Non-homologous End-joining Repair Research Articles

Tenascin C as a novel zinc finger protein 750 target regulating the immunogenicity via DNA damage in lung squamous cell carcinoma

Modulation of DNA damage repair in lung squamous cell carcinoma (LUSC) can result in the generation of neoantigens and heightened immunogenicity. Therefore, understanding DNA damage repair mechanisms holds significant clinical relevance for identifying targets for immunotherapy and devising therapeutic strategies. Our research has unveiled that the tumor suppressor zinc finger protein 750 (ZNF750) in LUSC binds to the promoter region of tenascin C (TNC), leading to reduced TNC expression. This modulation may impact the malignant behavior of tumor cells and is associated with patient prognosis. Additionally, single-cell RNA sequencing (scRNA-seq) of LUSC tissues has demonstrated an inverse correlation between ZNF750/TNC expression levels and immunogenicity. Manipulation of the ZNF750-TNC axis in vitro within LUSC cells has shown differential sensitivity to CD8+ cells, underscoring its pivotal role in regulating cellular immunogenicity. Further transcriptome sequencing analysis, DNA damage repair assay, and single-strand break analyses have revealed the involvement of the ZNF750-TNC axis in determining the preference for homologous recombination (HR) repair or non-homologous end joining (NHEJ) repair of DNA damage. with involvement of the Hippo/ERK signaling pathway. In summary, this study sheds light on the ZNF750-TNC axis's role in DNA damage repair regulation in LUSC, laying a groundwork for future translational research in immune cell therapy for LUSC.

The impact of developmental stage, tissue type, and sex on DNA double-strand break repair in Drosophila melanogaster.

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.

RUVBL1 ubiquitination by DTL promotes RUVBL1/2-β-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor β-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-β-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study’s discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-β-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-β-catenin provides a potential radiosensitization approach when treating breast cancer.

Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.

Abstract 2832: Mouse models advancing cancer research - The Jackson Laboratory

Abstract Genetically defined mice are a cornerstone of cancer research. The Jackson Laboratory (JAX) offers more than 13,500 unique mouse lines to the scientific community, many useful in advancing cancer research. Available are mouse models for specific cancers, xenograft recipients, immunodeficient platforms for PDX studies and multi-purpose tool strains having CRISPR/Cas9 utility, conditional/inducible expression (Cre-lox, FLP-frt, Tet-On/-Off), optogenetic function and calcium-sensing technologies. This poster will highlight some recently added models, more than 100 of which have cancer research applications. The portfolio of strains useful for transplantation/engraftment and “humanized” mouse research continues to expand and complement the NSG (NOD scid IL2rγc-/-) model as ideal tools for engraftment with a wide range of malignant or non-malignant human or mouse tissues. Fluorescent protein-expressing strains include Traffic Light Reporters for tracking of DNA repair pathways, Gene Editing Reporters for detection of homology directed repair (HDR) and non-homologous end joining (NHEJ) repair events, as well as several CRISPR Editing, Bxb1 Integrase Editing, Base Editor and Prime Editor tools. For example, the Rosa26PE2 mouse has Cre-inducible expression of a prime editor enzyme and a fluorescent reporter, allowing viral delivery of prime editing guide RNAs to introduce cancer mutations into the genome. Recently added CRISPR Array Repair Lineage Tracing (CARLIN and DARLIN) component strains allow researchers to achieve high barcode diversity in targeted cell populations. In addition to safeguarding each strain by cryopreservation, JAX has a comprehensive genetic quality control program that confirms mutation identity and genetic background, and screens for unwanted alleles (such as stray GFP, Cre, neo, etc.). Search the comprehensive collection of innovative mouse models using our improved JAX Mouse Search site (mice.jax.org). This resource includes models created by many generous donating institutions. Researchers are encouraged to donate their mouse lines via a very short Strain Submission form (jax.org/donate-a-mouse). Please also visit the JAX Oncology webpage for cancer research related strains and resources (jax.org/jax-mice-and-services/solutions-by-therapeutic-area/oncology). JAX is supported by the NIH, The Howard Hughes Medical Institute and other private charitable foundations. Citation Format: Jason Beckwith, Jennifer Merriam, Alicia Valenzuela, Darcy Pomerleau, Jennifer Kelmenson. Mouse models advancing cancer research - The Jackson Laboratory [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 2832.

