DNA-dependent Protein Kinase Complex Research Articles

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex

BackgroundThe DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID.MethodsWe investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/−, or Ku70+/− mice used in this study.ResultsA ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α − induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system.ConclusionsThis study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

Harnessing the cooperation between DNA-PK and cGAS in cancer therapies: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is central for the initiation of anti-tumoural immune responses. Enormous effort has been made to optimise the design and administration of STING agonists to stimulate tumour immunogenicity. However, in certain contexts the cGAS-STING axis fuels tumourigenesis. Here, we review recent findings on the regulation of cGAS expression and activity. We particularly focus our attention on the DNA-dependent protein kinase (DNA-PK) complex, that recently emerged as an activator of inflammatory responses in tumour cells. We propose that stratification analyses on cGAS and DNA-PK expression/activation status should be carried out to predict treatment efficacy. We herein also provide insights into non-canonical functions borne by cGAS and cGAMP, highlighting how they may influence tumourigenesis. All these parameters should be taken into consideration concertedly to choose strategies aiming to effectively boost tumour immunogenicity.

Human DNA-dependent protein kinase activation mechanism

DNA-dependent protein kinase (DNA-PK), a multicomponent complex including the DNA-PK catalytic subunit and Ku70/80 heterodimer together with DNA, is central to human DNA damage response and repair. Using a DNA-PK-selective inhibitor (M3814), we identified from one dataset two cryo-EM structures of the human DNA-PK complex in different states, the intermediate state and the active state. Here we show that activation of the kinase is regulated through conformational changes caused by the binding ligand and the string region (residues 802–846) of the DNA-PK catalytic subunit, particularly the helix-hairpin-helix motif (residues 816–836) that interacts with DNA. These observations demonstrate the regulatory role of the ligand and explain why DNA-PK is DNA dependent. Cooperation and coordination among binding partners, disordered flexible regions and mechanically flexible HEAT repeats modulate the activation of the kinase. Together with previous findings, these results provide a better molecular understanding of DNA-PK catalysis.

DNAR-05. A ROLE FOR TAZ/YAP IN DNA DAMAGE REPAIR IN GLIOBLASTOMA

Abstract Previous studies have shown that glioma stem-like cells (GSCs) evade conventional cytotoxic therapies and repopulate as recurrent tumors. A fuller understanding of the molecular mechanisms underlying the biology and therapy resistance of GSCs is required. Our group has shown that TAZ, a transcriptional co-factor is highly expressed in about 70% of glioblastomas (GBMs). TAZ and its paralog YAP are oncogenic drivers of brain tumor progression. GSCs overexpressing TAZ undergo a proneural (PN) to mesenchymal (MS) subtype transition, which is accompanied by aggressive phenotypes such as increased grade and treatment resistance in GSC xenografts. Although the oncogenic functions of TAZ/YAP are well established, the exact molecular mechanisms underlying TAZ/YAP mediated cell fate transition and therapy resistance in GBM are not fully understood. Here, we present novel functional interaction between TAZ/YAP and DNA-dependent protein kinase (DNA-PK) in response to exposure to ionizing radiation in GSCs. Mass-spectrometric analyses of TAZ-binding proteins showed that DNA-PK complex composed of the catalytic subunit (DNA-PKcs) and a Ku heterodimer of Ku70 and K80 interacted with TAZ. Immunoprecipitation of DNA-PK showed interaction of both YAP as well. Interaction of YAP/TAZ with DNA-PK complex and TEAD was enhanced by ionizing radiation. DNA-PKcs phosphorylated both YAP and TAZ, and phosphorylation level of these proteins were increased after irradiation, consistent with an increased interaction of these proteins. Intriguingly, siRNA knockdown of YAP and TAZ resulted in inactivation of DNA-PKcs, although protein stability of DNA-PKcs was not affected. Pharmacological inhibition of TAZ/YAPTEAD resulted in increased DNA damage and suppressed cell proliferation, and this effect was synergized by irradiation. We conclude that TAZ and YAP are integral components of DNA damage response and that pharmacological targeting of TAZ/YAP/TEAD pathway can overcome radiation resistance in GSCs.

Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein involved in DNA damage response (DDR) signaling that may mediate kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD) due to its pleiotropic effects on proliferation and survival. To test this hypothesis, the expression of DNA-PK in human ADPKD and the in vitro effects of DNA-PK inhibition in a three-dimensional model of Madin-Darby Canine Kidney (MDCK) cyst growth and human ADPKD cells were assessed. In human ADPKD, the mRNA expression for all three subunits of the DNA-PK complex was increased, and using immunohistochemistry, the catalytic subunit (DNA-PKcs) was detected in the cyst lining epithelia of human ADPKD, in a focal manner. In vitro, NU7441 (a DNA-PK kinase inhibitor) reduced MDCK cyst growth by up to 52% after long-term treatment over 6–12 days. Although human ADPKD cell lines (WT9-7/WT9-12) did not exhibit synthetic lethality in response to DNA-PK kinase inhibition compared to normal human kidney cells (HK-2), the combination of low-dose NU7441 enhanced the anti-proliferative effects of sirolimus in WT9-7 and WT9-12 cells by 17 ± 10% and 11 ± 7%, respectively. In conclusion, these preliminary data suggest that DNA-PK mediates kidney cyst growth in vivo without a synthetically lethal interaction, conferring cell-specificity in human ADPKD cells. NU7441 enhanced the anti-proliferative effects of rapamycin complex 1 inhibitors, but the effect was modest.

Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice.

Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non-stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs-mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.

Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes.

The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches.

Abstract 6402: DNA-PK inhibitor, M3814, as a radiation sensitizer in the treatment of neuroendocrine tumors

Abstract Despite significant advances in the understanding and managing gastroenteropancreatic neuroendocrine tumors (GEPNETs), the therapeutic success of treatments remains relatively poor. Recent FDA approval of Peptide Receptor Radionuclide Therapy (PRRT) has paved the way for radiation-based treatment for GEPNETs. PRRT delivers high doses of radiation to tumor cells in the body to destroy cells or slow their growth. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in non-homologous end-joining (NHEJ) repair after radiation therapy. A novel, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks the NHEJ repair pathway for DNA double strand breaks (DSB). Here, we investigated the feasibility of radiosensitizing neuroendocrine (NET) cells with the DNA-PK inhibitor M3814, both in vitro and in preclinical NET models. Methods: 96-well clonogenic assays were used to evaluate efficacy of M3814 in combination with radiation therapy (XRT) in vitro in QGP-1 and BON pancreatic NET cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence after M3814 treatment. Western blot was used to confirm activation of DNA-PKcs (pDNA-PKcs S2056) after M3814 treatment. The efficacy of M3814 in combination with XRT was evaluated in QGP-1 and BON human xenograft models in athymic nude mice. Tumor cells were injected subcutaneously into athymic nude mice, and treatment started when palpable tumors (∼200 mm3) were established. M3814 was given orally (100 mg/kg) 30 min prior to XRT, which was applied using a radiation therapy device calibrated to deliver 2 Gy daily for 4 d. Results: Clonogenic assays demonstrated lack of cytotoxicity of M3814 alone at doses below 1000 nM and a strong radiosensitizing effect at 500 nM in both QGP-1 and BON cell lines. Immunofluorescence analysis revealed high numbers of γH2AX foci, marking unrepaired DSBs inside the micronuclei. Western blot showed that activation of DNA-PKcs, induced by XRT treatment, was quickly suppressed by M3814 treatment. In combination with XRT, M3814 showed efficacy in both QGP-1 and BON xenografts as demonstrated by a strong potentiation of the effect of XRT. In all models, a dose of 2 Gy administered daily for 4 d, in combination with M3814, induced a significant tumor growth inhibition compared to XRT alone. M3814, alone or in combination with XRT, did not induce significant weight loss or visual signs of toxicity in the mice in any of the studies. Conclusions: Our results demonstrate a benefit of adding M3814 to XRT in NET treatment. Selective DNA-PK inhibition by M3814 provides a potent therapeutic strategy for disruption of NHEJ DSB repair and may offers a novel therapeutic approach in advanced NET. Citation Format: Piotr Rychahou, Aman Chauhan, Zeta Chow, Tadahidi Izumi, Quan Chen, Eun Y. Lee, Dana Napier, Lowell B. Anthony, Michael J. Cavnar, Charles Kunos, B. Mark Evers. DNA-PK inhibitor, M3814, as a radiation sensitizer in the treatment of neuroendocrine tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6402.

Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation.

As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

Ribosomal protein S3 is a novel negative regulator of non-homologous end joining repair of DNA double-strand breaks.

DNA double-strand breaks (DSBs) are one of the most serious types of DNA damage. However, multiple repair pathways are present in cells to ensure rapid and appropriate repair of DSBs. Pathway selection depends on several factors including cell type, cell cycle phase, and damage severity. Ribosomal protein S3 (rpS3), a component of the 40S small ribosomal subunit, is a multi-functional protein primarily involved in protein synthesis. rpS3 is also involved in the mediation of various extra-ribosomal pathways, including DNA damage processing and the stress response. Here, we report that rpS3 is a novel negative regulator of non-homologous end joining (NHEJ)-mediated repair of DSBs. We found that rpS3 interacts with the Ku heterodimers of the DNA-dependent protein kinase (DNA-PK) complex and slows down NHEJ ligation reactions, ultimately triggering p53-dependent cell death following treatment with high-dose ionizing radiation. After DSB formation, DNA-PK phosphorylates rpS3, which consequently reduces the binding of rpS3 to the Ku complex. We hypothesized that rpS3 may play a role in DSB repair by repressing NHEJ, while inducing other repair pathways, and by initiating DSB-induced cell death in response to severe DNA damage.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks.

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

Expression of the DNA-dependent protein kinase catalytic subunit is associated with the radiosensitivity of human thyroid cancer cell lines.

The prognosis and treatment of thyroid cancer depends on the type and stage of the disease. Radiosensitivity differs among cancer cells owing to their varying capacity for repair after irradiation. Radioactive iodine can be used to destroy thyroid cancer cells. However, patient prognosis and improvement after irradiation varies. Therefore, predictive measures are important for avoiding unnecessary exposure to radiation. We describe a new method for predicting the effects of radiation in individual cases of thyroid cancer based on the DNA-dependent protein kinase (DNA-PK) activity level in cancer cells. The radiation sensitivity, DNA-PK activity, and cellular levels of DNA-PK complex subunits in five human thyroid cancer cell lines were analyzed in vitro. A positive correlation was observed between the D10 value (radiation dose that led to 10% survival) of cells and DNA-PK activity. This correlation was not observed after treatment with NU7441, a DNA-PK–specific inhibitor. A significant correlation was also observed between DNA-PK activity and expression levels of the DNA-PK catalytic subunit (DNA-PKcs). Cells expressing low DNA-PKcs levels were radiation-sensitive, and cells expressing high DNA-PKcs levels were radiation-resistant. Our results indicate that radiosensitivity depends on the expression level of DNA-PKcs in thyroid cancer cell lines. Thus, the DNA-PKcs expression level is a potential predictive marker of the success of radiation therapy for thyroid tumors.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

Fumarase: From the TCA Cycle to DNA Damage Response and Tumor Suppression.

Fumarase is an enzyme of the tricarboxylic acid (TCA) cycle in mitochondria, but in recent years, it has emerged as a participant in the response to DNA double strand breaks (DSBs) in the nucleus. In fact, this enzyme is dual-targeted and can be also readily detected in the mitochondrial and cytosolic/nuclear compartments of all the eukaryotic organisms examined. Intriguingly, this evolutionary conserved cytosolic population of fumarase, its enzymatic activity and the associated metabolite fumarate, are required for the cellular DNA damage response (DDR) to double-strand breaks. Here we review findings from yeast and human cells regarding how fumarase and fumarate may precisely participate in the DNA damage response. In yeast, cytosolic fumarase is involved in the homologous recombination (HR) repair pathway, through its function in the DSB resection process. One target of this regulation is the resection enzyme Sae2. In human cells, fumarase is involved in the non-homologous end joining (NHEJ) repair pathway. Fumarase is phosphorylated by the DNA-dependent protein kinase (DNA-PK) complex, which induces the recruitment of fumarase to the DSB and local generation of fumarate. Fumarate inhibits the lysine demethylase 2B (KDM2B), thereby facilitating the dimethylation of histone H3, which leads to the repair of the break by the NHEJ pathway. Finally, we discuss the question how fumarase may function as a tumor suppressor via its metabolite substrate fumarate. We offer a number of models which can explain an apparent contradiction regarding how fumarate absence/accumulation, as a function of subcellular location and stage can determine tumorigenesis. Fumarate, on the one hand, a positive regulator of genome stability (its absence supports genome instability and tumorigenesis) and, on the other hand, its accumulation drives angiogenesis and proliferation (thereby supporting tumor establishment).

