Non-homologous End Joining Repair Pathway Research Articles

Inhibition of SETMAR-H3K36me2-NHEJ repair axis in residual disease cells prevents glioblastoma recurrence.

Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell-specific survival mechanism essential for GBM relapse. Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

Long noncoding RNA lnc-RI regulates DNA damage repair and radiation sensitivity of CRC cells through NHEJ pathway.

A percentage of colorectal cancer (CRC) patients display low sensitivity to radiotherapy, which affects its therapeutic effect. Cancer cells DNA double-strand breaks (DSBs) repair capacity is crucial for radiosensitivity, but the roles of long noncoding RNAs (lncRNAs) in this process are largely uncharacterized. This study aims to explore whether lnc-RI regulates CRC cell growth and radiosensitivity by regulating the nonhomologous end-joining (NHEJ) repair pathway. CRC cells in which lnc-RI has been silenced showed lower cell growth and higher apoptosis rates due to increased DSBs and cell cycle arrest. We found that miR-4727-5p targets both lnc-RI and LIG4 mRNA and inhibit their expression. CRC cells showed increased radiosensitivity when lnc-RI was silenced. These results reveal novel roles for lnc-RI in both DNA damage repair and radiosensitivity regulation in CRC cells. Our study revealed that lnc-RI regulates LIG4 expression through lnc-RI/miR-4727-5p/LIG4 axis and regulates NHEJ repair efficiency to participate in DNA damage repair. The level of lnc-RI was negatively correlated with the radiosensitivity of CRC cells, indicates that lnc-RI may be a potential target for CRC therapy. We also present the first report of the function of miR-4727-5p.

Positive newborn screen: a case of a novel variant in DCLRE1C in a patient with SCID

Background: Artemis enzyme, encoded by the DCLRE1C gene, is essential to V(D)J recombination in both T and B lymphocytes. Artemis functions as an important component of the nonhomologous end-joining DNA double-strand break repair pathway. Artemis deficiency leads to a T-B-NK+ severe combined immune deficiency (SCID) associated with radiosensitivity. Clinical presentation: We present a case of a positive newborn screen for SCID in a patient who was subsequently shown to have a T-B-NK+ phenotype. Further immune evaluation showed profound T and B lymphopenia, near-absent response to mitogen stimulation, and absent immunoglobulins A and M. Genetic investigation demonstrated a novel and putative pathogenic variant in the DCLRE1C gene. Conclusion: This case identifies a novel variant in the DCLRE1C gene in a patient with SCID identified by newborn screening. Statement of novelty: This case report identifies a novel variant in the DCLRE1C gene in a patient with T-B-NK+ SCID.

Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex

Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.

A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis.

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway.

Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, and the development of radiosensitizers is a current key challenge in radiobiology to maximize RT efficiency. In this study, the radiosensitizing effect of a natural compound from the withanolide family, withanolide D (WD), was assessed. Clonogenic assays showed that a 1 h WD pretreatment (0.7 μM) before irradiation decreased the surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieved its radiosensitizing effect, we then assessed whether WD could promote radiation-induced DNA damages and inhibit double-strand breaks (DSBs) repair in SKOV3 cells. Comet and γH2AX/53BP1 foci formation assays confirmed that DSBs were higher between 1 and 24 h after 2 Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induced the persistence of radiation-induced DNA damages. Immunoblotting was then performed to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PKc, ATM, and their phosphorylated forms appeared to be inhibited 24 h post-irradiation in WD-treated samples. XRCC4 expression was also down-regulated while RAD51 expression did not change compared to vehicle-treated cells suggesting that only non-homologous end joining (NHEJ) pathways was inhibited by WD. Mitotic catastrophe (MC) was then investigated in SKOV3, a p53-deficient cell line, to assess the consequence of such inhibition. MC was induced after irradiation and was predominant in WD-treated samples as shown by the few numbers of cells pursuing into anaphase and the increased amount of bipolar metaphasic cells. Together, these data demonstrated that WD could be a promising radiosensitizer candidate for RT by inhibiting NHEJ pathway and promoting MC. Additional studies are required to better understand its efficiency and mechanism of action in more relevant clinical models.

Abstract A095: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku

Abstract Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked. The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of moieties and optimized pharmacophore for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand break inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway. We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement. Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions. Citation Format: John Turchi, Katherine S Pawelczak, Navnath Gavande, Pamela S VanderVere-Carozza. Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A095. doi:10.1158/1535-7163.TARG-19-A095

ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining

Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients’ stratification and for the development of personalised therapeutic strategies.

Association of a Pathway-Specific Genetic Risk Score With Risk of Radiation-Associated Contralateral Breast Cancer

Radiation therapy for breast cancer is associated with increased risk of a second primary contralateral breast cancer, but the genetic factors modifying this association are not well understood. To determine whether a genetic risk score comprising single nucleotide polymorphisms in the nonhomologous end-joining DNA repair pathway is associated with radiation-associated contralateral breast cancer. This case-control study included a case group of women with contralateral breast cancer that was diagnosed at least 1 year after a first primary breast cancer who were individually matched to a control group of women with unilateral breast cancer. Inclusion criteria were receiving a first invasive breast cancer diagnosis prior to age 55 years between 1985 and 2008. Women were recruited through 8 population-based cancer registries in the United States, Canada, and Denmark as part of the Women's Environment, Cancer, and Radiation Epidemiology Studies I (November 2000 to August 2004) and II (March 2010 to December 2012). Data analysis was conducted from July 2017 to August 2019. Stray radiation dose to the contralateral breast during radiation therapy for the first breast cancer. A novel genetic risk score comprised of genetic variants in the nonhomologous end-joining DNA repair pathway was considered the potential effect modifier, dichotomized as high risk if the score was above the median of 74 and low risk if the score was at or below the median. The main outcome was risk of contralateral breast cancer associated with stray radiation dose stratified by genetic risk score, age, and latency. A total of 5953 women were approached for study participation, and 3732 women (62.7%) agreed to participate. The median (range) age at first diagnosis was 46 (23-54) years. After 5 years of latency or more, among women who received the first diagnosis when they were younger than 40 years, exposure to 1.0 Gy (to convert to rad, multiply by 100) or more of stray radiation was associated with a 2-fold increased risk of contralateral breast cancer compared with women who were not exposed (rate ratio, 2.0 [95% CI, 1.1-3.6]). The risk was higher among women with a genetic risk score above the median (rate ratio, 3.0 [95% CI, 1.1-8.1]), and there was no association among women with a genetic risk score below the median (rate ratio, 1.3 [95% CI, 0.5-3.7]). Among younger women with a high genetic risk score, the attributable increased risk for contralateral breast cancer associated with stray radiation dose was 28%. This study found an increased risk of contralateral breast cancer that was attributable to stray radiation exposure among women with a high genetic risk score and who received a first breast cancer diagnosis when they were younger than 40 years after 5 years or more of latency. This genetic risk score may help guide treatment and surveillance for women with breast cancer.

An unorthodox partnership in DNA repair pathway choice.

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng etal [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

Nickel Carcinogenesis Mechanism: DNA Damage.

Nickel (Ni) is known to be a major carcinogenic heavy metal. Occupational and environmental exposure to Ni has been implicated in human lung and nasal cancers. Currently, the molecular mechanisms of Ni carcinogenicity remain unclear, but studies have shown that Ni-caused DNA damage is an important carcinogenic mechanism. Therefore, we conducted a literature search of DNA damage associated with Ni exposure and summarized known Ni-caused DNA damage effects. In vitro and vivo studies demonstrated that Ni can induce DNA damage through direct DNA binding and reactive oxygen species (ROS) stimulation. Ni can also repress the DNA damage repair systems, including direct reversal, nucleotide repair (NER), base excision repair (BER), mismatch repair (MMR), homologous-recombination repair (HR), and nonhomologous end-joining (NHEJ) repair pathways. The repression of DNA repair is through direct enzyme inhibition and the downregulation of DNA repair molecule expression. Up to now, the exact mechanisms of DNA damage caused by Ni and Ni compounds remain unclear. Revealing the mechanisms of DNA damage from Ni exposure may contribute to the development of preventive strategies in Ni carcinogenicity.

Evolution of CRISPR towards accurate and efficient mammal genome engineering

The evolution of genome editing technology based on CRISPR (clustered regularly interspaced short palindromic repeats) system has led to a paradigm shift in biological research. CRISPR/Cas9-guide RNA complexes enable rapid and efficient genome editing in mammalian cells. This system induces double-stranded DNA breaks (DSBs) at target sites and most DNA breakages induce mutations as small insertions or deletions (indels) by non-homologous end joining (NHEJ) repair pathway. However, for more precise correction as knock-in or replacement of DNA base pairs, using the homology-directed repair (HDR) pathway is essential. Until now, many trials have greatly enhanced knock-in or substitution efficiency by increasing HDR efficiency, or newly developed methods such as Base Editors (BEs). However, accuracy remains unsatisfactory. In this review, we summarize studies to overcome the limitations of HDR using the CRISPR system and discuss future direction.

Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.

Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-β (Pol-β), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-β and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-β complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-β knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-β and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.

