-PKcs-i and Heat Shock Protein 90 inhibitor, HSP90-i) in cervical cancer cell lines. Survival curves of SiHa and HeLa cells, concerning the combined effects of radiation with hyperthermia and PARP1-i, DNA-PKcs-i or HSP90-i, were analyzed using the linear-quadratic model: S(D)/S(0) = exp − (αD + βD2). The values of the linear-quadratic (LQ) parameters α and β, determine the effectiveness at low and high doses, respectively. The effects of these sensitizing agents on the LQ parameters are compared to evaluate dose-dependent differences in radio enhancement. Combination of radiation with hyperthermia, PARP1-i and DNA-PKcs-i significantly increased the value of the linear parameter α. Both α and β were significantly increased for HSP90-i combined with hyperthermia in HeLa cells, though not in SiHa cells. The Homologous Recombination pathway is inhibited by hyperthermia. When hyperthermia is combined with DNA-PKcs-i and PARP1-i, the Non-Homologous End Joining or Alternative Non-Homologous End Joining pathway is also inhibited, leading to a more potent radio enhancement. The observed increments of the α value imply that significant radio enhancement is obtained at clinically-used radiotherapy doses. Furthermore, the sensitizing effects of hyperthermia can be even further enhanced when combined with other molecular targeting agents.

Doxorubicin and vincristine affect undifferentiated rat spermatogonia.

Anticancer drugs, such as alkylating agents, can affect male fertility by targeting the DNA of proliferative spermatogonial stem cells (SSC). Therefore, to reduce such side effects, other chemotherapeutics are used. However, less is known about their potential genotoxicity on SSC. Moreover, DNA repair mechanisms in SSC are poorly understood. To model treatments deprived of alkylating agents that are commonly used in cancer treatment, we tested the impact of exposure to doxorubicin and vincristine, alone or in combination (MIX), on a rat spermatogonial cell line with SSC characteristics (GC-6spg). Vincristine alone induced a cell cycle arrest and cell death without genotoxic impact. On the other hand, doxorubicin and the MIX induced a dose-dependent cell death. More importantly, doxorubicin and the MIX induced DNA breaks, measured by the COMET assay, at a non-cytotoxic dose. To elucidate which DNA repair pathway is activated in spermatogonia after exposure to doxorubicin, we screened the expression of 75 genes implicated in DNA repair. Interestingly, all were expressed constitutively in GC-6spg, suggesting great potential to respond to genotoxic stress. Doxorubicin treatments affected the expression of 16 genes (>1.5 fold change; P < 0.05) involved in cell cycle, base/nucleotide excision repair, homologous recombination and non-homologous end joining (NHEJ). The significant increase in CDKN1A and XRCC1 suggest a cell cycle arrest and implies an alternative NHEJ pathway in response to doxorubicin-induced DNA breaks. Together, our results support the idea that undifferentiated spermatogonia have the ability to respond to DNA injury from chemotherapeutic compounds and escape DNA break accumulation.

Linear Energy Transfer-Dependent Change in Rice Gene Expression Profile after Heavy-Ion Beam Irradiation.

A heavy-ion beam has been recognized as an effective mutagen for plant breeding and applied to the many kinds of crops including rice. In contrast with X-ray or γ-ray, the heavy-ion beam is characterized by a high linear energy transfer (LET). LET is an important factor affecting several aspects of the irradiation effect, e.g. cell survival and mutation frequency, making the heavy-ion beam an effective mutagen. To study the mechanisms behind LET-dependent effects, expression profiling was performed after heavy-ion beam irradiation of imbibed rice seeds. Array-based experiments at three time points (0.5, 1, 2 h after the irradiation) revealed that the number of up- or down-regulated genes was highest 2 h after irradiation. Array-based experiments with four different LETs at 2 h after irradiation identified LET-independent regulated genes that were up/down-regulated regardless of the value of LET; LET–dependent regulated genes, whose expression level increased with the rise of LET value, were also identified. Gene ontology (GO) analysis of LET-independent up-regulated genes showed that some GO terms were commonly enriched, both 2 hours and 3 weeks after irradiation. GO terms enriched in LET-dependent regulated genes implied that some factor regulates genes that have kinase activity or DNA-binding activity in cooperation with the ATM gene. Of the LET-dependent up-regulated genes, OsPARP3 and OsPCNA were identified, which are involved in DNA repair pathways. This indicates that the Ku-independent alternative non-homologous end-joining pathway may contribute to repairing complex DNA legions induced by high-LET irradiation. These findings may clarify various LET-dependent responses in rice.