Inactivation of DNA-PK by knockdown DNA-PKcs or NU7441 impairs non-homologous end-joining of radiation-induced double strand break repair.

The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in non-homologous end-joining (NHEJ) repair. We investigated the mechanism of NU7441, a highly selective DNA-PK inhibitor, in NHEJ-competent mouse embryonic fibroblast (MEF) cells and NHEJ-deficient cells and explored the feasibility of its application in radiosensitizing nasopharyngeal carcinoma (NPC) cells. We generated wild-type and DNA-PKcs−/− MEF cells. Clonogenic survival assays, flow cytometry, and immunoblotting were performed to study the effect of NU7441 on survival, cell cycle, and DNA repair. NU7441 profoundly radiosensitized wild-type MEF cells and SUNE-1 cells, but not DNA-PKcs−/− MEF cells. NU7441 significantly suppressed radiation-induced DSB repair post-irradiation through unrepaired and lethal DNA damage, the cell cycle arrest. The effect was associated with the activation of cell cycle checkpoints. The present study revealed a mechanism by which inhibition of DNA-PK sensitizes cells to irradiation suggesting that radiotherapy in combination with DNA-PK inhibitor is a promising paradigm for the management of NPC which merits further investigation.

RETRACTED: The effect of DNA-PKcs gene silencing on proliferation, migration, invasion and apoptosis, and in vivo tumorigenicity of human osteosarcoma MG-63 cells

The purpose of this study was to explore the role by which the DNA-dependent protein kinase complex catalytic subunit (DNA-PKcs) influences osteosarcoma MG-63 cell apoptosis, proliferation, migration and invasion. Osteosarcoma tissues and adjacent normal tissues were obtained from 57 osteosarcoma patients. Human osteosarcoma MG-63 cells were assigned into designated groups including the blank, siRNA-negative control (NC) and siRNA-DNA-PKcs groups. RT-qPCR and Western blotting methods were employed to evaluate the mRNA and protein expressions of DNA-PKcs. A cell counting kit-8 (CCK-8) assay was performed to assess cell viability. The evaluation of cell migration and invasion were conducted by means of Scratch test and Transwell assay. Flow cytometry with PI and annexin V/PI double staining was applied for the analysis of the cell cycle and apoptosis. Twenty-Four Balb/c nude mice were recruited and randomly divided into the blank, siRNA-NC and siRNA-DNA-PKcs groups. Tumorigenicity of the Balb/c nude mice was conducted to evaluate the rate of tumor formation, as well as for the assessment of tumor size and weight, and confirm the number of lung metastatic nodules in the mice post transfection. Osteosarcoma tissues were found to possess greater expression of DNA-PKcs than that of the adjacent normal tissues. DNA-PKcs expression in osteosarcoma tissues were correlated with the clinical stage and metastasis. Compared with the blank and siRNA-NC groups, proliferation, miration, as well as the invasion abilities of the MG-63 cells increased. Furthermore, an increase in apoptosis and cells at the G1 stage in the MG-63 cells was observed, while there were reductions in the cells detected at the S stage. The mRNA and protein expressions of CyclinD1, PCNA, Bcl-2 decreased while those of Bax increased in the siRNA-DNA-PKcs group. The tumor formation rate, tumor diameter, weight and lung metastatic nodules among the nude mice in the siRNA-DNA-PKcs group were all lower than those in the blank and siRNA-NC groups. The observations and findings of the study suggested that the silencing of DNA-PKcs inhibits the proliferation, migration and invasion, while acting to promote cell apoptosis in MG-63 cells and osteosarcoma growth in nude mice.

Cryo-EM structure of the DNA-PK holoenzyme

DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.

Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificated when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells.