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Simple Summary Despite the introduction of targeted therapies against genomic drivers in non-small cell lung cancer (NSCLC), e.g., mutated epidermal growth factor receptor (EGFR) and EML4-ALK fusion or immune checkpoint blockade, many patients are receiving radiation therapy (RT). Thus, RT is important for the treatment of advanced NSCLC, where it is used alone or combined with targeted agents or chemotherapy. Unfortunately, a large fraction of the NSCLC cases show resistance to ionizing radiation (IR), calling for further understanding of the underlying mechanisms and novel ways to IR sensitization. Here, we demonstrate a novel function of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) pathway that controls cellular IR response. Moreover, we show that EphA2 can be targeted for IR sensitization of resistant NSCLC cells. Thus, we suggest that further understanding of this mechanism may allow for new RT sensitization approaches to treat LC. Abstract Ephrin (EFN)/Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.

Ephrin (EFN)/ Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.

Simple Summary Germ cell tumors are a model of curable solid tumors due to their unique sensitivity to cisplatin-based chemotherapy. Patients are typically young adults, and despite high cure rate, about 20% of them do not achieve remission or relapse, and 50% of them succumb to the disease. The mechanisms behind their resistance to therapy are largely unknown. By using Testicular Germ Cell Tumor (TGCT) cell lines as a model, we investigated the mechanism of acquired resistance to cisplatin. We demonstrated that resistance occurred by a fine modulation of the DNA repair pathway choice. Namely, in resistant cells, repair of double-strand breaks by non-homologous end joining was dampened by the reduced expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs). However, cisplatin-induced damage was repaired efficiently by homologous recombination. Additionally, we demonstrate that pharmacological inhibition of poly (ADP-ribose) polymerase (PARP) combined with cisplatin had an additive/synergistic effect on cisplatin-resistant cells, which represents the proof of concept for introducing PARP inhibitors in salvage therapy. Abstract Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line’s resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.

Long non-coding RNAs (lncRNAs) play important roles in cancer development and progression; however, their contributions to gastric cancer metastasis remain largely unknown. By lncRNA microarray screening, our study showed that 453 lncRNAs are dysregulated in gastric cancer tissues with or without lymph node metastasis, of which lnc-LEMGC ranks as one of the most significantly downregulated lncRNAs. Lnc-LEMGC inhibited cell migration and invasion both in vitro and in vivo, by combining with protein DNA-PKcs. Importantly, nucleotides 1300-1800 of lnc-LEMGC prevented DNA-PKcs phosphorylation of serine 2056 and partially abrogated the effects of downstream effectors, ErbB1, SRC and protein tyrosine kinase 2 (FAK), in the epidermal growth factor receptor (EGFR) pathway. The results of this study extend our knowledge of lncRNA's molecular mechanisms, in which lnc-LEMGC functions by directly suppressing the phosphorylation of its combined protein DNA-PKcs and inactivating the DNA-PKcs downstream EGFR signaling.

Combining targeted therapeutic agents is an attractive cancer treatment strategy associated with high efficacy and low toxicity. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is an essential factor in DNA damage repair. Studies from us and others have revealed that DNA-PKcs also plays an important role in normal mitosis progression. Histone deacetylase (HDACs) inhibitors commonly lead to mitotic aberration and have been approved for treating various cancers in the clinic. We showed that DNA-PKcs depletion or kinase activity inhibition increases cancer cells’ sensitivity to HDACs inhibitors in vitro and in vivo. DNA-PKcs deficiency significantly enhances HDACs inhibitors (HDACi)-induced mitotic arrest and is followed by apoptotic cell death. Mechanistically, we found that DNA-PKcs binds to HDAC6 and facilitates its acetylase activity. HDACi is more likely to impair HDAC6-induced deacetylation of HSP90 and abrogate HSP90’s chaperone function on Aurora A, a critical mitotic kinase that regulates centrosome separation and mitotic spindle assembly in DNA-PKcs-deficient cells. Our current work indicates crosstalk between DNA-PKcs and HDACs signaling pathways, and highlights that the combined targeting of DNA-PKcs and HDACs can be used in cancer therapy. Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit, HDACs, Histone deacetylases, DSBs, DNA double-strand breaks, ATM, ataxia telangiectasia mutated, ATR, ATM-Rad3-related

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg−/−) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper.

Background Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. Methods Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by γH2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (~ 80% vs. ~ 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. Conclusions The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism.

