Major DNA Double-strand Breaks Repair Pathways Research Articles

New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement.

Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G2-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy.

Understanding the DNA double-strand break repair and its therapeutic implications

Repair of DNA double-strand breaks (DSBs) and its regulation are tightly integrated inside cells. Homologous recombination, nonhomologous end joining and microhomology mediated end joining are three major DSB repair pathways in mammalian cells. Targeting proteins associated with these repair pathways using small molecule inhibitors can prove effective in tumors, especially those with deregulated repair. Sensitization of cancer to current age therapy including radio and chemotherapy, using small molecule inhibitors is promising and warrant further development. Although several are under clinical trial, till date no repair inhibitor is approved for commercial use in cancer patients, with the exception of PARP inhibitors targeting single-strand break repair. Based on molecular profiling of repair proteins, better prognostic and therapeutic output can be achieved in patients. In the present review, we highlight the different mechanisms of DSB repair, chromatin dynamics to provide repair accessibility and modulation of inhibitors in association with molecular profiling and current gold standard treatment modalities for cancer.

The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

Abstract 5499: Cell cycle-dependent, comprehensive mathematical modeling of the role of DNA repair in response to radiotherapy for prostate cancer

Abstract The purpose of this study is to develop a comprehensive mathematical model of DNA double-strand break (DSB) repair in a cell cycle-dependent manner to analyze the improved radiosensitivity of prostate cancer (PCa) cells to ionizing radiation (IR) when radiation treatment is combined with androgen deprivation therapy (ADT). The effectiveness of the combination treatment, which is currently a standard treatment for PCa, depend on DSB repair capacity of the cells as these are the main lesions following IR. It is reported in the literature that the major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are both impaired after ADT, which then resulted in increased radiosensitivity and better IR treatment outcomes in PCa. In our previous work, we have developed quantitative models for NHEJ and HR individually to analyze the mechanism of the effect of ADT on IR treatment outcomes through impaired repair dynamics. The present work combines these two models in a cell cycle-dependent manner in order to develop a comprehensive model to analyze the repair dynamics after IR only and IR+ADT treatments. NHEJ is the major pathway, whereas HR is restricted to S- or G2-phases of the cell cycle after DNA replication has been completed and we have incorporated the cell cycle dependent contributions of the NHEJ and HR models in our comprehensive model. The literature data used in the development of these previous models were from both in vitro experiments as well as clinical data from PCa patients. In our comprehensive model, we have used the data from the literature to determine the distribution of the initial DSBs for cells in different cell cycle phases as the damage depends on the cell cycle phase at the time of radiation. We have incorporated the data on the percent contributions of NHEJ and HR repair in different cell cycle phases into the model and calculated the repair outcomes from NHEJ and HR models according to these ratios. Cell cycle arrest is implemented in relation to the amount of remaining DSBs in each cell cycle phase after repair. Using the number of unrepaired DSBs, we have calculated the proportion of cells that would progress to the next cell cycle phase as well as the proportion for which the cell death mechanism is triggered. The simulation results showed that ADT combined with IR has enhanced the treatment outcome. The cell survival rate was lower for the combination treatment case (55.7% compared to 62.1 % of IR only case). The cell proliferation was also significantly slower (85-95h doubling time compared to 35-45h of IR only case). The results agreed well with the experimental data that showed 30h and 95.5 h of doubling times for IR only and IR+ADT treatment respectively. The comprehensive model outcomes show that impaired NHEJ and HR dynamics as a result of ADT have the potential to enhance the IR treatment outcomes for PCa patients. Citation Format: Mengdi Qian, Alexandru Almasan, Evren Gurkan-Cavusoglu. Cell cycle-dependent, comprehensive mathematical modeling of the role of DNA repair in response to radiotherapy for prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5499.

Harnessing DNA Double-Strand Break Repair for Cancer Treatment.