Abstract 567: Fisetin sensitizes triple negative breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) represents a highly aggressive subtype, constituting approximately 20% of all breast cancer cases. Y-box binding protein-1 (YB-1) is often overexpressed in various tumor types and has been implicated in conferring resistance to cell death induced by radiotherapy. Fisetin, a flavonoid compound with demonstrated anti-cancer properties, operates in part through inhibiting phosphorylation of YB-1 by p90 ribosomal S6 kinase (RSK). In this study, we investigated the potential synergistic effects of combining fisetin with radiotherapy in TNBC. The activation status of the RSK signaling pathway was assessed in both total cell lysate and subcellular fractions using Western blotting. A standard clonogenic assay was employed to evaluate post-irradiation cell survival. To investigate the frequency of double-strand breaks (DSBs) and chromosomal aberrations, γH2AX foci assay and 3-color fluorescence in situ hybridization analyses were conducted, respectively. The DSB repair pathways targeted by fisetin were examined in cells expressing genomically integrated reporter constructs by quantifying green fluorescence protein expression through flow cytometry. Apoptosis and autophagy were measured using flow cytometric quantification of sub-G1 cells and the assessment of LC3-II protein expression, respectively. Kinase array and phosphoproteomics analyses were carried out to study the potential impact of fisetin on the signaling involved in the DNA damage response (DDR) after irradiation. We demonstrated that fisetin mimics the effects of RSK pharmacological inhibitors in terms of effect on YB-1 phosphorylation. Treatment with fisetin increased frequency of DSB in non-irradiated cells and impaired repair of DSB after irradiation. Fisetin attenuated DSB repair by suppressing the classical non-homologous end-joining and homologous recombination repair pathways. The frequency of chromosomal aberration was enhanced by fisetin treatment. The effect of fisetin on inhibiting DSB repair was partially YB-1 dependent. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. Fisetin neither inhibited DSB repair nor radiosensitized normal human fibroblast HSF7 cells. The radiosensitizing effect of fisetin was not attributed to enhancing apoptosis or modulating autophagy. In summary, combining fisetin with radiotherapy may enhance TNBC radiotherapy outcomes. -This research was supported by a grant from the German Research Council (DFG,TO 685/2-3). Citation Format: Shayan Khozooei, Soundaram Veerappan, Mahmoud Toulany. Fisetin sensitizes triple negative breast cancer cells to radiation [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 567.

Abstract 5825: Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer

Abstract Rare cell populations manage to evade the effects of cancer therapies, ultimately leading to drug resistance and subsequent cancer relapse. These resilient populations, often referred to as "persisters," exhibit distinct molecular characteristics when compared to the broader tumor population. In the current standard of clinical care for non-small cell lung cancer (NSCLC), patients typically undergo a series of standard treatments, such as surgery followed by chemotherapy/immunotherapy, and then targeted therapy, based on the disease stages. This continues until drug resistance emerges, with little attention given to the state of these persister cells in response to these treatments. In the case of drug resistance in KRAS-mutated NSCLC, ongoing clinical investigations are exploring the combined efficacy of KRAS G12Ci inhibitors with drugs that target mitogen-activated protein kinase (MAPK) feedback reactivation, immunotherapy, and chemotherapy. Despite the myriad of combination therapies, persister populations invariably emerge. The manner in which these persisters evolve to withstand therapies remains elusive. We employed a DNA barcode lineage tracing analysis coupled with mathematical modeling to decode persister evolution to therapies targeting different pathways, e.g., KRAS+ SHP2, KRAS+CDK4/6, and KRAS+MCL-1. Our model reveals that rapidly emerging persister populations are predetermined and do not exhibit clear genetic alterations. In some cells, common persisters exist in response to different therapies. Single-cell ATAC-RNA sequencing showed that these persisters are enriched in chromatin-binding of transcription factors that regulate classic non-homologous end-joining (cNHEJ) DNA repair (ZNF384), interferon response (IRF1), and stem-like characteristics (FOXP1), along with the activation of an IFN-γ transcriptional signature. These findings underscore the connection between specific DNA repair processes, chromosomal instability, and resistance to KRAS G12Ci. In other cells, however, distinct persister populations exist to different therapies, leading to the hypothesis that these diverse persister populations can be targeted orthogonally or sequentially to achieve tumor regression. In summary, our study unveils the heterogeneity in persister evolution trajectories in KRAS NSCLC. Our platform identifies targetable persister populations in NSCLC models and offers a proof-of-concept for non-standard strategies to combat drug resistance based on persister cell states. Citation Format: Chendi Li, Christopher J. Graser, Qian Qin, Anahita Nimbalkar, Usman Syed, Barbara Karakyriakou, Sarah Clark, Anne Y. Saiki, Paul E. Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron Hata. Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer [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 5825.

