Non-homologous End-joining Process Research Articles

Abstract 4540: SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo

Abstract Cancer therapies such as ionizing radiation or topoisomerase II inhibitors (for example, doxorubicin) induce DNA double-strand breaks (DSBs), which can subsequently be repaired by homologous recombination or non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine protein kinase complex, is a pivotal component of the NHEJ process that maintain genome integrity. The critical role of DNA-PK in DNA damage response (DDR) and the dysregulated DNA-PK expression in cancer make it an intriguing therapeutic target for cancer, especially when combined with DSBs agents. Here, we identified a highly potent and selective DNA-PK inhibitor, SY-7021, with an IC50 value of 0.242 nM on DNA-PK kinase and high selectivity on ATM and ATR (greater than 400-fold) in biochemical assays. In a reporter assay for NHEJ repair, SY-7021 was able to dose-dependently reduce cellular NHEJ efficiency. Functionally, SY-7021 inhibited cell proliferation in multiple cancer cell lines alone or combined with doxorubicin. Basically, combination of SY-7021 and doxorubicin induced significant G2/M phase arrest and cell apoptosis, as well as enhanced the phosphorylation on Ser139 of γH2AX, Tyrosine 68 of CHK2 and Serine15 of p53 in MDA-MB-468 cells. In a subcutaneous NCI-H1703 xenograft tumor model, SY-7021 administered orally twice daily achieved dose-dependent tumor growth inhibition in vivo, with a tumor growth inhibition (TGI) of 105.6% at 60 mg/kg and no significant weight loss was observed. In addition, SY-7021 demonstrates good PK properties and acceptable safety profiles in in vivo studies. Collectively, SY-7021, a potent and selective DNA-PK inhibitor, shows significant inhibition on tumor growth in in vitro and in vivo studies, providing a rationale treatment for multiple tumors in monotherapy or in combination with other agents. Citation Format: Kai Zhang, Zhihua Liu, Yuhao Gao, Chang Lu, Shikang Cheng, Xijie Liu, Hong Luo, Yinghui Sun. SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo [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 4540.

FSP1 inhibition enhances olaparib sensitivity in BRCA-proficient ovarian cancer patients via a nonferroptosis mechanism

Poly ADP-ribose polymerase inhibitors (PARPis) exhibit promising efficacy in patients with BRCA mutations or homologous repair deficiency (HRD) in ovarian cancer (OC). However, less than 40% of patients have HRD, it is vital to expand the indications for PARPis in BRCA-proficient patients. Ferroptosis suppressor protein 1 (FSP1) is a key protein in a newly identified ferroptosis-protective mechanism that occurs in parallel with the GPX4-mediated pathway and is associated with chemoresistance in several cancers. Herein, FSP1 is reported to be negatively correlated with the prognosis in OC patients. Combination therapy comprising olaparib and iFSP1 (a FSP1 inhibitor) strongly inhibited tumour proliferation in BRCA-proficient OC cell lines, patient-derived organoids (PDOs) and xenograft mouse models. Surprisingly, the synergistic killing effect could not be reversed by ferroptosis inhibitors, indicating that mechanisms other than ferroptosis were responsible for the synergistic lethality. In addition, cotreatment was shown to induce increased γH2A.X foci and to impair nonhomologous end joining (NHEJ) activity to a greater extent than did any single drug. Mass spectrometry and immunoprecipitation analyses revealed that FSP1 interacted with Ku70, a classical component recruited to and occupying the end of double-strand breaks (DSBs) in the NHEJ process. FSP1 inhibition decreased Ku70 PARylation, impaired subsequent DNA-PKcs recruitment to the Ku complex at DSB sites and was rescued by restoring PARylation. These findings unprecedentedly reveal a novel role of FSP1 in DNA damage repair and provide new insights into how to sensitize OC patients to PARPi treatment.

Structure and mechanism in non-homologous end joining

DNA double-stranded breaks (DSBs) are a particularly challenging form of DNA damage to repair because the damaged DNA must not only undergo the chemical reactions responsible for returning it to its original state, but, additionally, the two free ends can become physically separated in the nucleus and must be bridged prior to repair. In nonhomologous end joining (NHEJ), one of the major pathways of DSB repair, repair is carried out by a number of repair factors capable of binding to and directly joining DNA ends. It has been unclear how these processes are carried out at a molecular level, owing in part to the lack of structural evidence describing the coordination of the NHEJ factors with each other and a DNA substrate. Advances in cryo-Electron Microscopy (cryo-EM), allowing for the structural characterization of large protein complexes that would be intractable using other techniques, have led to the visualization several key steps of the NHEJ process, which support a model of sequential assembly of repair factors at the DSB, followed by end-bridging mediated by protein-protein complexes and transition to full synapsis. Here we examine the structural evidence for these models, devoting particular attention to recent work identifying a new NHEJ intermediate state and incorporating new NHEJ factors into the general mechanism. We also discuss the evolving understanding of end-bridging mechanisms in NHEJ and DNA-PKcs’s role in mediating DSB repair.

Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.

Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs),incontrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.

Targetedmutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects.

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome-editing platforms employ sequence-specific nucleases (SSNs) to generate DNA double-stranded breaks at their target gene. Such DNA breaks are typically repaired via the error-prone nonhomologous end-joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end-use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN-mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.

Strong suppression of gene conversion with increasing DNA double-strand break load delimited by 53BP1 and RAD52.

In vertebrates, genomic DNA double-strand breaks (DSBs) are removed by non-homologous end-joining processes: classical non-homologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ); or by homology-dependent processes: gene-conversion (GC) and single-strand annealing (SSA). Surprisingly, these repair pathways are not real alternative options restoring genome integrity with equal efficiency, but show instead striking differences in speed, accuracy and cell-cycle-phase dependence. As a consequence, engagement of one pathway may be associated with processing-risks for the genome absent from another pathway. Characterization of engagement-parameters and their consequences is, therefore, essential for understanding effects on the genome of DSB-inducing agents, such as ionizing-radiation (IR). Here, by addressing pathway selection in G2-phase, we discover regulatory confinements in GC with consequences for SSA- and c-NHEJ-engagement. We show pronounced suppression of GC with increasing DSB-load that is not due to RAD51 availability and which is delimited but not defined by 53BP1 and RAD52. Strikingly, at low DSB-loads, GC repairs ∼50% of DSBs, whereas at high DSB-loads its contribution is undetectable. Notably, with increasing DSB-load and the associated suppression of GC, SSA gains ground, while alt-EJ is suppressed. These observations explain earlier, apparently contradictory results and advance our understanding of logic and mechanisms underpinning the wiring between DSB repair pathways.

Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

BackgroundThe mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis.ResultsIn the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ.ConclusionsOur findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed.

Roles of cell cycle phases and DNA end structures in determining requirements for the Yku, Mrx and Ligase IV complexes in nonhomologous end‐joining repair of plasmid DNA

Deoxyribonucleic acid (DNA) can be damaged through a variety of different damaging agents such as free radicals, radiation and environmental chemicals. Types of damage include single‐strand lesions such as base or sugar modifications and more deleterious lesions such as interstrand crosslinks and DNA double‐stranded breaks (DSBs). Unrepaired or misrepaired DSBs in a cell can lead to mutations and chromosome rearrangements, potentially leading to loss of cell function and even cell death. There are two independent pathways used to repair DSBs in DNA; these pathways are homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ involves the functions of three major protein complexes. In the budding yeast Saccharomyces cerevisiae, these assemblages include the Yku, Mrx and DNA ligase IV complexes. Each complex has a specific function. Yku (Yku70 and Yku80) protects the broken ends from degradation and recruits other repair proteins. Mrx, composed of Mre11, Rad50 and Xrs2, may act to tether the ends together and perform nuclease processing of the ends if necessary. The ligase IV complex, containing Dnl4 and Lif1 and possibly Nej1, performs the final step, ligating the two broken ends of DNA thus ending the NHEJ process.Repair of DSBs by NHEJ is frequently analyzed using plasmid‐based assays. In these experiments, a plasmid with a single DSB is transformed into cells and the efficiency of repair/recircularization inside cells is monitored. The region around the DSB site is designed so that it lacks homology with the host cell's chromosomal DNA to prevent repair by HR. An uncut plasmid is usually also transformed into the cells to normalize repair efficiencies to simple cell transformation efficiencies. Many past studies involving NHEJ repair using plasmid assays have observed that repair in yku, mrx, and dnl4 mutants is reduced similarly, typically 10–30 fold, suggesting equal importance of the three major complexes in the pathway. Using a high efficiency yeast transformation method involving treatment with dithiothreitol (DTT) and inclusion of a rich broth recovery step, we observed that repair of broken plasmids with 5′ single‐stranded DNA overhangs (4 nt long) was strongly reduced in yku mutants (yku70 or yku80) and DNA ligase IV mutants (dnl4, lif1, or nej1), but not in mrx strains (mre11, rad50 or xrs2). The accuracy of repair was also strikingly different among mrx cells vs. yku and dnl4 mutants. By contrast, efficiency and accuracy of repair of plasmids containing 3′ overhangs was reduced to similar levels in yku70, mre11 and dnl4 mutants. Since NHEJ repair efficiency is most active in G1 phase, we have also assessed the differential repair of broken plasmids in yku, mrx and dnl4 mutants in cultures with different proportions of G1 cells. These experiments have included mid‐log phase, early stationary phase, stationary phase and alpha factor‐arrested G1 phase cells. Work utilizing linear plasmids containing different end sequences and structures will be described.Support or Funding InformationNational institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4198: Highly potent and selective DNA-PK inhibitor M3814 with sustainable anti-tumor activity in combination with radiotherapy