DNA double-strand breaks (DSBs) are highly deleterious, with a single unrepaired DSB being sufficient to trigger cell death. Compared to healthy cells, cancer cells have a higher DSB burden due to oncogene-induced replication stress and acquired defects in DNA damage response (DDR) mechanisms. Consequently, hyperproliferating cancer cells rely on efficient DSB repair for their survival. Moreover, augmented DSB repair capacity is a major cause of radio- and chemoresistance and, ultimately, cancer recurrence. Although inherited DDR defects can predispose individuals to develop certain cancers, the very same vulnerability may be therapeutically exploited to preferentially kill tumor cells. A paradigm for DNA repair targeted therapy has emerged in cancers that exhibit mutations in BRCA1 or BRCA2 tumor suppressor genes, conferring a strong defect in homologous recombination, a major and error-free DSB repair pathway. Clinical validation of such approaches, commonly described as synthetic lethality (SL), has been provided by the regulatory approval of poly(ADP-ribose) polymerase 1 inhibitors (PARPi) as monotherapy for BRCA1/2-mutated breast and ovarian tumors. In this review, we will describe the different DSB repair mechanisms and discuss how their specific features could be exploited for cancer therapy. A major emphasis is put on advances in combinatorial treatment modalities and SL approaches arising from DSB repair pathway interdependencies.

Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells.

DNA double‐strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs), structural components [X‐ray repair cross‐complementing protein 4 (XRCC4), XRCC4‐like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia‐telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA‐PKcs, DNA‐PKcs/XRCC4, and DNA‐PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB‐inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA‐PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA‐PKcs. Moreover, ATM possesses overlapping functions with DNA‐PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ‐deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells.

Targeting DNA Double-Strand Break (DSB) Repair to Counteract Tumor Radio-resistance

During the last decade, advances of radiotherapy (RT) have been made in the clinical practice of cancer treatment. RT exerts its anticancer effect mainly via leading to the DNA Double-Strand Break (DSB), which is one of the most toxic DNA damages. Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) are two major DSB repair pathways in human cells. It is known that dysregulations of DSB repair elicit a predisposition to cancer and probably result in resistance to cancer therapies including RT. Therefore, targeting the DSB repair presents an attractive strategy to counteract radio-resistance. In this review, we describe the latest knowledge of the two DSB repair pathways, focusing on several key proteins contributing to the repair, such as DNA-PKcs, RAD51, MRN and PARP1. Most importantly, we discuss the possibility of overcoming radiation resistance by targeting these proteins for therapeutic inhibition. Recent tests of DSB repair inhibitors in the laboratory and their translations into clinical studies are also addressed.

Characterization of DNA double-strand break repair pathways in diffuse large B cell lymphoma.

Efficient DNA repair is indispensable for maintaining genomic integrity in humans. Cancer associated deletions and mutations are mainly due to misrepaired DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ) and homologous recombination (HR) are two major DSB repair pathways in humans. An error prone, alternative NHEJ pathway that utilizes microhomology was also reported in cancer cells and to a lesser extent in normal cells. In the present study, we evaluated the efficiency of various DSB repair pathways in the most common lymphoma, the diffuse large B cell lymphoma (DLBCL). Here we show that DNA repair through c-NHEJ pathway is limited in SUDHL8, a cell line derived from a DLBCL patient. Unlike c-NHEJ, microhomology mediated end joining (MMEJ) was predominant at physiological temperature. Consistent with the observation, expression level of repair proteins such as LIGASE I, LIGASE III, PARP1, CtIP, and MRE11 was higher in DLBCL cells when compared to c-NHEJ proteins. Further, inhibition of LIGASE I or MRE11, led to reduction in the efficiency of MMEJ in DLBCL cells. Besides, HR-mediated DSB repair occurring through gene conversion was observed. Thus, our results reveal the predominance of MMEJ over c-NHEJ in repairing DSBs in DLBCL cells, while error-free repair through HR was also evident.

Altered DNA repair; an early pathogenic pathway in Alzheimer\u2019s disease and obesity

Unrepaired DNA double-strand breaks (DSBs) are lethal. The present study compared the extent of DSBs, neuronal apoptosis, and status of two major DSB repair pathways - homologous combinational repair (HR) and nonhomologous end-joining (NHEJ) - in hippocampus of 5–6 month and 16–18 month-old wild-type and APP/PSEN1 mice fed control diet or high fat diet (60% fat from lard). We performed immunohistochemical staining and quantification for nuclear foci formation of γ-H2AX for DSBs, RAD51, and 53BP1, which represent the functional status of HR and NHEJ, respectively. Increased γ-H2AX and caspase-3 staining indicated greater DSBs and associated neuronal apoptosis in APP/PSEN1 mice at both ages studied. RAD51-positive foci were fewer in APP/PSEN1 indicating that HR processes may be diminished in these mice, although NHEJ (53BP1 staining) appeared unchanged. High fat diet in young wild-type mice led to similar changes to those observed in APP/PSEN1 mice (γ-H2AX and caspase-3 staining, and fewer RAD51-positive foci). Overall, these data suggest that APP/PSEN1- and high fat diet-associated early accumulation of DSBs and neuronal cell death, resulted at least in part, from inhibition of HR, one of the major DSB repair pathways.

Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.

Abstract 2478: PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers

Abstract PARP inhibitors (PARPi) have exhibited clinical success in inherited breast cancers with mutations in homologous recombination (HR) DNA double strand break (DSB) repair genes BRCA 1/2 by synthetic lethality. Novel PARPi have been shown to function not only by catalytic inhibition of PARP 1/2 activity, but also by “trapping” of PARP to sites of DNA damage. Trapping of PARP is a response generated by PARPi that is independent of the inhibition of PARP’s catalytic activity, and leads to lethal unrepaired DSBs during replication. Importantly, PARP trapping ability of PARPi correlates with its cytotoxic effect. In this study, we investigated whether PARP trapping has distinct effects on regulation of DNA DSB repair in breast cancer cells. Major DSB repair pathways in mammalian cells include HR and non-homologous end joining (c-NHEJ). Alternative NHEJ (ALT-NHEJ) is a recently discovered and highly error-prone pathway which requires DNA resection like HR, and utilizes PARP1. Our hypothesis is that PARP1 catalytic inhibition vs ‘trapping’ leads to distinct outcomes on DSB repair, highlighting inhibitor efficacy. To test our hypothesis, we utilized MCF10A (non-tumorigenic), MDA-MB-231 (TNBC/BRCA+), and SUM149PT (TNBC/BRCA-) breast cancer cell lines with stably integrated GFP-reporter plasmids designed to assess interchromosomal activities of the specific DSB repair pathways. For our analysis, we used varying concentrations of Talazoparib (MDV3800), which is a potent PARP trapping agent when used in combination with DNA damaging agents such as methyl methanesulfonate (MMS). First, we determined optimal conditions to induce PARP trapping in cells without leading to significant toxicity. As observed by others, combination treatments with of MMS/MDV3800 led to a significant increase in PARP trapping relative to treatments with individual drugs. Studies were also performed with a potent inhibitor of coenzyme NAD+ (Daporinad/FK866) that blocks poly-ADP ribosylation activity in combination of MMS. We observed significant PARP trapping with FK866/MMS combination treatment, indicating that PARP depletion under the conditions of DNA damage also leads to potent PARP trapping. Next, we investigated the effects of these inhibitors on DSB repair. Surprisingly, combination MDV3800/MMS lead to an overall decrease in HR and increase in ALT NHEJ activity. These results were observed in both our BRCA+/- lines. Similar results are observed between the repair activities when using FK866/MMS treatments. Overall, our results suggest that PARP inhibitors with potent PARP trapping activity increases ALT NHEJ, which may potentially compensate for lack of HR. Studies are ongoing to determine whether targeting ALT NHEJ may increase cytotoxicity in these cells. Citation Format: Bryan M. Pelkey, Pratik Nagaria, and Feyruz V. Rassool. PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers [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 2478. doi:10.1158/1538-7445.AM2017-2478

Abstract B10: The role of Down syndrome's DYRK1A kinase in repair of the DNA double strand breaks