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.

Abstract 2874: Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1

Abstract The efficacy of image-guided radiotherapy (RT) has been linked to directing increased chromosomal double strand breaks (DSBs) to tumor cells while sparing surrounding normal tissue. DSBs can block cell cycle progression and promote mitotic catastrophe, cell death and senescence. End processing of persistent DSBs releases single strand DNA (ssDNA), driving cGAS/STING signaling and anti-tumor immune response. DSB repair, whether by non-homologous end-joining (NHEJ) or homologous recombination (HR), is a resistance mechanism, supporting cell survival, repopulation and immune evasion. Decades of effort to block DSB repair to enhance benefits of RT has yet to produce approved agents. Toxicity due to lack of cancer specificity may prevent the current generation of targeted agents from reaching the clinic. An answer may come from examining cancer metabolic reprogramming as a determinant of intrinsic radiation resistance. Along with other glucose metabolites, the Warburg effect increases N-acetyl glucosamine (GlcNAc) biosynthesis, which not only supports N-glycosylation but also modification of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). OGT-dependent O-GlcNAcylation confers radiation resistance, in part by accelerating DSB repair. OGT regulates γH2AX, 53BP1 and BRCA1 foci kinetics and suppresses ssDNA formation at DSB ends, limiting HR but favoring NHEJ for S/G2 DSB repair. Blocking OGT induces radiation sensitization in vitro and in vivo. In cells, OGT inhibition results in hyperresection, persistent RPA and RAD51 foci and accumulation of cytosolic DNA. One mediator may be the histone methyltransferase EZH2, which is rapidly recruited to deposit H3K27me3 at DSBs, promoting NHEJ repair. Irradiation of EZH2 KO cells results in hyperresection that cannot be suppressed by O-GlcNAcylation. Cancer cells may also accumulate NAD+, a substrate for PARP1 incorporated into poly-ADP-ribose to initiate DSB repair. Much like OGT inhibition, blocking PARP1 induces hyperresection and shifts repair toward HR and away from NHEJ. Importantly, increasing O-GlcNAcylation can suppress ssDNA formation even in PARP1 KO cells. In turn, inhibiting PARP1 enhances hyperesection in EZH2 KO cells, leading to a marked increase in cytosolic ssDNA. Our data assign complementary roles for EZH2 and PARP1 in DSB repair pathway choice by independently limiting 5' end resection. This directs DSBs to rapid, imprecise repair by NHEJ over slow and precise repair by HR. An implication is that PARP inhibitor resistance due to the Warburg effect may be mediated by EZH2. Driving hyperresection by targeting O-GlcNAcylation or H3K27 trimethylation along with PARP inhibition may saturate the capacity of HR and produce irreversible damage in cancer cells. Targeting impacts of metabolic reprogramming on DSB repair may not only enhance the therapeutic index of radiation but also sharpen synthetic lethality with HR deficiency. Citation Format: Elena E. Efimova, Sera Averbek, Donald J. Wolfgeher, Ding Wu, Yue Liu, Stephen J. Kron. Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1 [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 2874.

Non-homologous End-joining Genotype, mRNA Expression, and DNA Repair Capacity in Childhood Acute Lymphocytic Leukemia.

The capacity for non-homologous end-joining (NHEJ) repair plays a pivotal role in maintaining genome stability and in carcinogenesis. However, there is little literature on the involvement of NHEJ-related genes in childhood acute lymphocytic leukemia (ALL). Our study aimed to elucidate the impact of polymorphisms of X-ray repair cross-complementing group 4 (XRCC4) (rs6869366, rs2075685, rs2075686, rs28360071, rs3734091, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and DNA ligase IV (LIG4) rs1805388, on the odds of childhood ALL. Genotypes NHEJ-related genes of 266 cases and 266 controls were determined, and the genotype-phenotype correlation was investigated by examining mRNA transcript expression and the capacity for overall and precise NHEJ repair. The variant genotypes of XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 were significantly associated with increased odds of childhood ALL. Further analysis based on susceptibility genotypes showed no significant differences in mRNA transcript expression levels among childhood ALL cases with various putative high-risk genotypes, except XRCC6 rs5751129. Moreover, the overall NHEJ repair capacity was similar among carriers of different XRCC4, XRCC5, and XRCC6 genotypes. However, it is worth noting that individuals carrying the variant C allele at XRCC6 rs5751129 exhibited lower precise NHEJ repair capacity compared to those with the wild-type T allele. Our study identified significant associations between XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 genotypes and childhood ALL. Notably, lower transcriptional expression and reduced precise NHEJ repair capacity were observed in patients carrying the C allele of XRCC6 rs5751129. Further investigations are required to gain deeper insights into childhood ALL development.