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking core NHEJ factors Ku70 and Ku80. Inactivation of other core NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of one accessory NHEJ gene,e.g. Paxx, Mri and Dna-pkcs, results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of accessory NHEJ on early stages of neurodevelopment, we isolated neural stem and progenitors cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf,Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that accessory NHEJ factors are important for maintaining the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking core NHEJ factors Ku70 and Ku80. Inactivation of other core NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of one accessory NHEJ gene, e.g. Paxx, Mri and Dna-pkcs, results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of accessory NHEJ on early stages of neurodevelopment, we isolated neural stem and progenitors cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that accessory NHEJ factors are important for maintaining the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damage throughout the cell cycle and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on the early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XRCC4-like factor (XLF), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX) maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

Radio-resistance mechanisms limit the benefit of radiation therapy (RT) for melanoma brain metastases. A key pathway for radiation-induced DNA double-strand break repair is non-homologous end joining, which is critically mediated by DNA-dependent protein kinase (DNA-PKcs). Here we evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells at concentrations of ≥300 nM, and a minimum of 16 h exposure with ~300 nM M3814 was required for effective sensitization. M3814 inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. Persistent γH2AX foci were observed in 28% M12 cells at 24 hours after co-treatment with M3814 and RT as compared to 12% cells following RT alone. In vivo pharmacokinetic analysis after single oral dose of 20 mg/kg M3814, showed reasonably short half-life (~2.44 hours) and poor brain distribution in wild-type FBV mice (Kpuu, 0.027). Consistent with an efflux liability, brain distribution of M3814 in triple knockout mice for BCRP/MDR1A/B was ~11 fold higher (Kpuu, 0.215). Compared to normal brain, much higher M3814 concentrations were detected in intracranially implanted M12 tumors (~23 fold and ~20 fold) 2 and 6 hours after a single oral dose of 50mg/kg respectively. The relative exclusion of M3814 from normal brain as compared to brain metastases suggests that this drug may have a favorable toxicity profile when combined with radiation for treatment of melanoma brain metastases, and this hypothesis is being tested in ongoing efficacy studies.

Steroid hormone signaling induces vast gene expression programs which necessitate the local formation of transcription factories at regulatory regions and large-scale alterations of the genome architecture to allow communication among distantly related cis-acting regions. This involves major stress at the genomic DNA level. Transcriptionally active regions are generally instable and prone to breakage due to the torsional stress and local depletion of nucleosomes that make DNA more accessible to damaging agents. A dedicated DNA damage response (DDR) is therefore essential to maintain genome integrity at these exposed regions. The DDR is a complex network involving DNA damage sensor proteins, such as the poly(ADP-ribose) polymerase 1 (PARP-1), the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the ataxia–telangiectasia-mutated (ATM) kinase and the ATM and Rad3-related (ATR) kinase, as central regulators. The tight interplay between the DDR and steroid hormone receptors has been unraveled recently. Several DNA repair factors interact with the androgen and estrogen receptors and support their transcriptional functions. Conversely, both receptors directly control the expression of agents involved in the DDR. Impaired DDR is also exploited by tumors to acquire advantageous mutations. Cancer cells often harbor germline or somatic alterations in DDR genes, and their association with disease outcome and treatment response led to intensive efforts towards identifying selective inhibitors targeting the major players in this process. The PARP-1 inhibitors are now approved for ovarian, breast, and prostate cancer with specific genomic alterations. Additional DDR-targeting agents are being evaluated in clinical studies either as single agents or in combination with treatments eliciting DNA damage (e.g., radiation therapy, including targeted radiotherapy, and chemotherapy) or addressing targets involved in maintenance of genome integrity. Recent preclinical and clinical findings made in addressing DNA repair dysfunction in hormone-dependent and -independent prostate and breast tumors are presented. Importantly, the combination of anti-hormonal therapy with DDR inhibition or with radiation has the potential to enhance efficacy but still needs further investigation.

Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical non-homologous end-joining pathway, but it may have roles in modulating gene expression and thus, the overall cellular response to DNA damage. Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Downregulation of pathways involved in biosynthesis were observed in both genotypes, but upregulated biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trial as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

Summary DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 A at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 A overall and 3.2 A for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is encoded by the protein kinase, DNA-activated, catalytic polypeptide ( PRKDC) gene. DNA-PKcs plays a major role in nonhomologous end joining DNA repair, and it has been identified to be an important factor in tumor progression and metastasis. DNA-PKcs may have opposite effects in diseases, depending on the cell and tissue types. In this review, we discuss its role in various tumors. High levels of DNA-PKcs are directly associated with prognosis, neoplasm recurrence rates, and overall survival. Our results suggest that DNA-PKcs may serve as a therapeutic target for advanced malignancies.

The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and recruits and activates DNA-PK. DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs5A mouse model carrying an alanine substitution at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocation, excess end-resection, and preferential usage of micro-homology – all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.Key pointsLoss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (&beta, HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that &beta, HPV promotes tumorigenesis, while others suggest that &beta, HPV protects against skin cancer. Most of the molecular evidence that &beta, HPV impairs DNA repair has been gained via characterization of the E6 protein from &beta, HPV 8 (&beta, HPV 8E6). Moreover, &beta, HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, &beta, HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, &beta, HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, &beta, HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, &beta, HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of &beta, HPV 8E6.

The DNA damage response (DDR) is orchestrated by three apical signalling kinases: ATM, ATR and DNA-PKcs. Despite their central roles, structural and biochemical understanding has remained limited, mainly due to their large size. Recent advances in cryo-electron microscopy allowed for the structural analysis of these kinases, revealing their overall architecture and providing high resolution structures of their active sites. Combined with novel biochemical insights it is now possible to dissect the elements that are important for activation. In this review we discuss the recent structures of these kinases, the possible mechanisms to regulate their activity, substrate recognition and emerging insights in the elements required for kinase activation.