Abstract The function of DYRK1A protein kinase is regulated by its gene dosage whereby both gains and losses of one copy of DYRK1A gene on chromosome 21 result in developmental abnormalities. In order to better understand the function and regulation of DYRK1A, we applied a highly sensitive MudPIT proteomic approach to identify DYRK1A-interacting proteins in human cells. Four biological replicate MudPIT experiments were performed and the proteins reproducibly detected in the DYRK1A immunoprecipitates but not in the controls, were identified. Six proteins detected in all four biological replicate experiments were also most highly enriched in the DYRK1A immunoprecipitates, suggesting that these proteins form stable and abundant complexes with DYRK1A. One of these proteins, RNF169, has been recently characterized as a component of ubiquitin-mediated cascade involved in the repair of DNA double-strand breaks (DSBs). DSBs are deleterious DNA lesions that are repaired by hierarchical and orchestrated recruitment of multiple different proteins to a modified chromatin in the vicinity of the DNA damage sites. Presence of specific chromatin marks including ubiquitination regulates the choice between two major DSB repair pathways: homologous recombination repair (HRR) and non-homologous end joining (NHEJ), mediated by recruitment of chromatin-binding DNA damage response proteins including 53BP1 and RNF169. Binding of 53BP1 could prevent the resection of the DNA strands near the damage site necessary for the HRR while RNF169 is thought to limit the 53BP1 accumulation and therefore, to promote the HRR. To determine whether DYRK1A plays a role in these processes, we knocked out its expression in human and mouse cell lines using CRISPR-Cas9 approach. We found that initial accumulation of 53BP1 at the gamma-irradiation induced foci (IRIFs) was similar in the DYRK1A-null and in the control cells. However, both the number of the 53BP1 IRIFs and their persistence over time were significantly reduced in the cell lines that lacked DYRK1A. This effect was dependent on the presence of RNF169, suggesting that DYRK1A regulates the ability of RNF169 to limit 53BP1's accumulation at the DSBs. Next, we sought to determine the mechanism of this regulation and found that RNF169 is phosphorylated by DYRK1A at two sites located in a highly conserved domain with no known function. Interestingly, the phospho mimetic mutant of RNF169 displayed a decreased ability to inhibit 53BP1 IRIF formation when compared to the wild type or the non-phosphorylatable RNF169 alleles. We also determined the effect of DYRK1A overexpression on the recruitment of RNF169 and 53BP1 to the sites of IR-induced DNA damage. Surprisingly, accumulation of both RNF169 and 53BP1 in the IRIFs was diminished upon overexpression of active, but not the kinase-inactive DYRK1A. Domain-mapping of DYRK1A-RNF169 binding demonstrated that the ability of DYRK1A to abolish the RNF169 IRIF formation is independent of their interaction, suggesting that overexpression of functional DYRK1A could affect other factors in the cell that play a role in the DNA damage response. Since loss of DYRK1A could be relevant to cancer due to its widespread gene copy number losses, we determined the effect of DYRK1A loss on the ability of the cells to repair their DNA. Using the DR-GFP reporter of HRR and the neutral comet assays, we found that CRISPR-Cas9-mediated depletion of DYRK1A results in an increased efficiency of the DNA DSB repair. Our findings implicate DYRK1A in the critical processes of DNA damage response and characterize a novel function of this important protein kinase. Citation Format: Vijay R. Menon, Varsha Ananthapadmanabhan, Larisa Litovchick. The role of Down syndrome's DYRK1A kinase in repair of the DNA double strand breaks [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 B10.

DNA double-strand break repair inPenaeus monodonis predominantly dependent on homologous recombination

DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

TAS-116, a Novel Hsp90 Inhibitor, Selectively Enhances Radiosensitivity of Human Cancer Cells to X-rays and Carbon Ion Radiation.

Hsp90 inhibitors have been investigated as cancer therapeutics in monotherapy and to augment radiotherapy; however, serious adverse effects of early-generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here, we investigated the radiosensitizing effects of TAS-116 in low linear energy transfer (LET) X-ray and high LET carbon ion-irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of noncancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci and delayed the repair of DNA double-strand breaks (DSB). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and nonhomologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell-cycle progression marker, markedly increasing G2-M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared with either individual treatment. These results demonstrate that TAS-116 radiosensitizes human cancer cells to both X-rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell-cycle arrest. The promising results of combination TAS-116 + carbon ion radiotherapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. Mol Cancer Ther; 16(1); 16-24. ©2016 AACR.