Abstract 1301: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku

Abstract Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked. The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of pharmacophores and moieties for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand beaks inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway. We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement. Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions. Citation Format: John J. Turchi, Pamela S. VanderVere-Carozza, Navnath S. Gavande, Katherine S. Pawelczak. Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1301.

Abstract 2923: DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy

Abstract A novel, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks the non-homologous end-joining pathway for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We have previously shown that M3814 boosts ATM/p53 signaling and reinforces cell cycle checkpoint controls in irradiated p53 wild-type cancer cells, inducing irreversible premature senescence. However, in the absence of functional p53 this protective mechanism is disrupted, cancer cells progress through the cell cycle with unrepaired DSBs, leading to aberrant mitotic division and ultimately cell death. Here, we investigated the mechanistic consequences of DNA-PK inhibition in p53-dysfunctional cancer cells exposed to IR. We found that cell cycle progression with persistent DSB damage, promoted by M3814, causes severe chromosome aberrations, misalignment, and mis-segregation. This leads to the formation of micronuclei after each abnormal mitotic division. Live cell imaging and immunofluorescence analysis revealed high numbers of γH2AX foci, marking unrepaired DSBs inside the micronuclei, and lamin-B1-deficient micronuclei lamina. Cyclic GMP-AMP synthase (cGAS) was found to colocalize with micronuclei, suggesting that compromised laminar integrity exposes damaged DNA to the cytosol and activates the STING pathway. Gene expression analyses revealed activation of over 60 immune-related genes in cells exposed to IR+M3814, consistent with a STING-driven inflammatory response. Indeed, many cyto-/chemokines with immunomodulatory properties were detected in culture media from IR+M3814 treated cells. In addition, elevated expression of PD-L1 protein was found in irradiated p53-deficient cancer cells exposed to M3814. Together, these results provided a clear rationale for combination with PD-L1 inhibitory therapy. Murine p53-mutant cancer cells, MC38, were grown subcutaneously bilaterally in syngeneic mice. One of the tumors was exposed to 5 x 3.6 Gy daily IR fractions given locally and daily oral M3814 at previously established efficacious doses, followed by three applications (every 3-4 days) of the PD-L1 inhibitory antibody, avelumab. The other tumors were left unirradiated to monitor for abscopal effect. Study results demonstrated a superior benefit of adding M3814 to the IR/avelumab combination, including enhancement of the abscopal effect of IR/avelumab combination. Altogether, our results revealed that selective DNA-PK inhibition by M3814 provides a powerful new mechanism for induction of micronucleation and STING inflammatory signaling in p53-disfunctional cancer cells and offers a new approach to combination radio-immunotherapy of cancer. Citation Format: Michael Carr, Astrid Zimmermann, Yige Guo, Xiaohong Liu, Patrick Steiner, Susanne Hahn, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2923.

Abstract 4302: AR signaling promotes prostate carcinogenesis

Abstract Prostate cancer (PCa) is the most common non-skin cancer in American men. Each year, about 164,690 new patients will be diagnosed and about 29,430 men will die from this disease in 2018. Studies have identified several factors that increase the risk of PCa, including age, race, family history, and some diet factors. However, the exact cause of PCa remains unclear. Prostate is an androgen dependent organ and the growth of PCa also dependents on androgen and AR signaling. It is well established that AR signaling is the central player in promoting PCa progression. Here, we explore if AR signaling also plays a role in prostate carcinogenesis. We found that activation of AR signaling in normal prostate epithelial cells suppressed their cell proliferation in vitro but induced carcinogenesis in vivo. To explore the mechanisms that contribute to the differential effects of AR in vitro and in vivo, we examined if AR signaling induces genome instability in prostate epithelial cells, resulting in carcinogenesis and tumor progression. Senescence-associated beta-galactosidase staining results indicated that androgen treatment induced cellular senescence of prostate epithelial cells. Also, we found that the cells that survived the cellular senescence had altered genome ploidy and increased expression of active DNA-PK, suggesting that non-homologous end joining repair pathway is active in these cells. Our data support that AR signaling promotes genome instability in prostate epithelial cells, contributing to carcinogenesis. This may explain the high frequency of cancer developed in hormone-regulated organs such as prostate, mammary gland, and liver. Funding: This study was supported by: FWCC/Foundation Legacy Funds, DOD New Investigator Award, The Office of Research SEED awards, The Department of Biochemistry & Molecular Biology. Citation Format: Siyuan Cheng, Shu Yang, Zachary M. Connelly, Fenghua Chen, Xiuping Yu. AR signaling promotes prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4302.

CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.

EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells

Epstein-Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members, MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression of MLH1 correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.

Frequency of DNA end joining in trans is not determined by the predamage spatial proximity of double-strand breaks in yeast

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.