Abstract 3713: Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles

Abstract Heavy ion therapy is a promising approach for cancer treatment, particularly for locally advanced solid tumors. High linear energy transfer (LET) radiation induces greater lesions density in close proximity (clusters) compared that in low-LET radiation. Such clustered DNA damage is thought to be more difficult to repair than isolated DNA damage produced by equivalent doses of low-LET radiation and persist longer in irradiated cells. It has been known that homologous combination contributes to the repair of clustered DNA damage other than classic non-homologous end-joining (NHEJ). DNA double-strand breaks are also repaired by an alternative NHEJ pathway with PARP1, DNA ligase III and Mre11. This alternative pathway is error prone and contributes to genomic instability, such as chromosomal translocation and telomere fusion. Genetic analysis of tumors in TCGA database revealed that these genes are amplified in human tumors and their overexpression is correlated with poor overall survival of cancer patients. However, influence of alternative NHEJ pathways on the relative biological effectiveness (RBE) of heavy ions remains unclear. Here we investigated the effect of PARP-1 and DNA ligase III on RBE of heavy ion particles on cell survival and chromosomal aberration in breast cancer cells and pancreatic cancer cells. Cells were irradiated with X-ray, Carbon (290 MeV/n, 13 KeV/μM) and Iron (1 GeV/n, 150 KeV/μM). We found that overexpression of PARP-1 and DNA ligase III increased RBE of carbon and iron. PARP inhibitor reduced chromosomal aberration induced by heavy ions. This study may provide predictor of radiation responsive of tumor cells from cancer patients that would lead to patient stratification for heavy ion therapy selection Citation Format: Huichen Wang, Premkuma Saganti. Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3713.

Deciphering relationship between microhomology and in-frame mutation occurrence in human CRISPR-based gene knockout.

To the Editor: CRISPR-based gene editing is widely implemented in various cell types and has great potential for molecular therapy.1 The CRISPR-Cas9 system creates sequence-specific double-strand DNA breaks that are repaired by a dominant error-prone nonhomologous end-joining (NHEJ) pathway, often resulting in gene inactivation by generating frameshift alleles.1,2,3,4,5,6,7 CRISPR-based gene knockout (KO) often produces in-frame variants that retain functionality, however, which reduces KO efficiency. Recently, Sangsu Bae et al pioneered studies to use microhomology prediction to improve CRISPR-based KO efficiency in cell lines, by in-silico selection of target sites to reduce in-frame mutations.2 They presented that the preference of the in-frame mutations at a given target site can be predicted by the microhomology profile, and an alternative NHEJ pathway, i.e., the microhomology-mediated end joining (MMEJ) occurs.2,3 A score was defined to predict the microhomology-based out-of-frame mutation preferences.2,4

Abstract 2854: Targeting error-prone alternative nonhomologous end joining (A-NHEJ) DNA repair pathway to treat ESR1-mediated therapy resistance

Abstract Endocrine therapies for estrogen receptor (ESR1) positive breast cancer involve modulation of ESR1 signaling using either antiestrogens (AE) or aromatase inhibitors (AI). Despite initial positive response, most patients develop resistance to these drugs and therapy resistance is a major clinical problem. Emerging findings suggest that during the progression of ESR1 positive breast cancer, the cancer cell increasingly becomes dependent on the error-prone alternative-non-homologous end joining (A-NHEJ) DNA repair pathway for double strand break (DSB) repair leading to an increase in chromosomal abnormalities contributing to hormonal therapy resistance. The mechanism by which ESR1 pathway regulates A-NHEJ remains unknown. Recent studies from our lab discovered that ESR1 coregulator Proline-, Glutamic acid-, and Leucine-rich Protein-1 (PELP1) is phosphorylated by the DNA damage response kinases and plays a key role in the A-NHEJ pathway. In this study, we dissected the mechanism by which PELP1 regulates the A-NHEJ pathway and developed a novel inhibitor of PELP1-A-NHEJ axis. Using plasmid based repair assay, we provided evidence that PELP1 regulates degree of end-resection that is essential for A-NHEJ pathway mediated DNA repair. Immunoprecipitation assays revealed that PELP1 interacts with exonuclease protein, Mre11 that plays an essential role in A-NHEJ pathway. Using PELP1 domain deletions and GST pull down assays, we identified C-terminus of PELP1 as the region that interacts with key components of the A-NHEJ pathway. We have rationally screened a random peptide library composed of 10 million peptides, and identified a tight-binding peptide to the C-terminus of PELP1 that interferes with PELP1- Mre11 interaction. Using emerging stapled peptide technology, we converted this peptide to a stable cell permeable Peptide Inhibitor of PELP1 (sPIP3). sPIP3 bound to PELP1 with high affinity, interfered PELP1-Mre11 interaction, reduced A-NHEJ repair frequency in reporter based assays and significantly reduced frequency of chromosomal translocation. In MTT based assays, sPIP3 demonstrated significant cytotoxic effect on breast cancer cells with minimal effect on normal cells. Further, sPIP3 significantly reduced survival and promoted apoptosis of hormonal therapy resistant breast cancer model cells. Collectively, these findings demonstrate the therapeutic potential of sPIP3 which represents a novel drug for the treatment of therapy resistant breast cancer by inhibiting error prone A-NHEJ pathway. Citation Format: Samaya Krishnan, Binoj Nair, Gangadhara Sareddy, Monica Mann, Ratna K. Vadlamudi. Targeting error-prone alternative nonhomologous end joining (A-NHEJ) DNA repair pathway to treat ESR1-mediated therapy resistance. [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 2854. doi:10.1158/1538-7445.AM2015-2854

Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4-Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.