Abstract LB-164: DNA ligase IV modulates the cellular response to DNA replication stress

Abstract Correctly repairing DNA damage is crucial to the survival and genomic integrity of cells. Double strand breaks (DSBs) are particularly problematic DNA damage; failure to properly repair DSBs can precede genomic instability leading to cancer. Because of the high proliferative index of cancer cells, replication toxins are successfully used to treat cancer. The two major DNA DSB repair pathways are homologous recombination and nonhomologous end-joining (NHEJ). DNA ligase IV is a central component of NHEJ, performing the final ligation step to reseal the broken DNA ends, in concert with XRCC4. We determined cells lacking the early acting NHEJ protein, and DNA damage response signaling kinase, DNA-PK, restarted DNA replication forks quicker than wild-type following replication stress. The role of other NHEJ proteins in replication fork restart is unclear. Our goal was to investigate whether ligase IV, a protein that appears to function exclusively in NHEJ, is involved in the replication stress response. We tested the hypothesis that ligase IV delays replication restart following exposure to replication toxins. Ligase IV knockdown was confirmed by qPCR and western blotting. siRNA knockdown of ligase IV alone reduced cell viability and it reduced EdU incorporation after replication stress. The opposite effects of DNA-PK and ligase IV on the speed of replication fork restart suggests that NHEJ, per se, does not directly affect replication fork restart. We previously demonstrated that DNA-PK regulates replication fork restart, at least in part, by phosphorylating RPA32, which in turn regulates ATR activation and downstream checkpoint functions. Ligase IV deficiency may influence replication fork restart indirectly, for example, by causing spontaneous DSBs to persist longer, limiting availability of repair factors needed for fork restart (saturation effect). A not mutually exclusive possibility is that fork restart is hindered by persistent DSBs due to checkpoint activation. Understanding how NHEJ proteins impact the replication stress response may provide opportunities for developing more effective cancer treatments. Citation Format: Grace Hooks, Yasmin Anchondo, Neelam Sharma, Jac Nickoloff, Amanda Ashley. DNA ligase IV modulates the cellular response to DNA replication stress. [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 LB-164.

Abstract 1644: NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC

Abstract Background: Despite optimal multimodality therapy, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. A common limitation is our inability to provide sufficient radiotherapy (RT) to eradicate tumor due to risk of toxicities in surrounding tissues. There is thus an unmet need for radiosensitizers that can improve the therapeutic index. Dimethylaminoparthenolide (DMAPT), an orally bioavailable small molecule NF-κB inhibitor, inhibits repair of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) and increases control of subcutaneous mouse NSCLC xenografts. While our prior work focused on inhibition of homologous recombination as a mechanism for radiosensitization, we sought to characterize the effect of DMAPT on a second major DSB repair pathway, non-homologous end-joining (NHEJ). Methods: NHEJ was assessed in NSCLC lines using the pEJ reporter and flow cytometry. The NF-κB super repressor (IκBαS32A,S36A) was used as a control. Immunofluorescence and Western blotting were used to assess NHEJ biomarkers including 53BP1, DNA-PKS2056, Ku70/80 and XRCC4. Cell fractionation was performed to assess Ku chromatin binding. Quantitative RT-PCR was performed to assess gene transcription. Ku complexes were purified to identify binding partners. Results: NSCLC cells treated with IR-sensitizing doses of DMAPT (5-15 μM) or the NF-κB super repressor showed significant decreases in NHEJ. DMAPT increased the persistence of 53BP1 foci, indicating a failure to complete NHEJ. Regardless of exogenous DNA damage, there was reduced Ku70 chromatin binding following DMAPT treatment. DMAPT-treated cells produced fewer distinct IR-induced DNA-PKS2056 foci. Further, there was decreased IR-induced XRCC4 chromatin recruitment, suggesting that repair was impaired prior to ligation. There was no change in Ku70/80 transcription following DMAPT and/or IR. However, Western blotting of purified Ku complexes showed that DMAPT treatment decreased Ku association with RNA binding partners including RPL19. We also observed decreased recruitment of DNA-PKcs to the Ku complex, suggesting decreased Ku affinity for DSBs. Conclusions: These results indicate that DMAPT radiosensitizes NSCLC by perturbing the binding affinity of Ku to RNA, DNA and its complex binding partners, thus blocking NHEJ. Further mechanistic investigation and analysis of NHEJ biomarkers in vivo is needed to identify the precise mechanism. Citation Format: Peter V. Deraska, Colin O’Leary, Jean-Bernard Lazaro, Christopher J. Sweeney, Alan D. D’Andrea, David Kozono. NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC. [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 1644.

Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair.

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B(-/-)) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.

Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells

To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) – 2 major DSB repair pathways – holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment.

Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.

Pre-exposure to ionizing radiation stimulates DNA double strand break end resection, promoting the use of homologous recombination repair.

The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.