Abstract Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely used classes of cancer therapeutics today. Double strand breaks (dsb) generated in DNA by radiation induce a multitude of cellular responses, including DNA repair, cell cycle arrest or cell death once the damage is left unrepaired. A complex set of molecular events are responsible for DNA repair mediated by two major repair mechanisms - homologous recombination (HR) or non-homologous end joining (NHEJ). DNA-PKcs together with its regulatory protein subunits Ku70 & Ku80, is an integral component of the NHEJ process and considered an attractive intervention point to inhibit DNA dsb repair. We have developed an orally bioavailable, highly potent & selective inhibitor of DNA-PK, M3814, for cancer therapy in combination with DNA damaging modalities such as radiation & radio-chemotherapy. Here, we present the optimization of initial hit matter & its structure-activity relationships leading to M3814, its chemical structure (first-time disclosure) & preclinical characterization using biochemical, cellular & human tumor xenograft models. M3814 sensitized multiple tumor cell lines to radiation therapy in vitro and strongly enhanced the anti-tumor activity of ionizing radiation in vivo with complete tumor regression by applying a clinically relevant fractionated radiation regimen. These effects are due to inhibition of DNA-PK protein kinase activity as demonstrated by the levels of DNA-PK autophosphorylation in human tumor cell lines & xenograft tumors. M3814 is currently being investigated in PhI/II clinical trials. Citation Format: Thomas Fuchss, Werner W. Mederski, Ulrich Emde, Hans-Peter Buchstallter, Frank Zenke, Astrid Zimmermann, Christian Sirrenberg, Lubo Vassilev, Lars Damstrup, Klaus Urbahns, Andree Blaukat. Highly potent and selective DNA-PK inhibitor M3814 with sustainable anti-tumor activity in combination with radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4198. doi:10.1158/1538-7445.AM2017-4198

Abstract 2492: DNA polymerase beta participates in DNA end-joining

Abstract DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA polymerase theta (Pol θ), an A-family polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family polymerases, mammalian DNA polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA polymerase beta (Pol β), another X-family polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair. Citation Format: Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. DNA polymerase beta participates in DNA end-joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2017-2492

Abstract A46: Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events

Abstract This study focuses on an unexpected role for particular nuclear pore proteins in the antagonism between the DNA repair factors BRCA1 and 53BP1, and the impact this has on the response to poly(A) ribose polymerase (PARP) inhibition. Repair of DNA double strand breaks, a cornerstone of genomic integrity, typically follows one of two distinct pathways: the high fidelity process of homologous recombination (HR) repair, in which BRCA1 plays a key role, and the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection and prevents HR from occurring. Conversely, BRCA1 binding counteracts 53BP1-mediated events to promote HR repair. While mutual antagonism between BRCA1 and 53BP1 has been appreciated for some time, factors that modulate this relationship are only beginning to be elucidated. The nuclear pore component Nup153 has previously been implicated in the DNA damage response, specifically in promoting nuclear import of 53BP1. To further explore the role of Nup153 in this context, we engineered an additional strong nuclear localization sequence (NLS) on 53BP1 and found that this overrides the requirement for Nup153 in nuclear import of 53BP1. While 53BP1-NLS is recruited normally to DNA damage foci in control-treated cells, its accumulation at these sites is still severely impaired in Nup153-depleted cells, indicating that Nup153 has an additional and distinct role in 53BP1 intranuclear targeting. These observations are further underscored by the additional finding that a second nuclear pore protein, Nup50, is also required for intranuclear targeting of 53BP1, yet makes no discernable contribution to 53BP1 nuclear import. Of note, depletion of a third nucleoporin (Tpr), which is part of the same structural element of the nuclear pore, did not alter this DNA damage response pathway. To gain insight into the pathway affected by Nup153 and Nup50 and to understand how their role fits into a clinically relevant context, we probed the consequences of reducing Nup153 and Nup50 levels in cells deficient in BRCA1 activity. We found that the requirement for Nup153 and Nup50 in promoting 53BP1 recruitment to damage foci is abrogated under these circumstances. In cells where HR repair is otherwise defective, however, robust recruitment of 53BP1 to DNA damage foci requires Nup153 and Nup50. Together, our results provide novel insight into 53BP1 regulation during the DNA damage response and are consistent with a model where Nup153 and Nup50 normally promote 53BP1 targeting by counteracting BRCA1-dependent events. These findings have important implications with regard to PARP inhibition, a therapeutic strategy thought to capitalize on defective HR repair by forcing inappropriate use of NHEJ, culminating in cellular toxicity. Accordingly, we found that decreased levels of Nup153 and Nup50 do not affect the 53BP1-dependent pathway in BRCA1-deficient cells exposed to PARP inhibitor treatment, but still impair this response in cells deficient for BRCA2 or when the ATM and ATR signaling pathways are inhibited. Our results indicate that Nup153 and Nup50, as factors that modulate 53BP1 activity, belong to a novel category of biomarkers that could be screened to make better predictions of response to PARP inhibitor treatment. Citation Format: Douglas R. Mackay, Katharine S. Ullman. Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A46.

Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events.

DNA double-strand breaks are typically repaired through either the high-fidelity process of homologous recombination (HR), in which BRCA1 plays a key role, or the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection. The nucleoporin Nup153 has been implicated in the DNA damage response, attributed to a role in promoting nuclear import of 53BP1. Here, we define a distinct requirement for Nup153 in 53BP1 intranuclear targeting to damage foci and report that Nup153 likely facilitates the role of another nucleoporin, Nup50, in 53BP1 targeting. The requirement for Nup153 and Nup50 in promoting 53BP1 recruitment to damage foci induced by either etoposide or olaparib is abrogated in cells deficient for BRCA1 or its partner BARD1, but not in cells deficient for BRCA2. Together, our results further highlight the antagonistic relationship between 53BP1 and BRCA1, and place Nup153 and Nup50 in a molecular pathway that regulates 53BP1 function by counteracting BRCA1-mediated events.

DNA-PK and P38 MAPK: A Kinase Collusion in Alzheimer's Disease?

The pathogenesis of Alzheimer's disease (AD), characterized by prevalent neuronal death and extracellular deposit of amyloid plaques, is poorly understood. DNA lesions downstream of reduced DNA repair ability have been reported in AD brains. Neurons predominantly use a mechanism to repair double-strand DNA breaks (DSB), which is non-homologous end joining (NHEJ). NHEJ requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK is a holoenzyme comprising the p460 kD catalytic subunit (DNA-PKcs) and its activator Ku, a heterodimer of p86 and p70 subunits. Ku first binds and then recruits DNA-PKcs to double-stranded DNA ends before NHEJ process begins. Studies have shown reduced NHEJ activity as well as DNA-PKcs and Ku protein levels in AD brains suggesting possible contribution of unrepaired DSB to AD development. However, normal aging brains also show reduced DNA-PKcs and Ku levels thus challenging the notion of any direct link between NHEJ and AD. Another kinase, p38 MAPK is induced by various DNA damaging agents and DSB itself. Increased DNA damage with aging could induce p38 MAPK and its induction may be sustained when DNA repair is compromised in the brain with reduced DNA-PK activity. Combined, these two events may potentially set the stage for an awry nervous system approaching AD.

A Life Investigating Pathways That Repair Broken Chromosomes.

Double-strand breaks (DSBs) pose a severe challenge to genome integrity; consequently, cells have developed efficient mechanisms to repair DSBs through several pathways of homologous recombination and other nonhomologous end-joining processes. Much of our understanding of these pathways has come from the analysis of site-specific DSBs created by the HO endonuclease in the budding yeast Saccharomyces cerevisiae. I was fortunate to get in on the ground floor of analyzing the fate of synchronously induced DSBs through the study of what I coined "in vivo biochemistry." I have had the remarkable good fortune to profit from the development of new techniques that have permitted an ever more detailed dissection of these repair mechanisms, which are described here.