A di-acetyl-decorated chromatin signature couples liquid condensation to suppress DNA end synapsis

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.

Expression of DNA Repair Genes in Ewing Sarcoma.

Ewing sarcoma is an aggressive mesenchymal malignancy commonly affecting children and young adolescents. The molecular basis of this neoplasia is well reported with the formation of the EWSR1/FLI1 fusion gene being the most common genetic finding. However, this fusion gene has not been targeted therapeutically nor is being used as a prognostic marker. Its relevance regarding the molecular steps leading to Ewing sarcoma genesis are yet to be defined. The generation of the oncogenic EWSR1/FLI1 fusion gene, can be attributed to the simultaneous introduction of two DNA double-strand breaks (DSBs). The scope of this study is to detect any association between DNA repair deficiency and the clinicopathological aspects of Ewing's sarcoma disease. We have conducted an expression analysis of 35 patients diagnosed with Ewing sarcoma concerning the genes involved in non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways. We have analyzed the expression levels of 6 genes involved in NHEJ (XRCC4, XRCC5, XRCC6, POLλ, POLμ) and 9 genes involved in HR (RAD51, RAD52, RAD54, BRCA1, BRCA2, FANCC, FANCD, DNTM1, BRIT1) using real time PCR. Age, sex, location of primary tumor, tumor size, KI67, mitotic count, invasion of adjacent tissues and treatment were the clinicopathological parameters included in the statistical analysis. Our results show that both these DNA repair pathways are deregulated in Ewing sarcoma. In addition, low expression of the xrcc4 gene has been associated with better overall survival probability (p=0.032). Our results, even though retrospective and in a small number of patients, highlight the importance of DSBs repair and propose a potential therapeutic target for this type of sarcoma.

CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.)

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Exploring DNA repair deficient CHO cell response to low dose rate radiation

IntroductionDNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. Materials and methodsChinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. ResultsNHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DiscussionThese findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.

Precision Genome Editing with CRISPR-Cas9.

The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.

Palmitoyl ascorbic acid glucoside enhanced cell survival with post irradiation treatment

Radiation exposure poses a significant threat to cellular integrity by inducing DNA damage through the generation of free radicals and reactive oxygen species. Ascorbic acid, particularly its derivative Palmitoyl Ascorbic Acid 2-Glucoside (PA2G), has demonstrated remarkable radioprotective properties. While previous research focused on its pre-irradiation application, this study explores the post-irradiation radiomitigation potential of PA2G. Our findings reveal that post-irradiation treatment with PA2G enhances cell survival and accelerates DNA repair processes, particularly the non-homologous end-joining (NHEJ) repair pathway. Notably, PA2G treatment reduces the frequency of lethal chromosomal aberrations and micronuclei formation, indicating its ability to enhance the repair of complex DNA lesions. Furthermore, PA2G is shown to play a role in potentially lethal damage repair (PLDR). These radioprotective effects are specific to NHEJ and ATM pathways, as cells deficient in these mechanisms do not benefit from PA2G treatment. This study highlights PA2G as a versatile radioprotector, both pre- and post-irradiation, with significant potential for applications in radiation therapy and protection, offering new insights into its mechanism of action. Further research is required to elucidate the precise molecular mechanisms underlying PA2G's radiomitigation effects and its potential clinical applications.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.

Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.

Disruption of DNA-PK-Mediated Cgas Retention on Damaged Chromatin Potentiates Doxorubicin-Induced Cgas/Sting-Dependent Anti-Multiple Myeloma Activity