The treatment resistance of cancer cells is a multifaceted process in which DNA repair emerged as a potential therapeutic target. DNA repair is predominantly conducted by nuclear events, yet, how extra-nuclear cues impact the DNA damage response is largely unknown. Here, using a high-throughput RNAi-based screen in three-dimensionally-grown cell cultures of head and neck squamous cell carcinoma (HNSCC), we identified novel focal adhesion proteins controlling DNA repair, including the intermediate filament protein, synemin. We demonstrate that synemin critically regulates the DNA damage response by non-homologous end joining repair. Mechanistically, synemin forms a protein complex with DNA-PKcs through its C-terminal tail domain for determining DNA repair processes upstream of this enzyme in an ATM-dependent manner. Our study discovers a critical function of the intermediate filament protein, synemin in the DNA damage response, fundamentally supporting the concept of cytoarchitectural elements as co-regulators of nuclear events.

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC.

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

Background DNA-dependent protein kinase (DNA-PK), consisting of a Ku heterodimer (Ku70/80) and a large catalytic subunit (DNA-PKcs), plays an important role in the repair of DNA double-strand breaks via non-homologous end-joining (NHEJ) in mammalian cells. Severe combined immunodeficient (scid) mice carry a mutation in the gene encoding DNA-PKcs and are sensitive to ionizing radiation. To examine the roles of DNA-PKcs in the generation of deletion mutations in vivo, we crossed scid mice with gpt delta transgenic mice for detecting mutations. Results The scid and wild-type (WT) gpt delta transgenic mice were irradiated with a single X-ray dose of 10 Gy, and Spi− mutant frequencies (MFs) were determined in the brain and spleen 2 days after irradiation. Irradiation with X-rays significantly enhanced Spi− MF in both organs in the scid and WT mice. The MFs in the brain of irradiated scid mice were significantly lower than those in WT mice, i.e., 2.9 ± 1.0 × 10− 6 versus 5.0 ± 1.1 × 10− 6 (P − 6 versus 4.8 ± 1.4 × 10− 6. Unirradiated scid and WT mice did not exhibit significant differences in MFs in either organ. Conclusions DNA-PKcs is unessential for the induction of deletion mutations in the spleen, while it plays a role in this in the brain. Therefore, the contribution of DNA-PKcs to NHEJ may be organ-specific.

Most cancer cells experience oncogene-induced replication stress and, as a result, exhibit high intrinsic activation of the ATR kinase. Although cancer cells often become more dependent on ATR for survival, the precise mechanism by which ATR signaling ensures cancer cell fitness and viability remains incompletely understood. Here, we find that intrinsic ATR signaling is crucial for the ability of cancer cells to promote DNA end resection, the first step in homology-directed DNA repair. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents the engagement of RAD51 as well as RAD52 at nuclear foci, leading to toxic DNA-PKcs signaling and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and engagement of RAD52. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.

Background and purpose Radiotherapy is the most important therapeutic measure against glioblastoma multiforme (GBM), which is regarded as the most common and highly lethal type of brain cancer. Nevertheless, most relapses originate in the close vicinity of the irradiated target volume. Genistein is a natural product that can suppress the invasive potential of cancer cells. In this study, DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-proficient and -deficient GBM cells were selected for in vitro and in vivo studies to investigate the inhibiting effects of genistein on radiation-induced invasion and migration and the corresponding mechanism. Materials and methods GBM cell lines with or without genistein pre-treatment were irradiated with X-rays. Cell survival was determined using colony formation assay and the rate of cellular proliferation was analyzed with a real-time cell electronic sensing system. For in vitro study, invasion and migration abilities were evaluated via wound-healing and transwell assays, while protein expression was determined with western blotting. Genistein interaction with DNA-PKcs was estimated with pull-down, recombinant and binding assays. For in vivo study, cells were stereotactically injected into NOD-SCID mice to establish tumors. Hematoxylin and eosin and immunohistochemistry were used to assess the invasive potential of GBM. Results X-ray irradiation enhanced the migration and invasion of DNA-PKcs-positive but not DNA-PKcs-negative GBM cells. It also activated the DNA-PKcs/Akt2/Rac1 signaling pathway, which contributed to GBM malignant progression by aggravating GBM cell invasive potential. The study successfully demonstrated that genistein can specifically bind to DNA-PKcs and block the DNA-PKcs/Akt2/Rac1 pathway, thereby effectively inhibiting radiation-induced invasion and migration of GBM cells in vitro and in vivo. The present study emphasized that radiation-induced invasive potential is initiated by DNA-PKcs, which is a well-known double strand breaks (DSB) repair protein, and determined the exact site for genistein binding to DNA-PKcs. Conclusion DNA-PKcs is not only a potential target for cancer therapy, but also a reliable biomarker for predicting radiation-induced invasion and migration of GBM cells. Thus, genistein might serve as a novel therapeutic strategy for treating GBM.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA-PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA-PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP2) response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast-specific conditional Prkdc-knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA-PKcs negatively regulates osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.