Abstract 2749: A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80

Abstract Purpose: Non-Homologous End-Joining (NHEJ) is the primary pathway for the repair of DNA double strand breaks (DSBs) in human and mammalian cells. NHEJ is frequently upregulated in diverse solid malignancies and is a determining factor in the process of carcinogenesis. In addition, upregulation of the NHEJ pathway can reduce the efficiency of anti-cancer therapies including radiation or chemotherapy. Inhibition of NHEJ by siRNA reduces the growth of NHEJ-upregulated tumors and alleviates radiation-refractory disease. The Ku70/80 heterodimer is the central scaffolding protein of the NHEJ pathway, which recruits and activates all other key NHEJ factors. Therefore, Ku70/80 protein is a logical target for disruption of the NHEJ process. Experimental Procedures: We report the identification of a prospective binding pocket in the Ku70/80 crystal structure, located in close proximity to the DNA-binding domain and the Ku70/80 heterodimer interface. Based on this pocket, an idealized virtual ligand (‘protomol’) was created and utilized to screen ‘in silico’ an extensive database of small molecule compounds for potential ligands. Nine of the highest ranking hits were chosen for biological evaluation. Results: We identified one compound (designated ‘Compound L’), which proved capable of disrupting the in vitro binding of Ku70/80 to a DNA substrate in a dose-dependent manner with an IC50 of 3.5 μM. In addition, Compound L showed dose-dependent inhibition of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKCS) activity, which requires Ku70/80 as a co-factor with an IC50 of 2.5 μM. Finally, we showed that Compound L synergistically sensitizes a glioblastoma cell line to ionizing radiation, with a marked sensitization at ∼20-25 μM. This latter observation clearly demonstrates the ability of Compound L to disrupt NHEJ-mediated repair of radiation-induced DSBs. Conclusions: Compound L is an active inhibitor of the Ku70/80 heterodimer. To the best of our knowledge, no such compound has been reported in the peer-reviewed literature. The structure of Compound L will be utilized as a scaffold for a ‘structure-activity relationship’ (SAR) based hit-to-lead drug development aimed at designing a novel class of anti-cancer agents targeting the Ku70/80 heterodimer. We predict Ku70/80 inhibitors would have applicability as single-modality drugs, following a synthetic lethality approach, or as sensitizing agents for concurrent radiation or chemotherapy in a variety of solid malignancies. Support: P30 CA023074. Citation Format: Daruka Mahadevan, Alfred C. Gallegos, Lauren N. Dominick, Laurence S. Cooke, Trace N. Bartels, Josef Vagner, Terry O. Matsunaga, Eric Weterings. A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80. [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 2749.

A novel small molecule inhibitor of the DNA repair protein Ku70/80.

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.

Abstract A43: DNA repair by non-homologous end joining induced gene silencing via DNA hypermethylation

Abstract Double strand breaks (DSBs) in DNA are repaired throughout the cell cycle and in non-dividing cells by the error-prone process of non-homologous end joining (NHEJ). In order to evaluate expression of a gene following NHEJ repair, we use a GFP reporter system to allow for targeted induction of a DSB in the genome of Hela cells. Prior to induction of the DSB, GFP is transcribed but inactive due to an extra exon interrupting the gene sequence, however cells that successfully repair the DSB express WT GFP that can be detected by fluorescent based assays. Following repair, two different GFP expression classes can be detected by flow cytometry: one that expresses GFP robustly (‘bright') and one that expresses GFP at a lower level (‘dim'). This is surprising given that these experiments are done in a cell line derived from a single clone. This phenomenon was confirmed using live cell imaging. Treatment with a hypomethylating drug causes some ‘dim' cells to be converted to ‘bright' cells, suggesting that the gene silencing that occurs in the ‘dim' population is a result of hypermethylation of the gene. Bisulfite deep sequencing confirms that areas up- and downstream of the break site are hypomethylated in ‘bright' cell populations and hypermethylated in ‘dim' cell populations. It can be concluded from these data that, following repair of a DSB by NHEJ, the epigenetic landscape of the repaired gene, specifically the methylation status of its CpGs, is altered in a way that changes the expression levels of the repaired gene. Though their cause is not fully understood, gene expression changes caused by epigenetic reprogramming are a hallmark of many different types of cancer. Given the increased reliance of cancer cells on DNA repair, a link between epigenetic revision and DNA repair could provide an explanation for changes in the epigenome that occur during cancer progression. Citation Format: Brittany N. Allen, Michal Masternak, Mark Muller. DNA repair by non-homologous end joining induced gene silencing via DNA hypermethylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A43.

Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p<0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1–8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p<0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins.

Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.