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM with multidrug resistance or extensive extramedullary plasmacytoma are difficult to manage and often treated with DNA damage-inducing chemotherapies, including topoisomerase II inhibitor doxorubicin. However, the efficacy of this treatment is still limited and requires further improvement. Targeting DNA damage response (DDR) may offer an opportunity for effective treatment of MM. In particular, in combination with DNA damage-inducing agents, this strategy has shown to improve chemo-radiotherapies in part by cGAS-STING-mediated innate immune response, which could be directly activated by an elevated level of cytosolic DNA due to DNA repair inhibition. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated to the cytoplasm upon DNA damage, leading to cGAS-STING activation. Here, after testing the potential of several DDR inhibitors in sensitizing MM cells (MMCs) to doxorubicin, we found that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of classical non-homologous end joining (c-NHEJ) repair, using DNA-PKcs inhibitor NU7441, synergistically enhanced doxorubicin-induced anti-MM effect by activation of cGAS-STING pathway, the combination index values at different combination doses were all less than 0.85. Knocking out either cGAS or STING in ARP-1 cells significantly reduced not only the transcription of IFNB1 and multiple ISGs but also the proportion of NU7441/doxorubicin-induced apoptotic cells. Stably overexpressing STING in U266 and RPMI-8226 cells significant upregulated the IFNB1 and ISGs levels, and made MMCs more susceptible to NU7441/doxorubicin-induced inhibition of cell proliferation. Despite a strong type I interferon (IFN) response induced by activation of cGAS-STING, apoptosis of MMCs caused by activated cGAS-STING was mediated in part by IRF3-NOXA-BCL2 axis, which is independent of type I IFN response. In addition to inducing intrinsic apoptosis of MMCs, activation of cGAS-STING signaling by NU7441/doxorubicin in MMCs also induced M1 polarization of macrophages (Mφs), which increased M1 marker CD86 of Mφs but decreased M2 marker CD163 and CD206, and reduced the IL-10 concentration secreted by Mφs, thus suppressing tumor-protecting potential of M2-like Mφs and improving bortezomib-induced apoptosis of MMCs. Mechanistic studies showed that the activation of cGAS-STING signaling was not due to the defective c-NHEJ following continuous DNA-PKcs inhibition, because NU7441 did not enhance the damaged DNA accumulation after doxorubicin induction detected by γH2AX immunoblot analysis and alkaline comet assay. Besides, deletion of XRCC4 in ARP-1 cells to directly impair c-NHEJ did not enhance doxorubicin-induced activation of cGAS-STING signaling, further excluding a role for impaired c-NHEJ. Instead, we found that cGAS was mainly localized in the nucleus and chromatin-bound in MMCs under mock treatment, and doxorubicin induced DNA damage and mobilized inactive cGAS into cytoplasm. Both mass spectrometry data and immunoblotting of cGAS immunocoprecipitates showed that cytoplasmic cGAS was associated with DNA-PK complex and nucleosomal histone H2A-H2B heterodimer, and DNA-PKcs inhibition promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of free cytosolic cGAS and DNA, activating cGAS-STING. Disruption of cytoplasmic cGAS-nucleosome binding by mutating cGAS R236 or R255 sites to E abolished the effect of NU7441 on doxorubicin-induced activation of cGAS-STING signaling. Analysis of Datasets MMRF CoMMpass and GSE2658 showed that MM patients with higher DNA-PKcs expression showed poorer prognosis than those with lower expression. Additionally, the DNA-PKcs expression was elevated with MM disease progression according to Datasets GSE5900, GSE13591 and MMRF CoMMpass, these indicated a possible involvement of DNA-PKcs in MM progression and provided a basis for targeting DNA-PKcs in MM treatment. Taken together, our study suggests that DNA-PKcs may help maintain the cGAS sequestration in damaged chromatin. Inhibition of DNA-PKcs may consequently disrupt this sequestration, inducing activation of cGAS-STING signaling and improving the efficacy of doxorubicin in treating MM.

Androgen signaling stabilizes genomes to counteract senescence by promoting XRCC4 transcription.

Aging is accompanied by a decreased DNA repair capacity, which might contribute to age-associated functional decline in multiple tissues. Disruption in hormone signaling, associated with reproductive organ dysfunction, is an early event of age-related tissue degeneration, but whether it impacts DNA repair in nonreproductive organs remains elusive. Using skin fibroblasts derived from healthy donors with a broad age range, we show here that the downregulation of expression of XRCC4, a factor involved in nonhomologous end-joining (NHEJ) repair, which is the dominant pathway to repair somatic double-strand breaks, is mediated through transcriptional mechanisms. We show that the androgen receptor (AR), whose expression is also reduced during aging, directly binds to and enhances the activity of the XRCC4 promoter, facilitating XRCC4 transcription and thus stabilizing the genome. We also demonstrate that dihydrotestosterone (DHT), a powerful AR agonist, restores XRCC4 expression and stabilizes the genome in different models of cellular aging. Moreover, DHT treatment reverses senescence-associated phenotypes, opening a potential avenue to aging interventions in the future.