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Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of the DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the higher localization of the DNA-PK into the nucleus. The finding that cocaine increases the nuclear localization of the DNA-PK provides further support to our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear localization of the DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study demonstrates that the cocaine-induced DNA-PK promotes the hyper-phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both the initiation and elongation phases, respectively, of HIV transcription. The cocaine-mediated enhancement of transcriptional initiation is supported by its activation of cyclin-dependent kinase 7 (CDK7). Additionally, the induction of transcriptional elongation is marked by higher LTR recruitment and the increased phosphorylation of CDK9, which indicates the stimulation of positive transcriptional elongation factor b (P-TEFb). We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observed that the phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the previously observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed the elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results unravel the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV. [ABSTRACT FROM AUTHOR]

Purpose: Glioblastomas are resistant to conventional therapies, including radiotherapy. Our previous study proved that epigenetic regulation influences the radiation response of glioma cells. This study evaluated the role of the acetyltransferase Tip60 on the radiation response. Material and Methods: Tip60 expression was down-regulated by transfecting specific siRNA's in A7 and MO59K cells with high and low expression of Tip60, respectively, and its effect on survival was assessed. DNA repair was analyzed by foci scoring (γH2AX, Rad51, 53BP1, pATM). The interaction of Tip60 with ATM and DNA-PK was investigated using the specific inhibitors KU55933 and NU7441, respectively. Results: Knockdown of Tip60 significantly (p <.001) reduced survival in both cell lines, but the effect was more pronounced in A7 cells. ATMi and DNA-PKi significantly reduced the surviving fraction following irradiation. However, no further effect of siTip60 on the radiosensitivity of ATMi treated A7 cells was observed. In contrast, DNA-PKi effectively enhanced the sensitizing effect of siTip60. Mechanistically, siTip60 reduced the number of initial Rad51 and ATM foci formation after irradiation and prevented their dissolution at 24 h. siTip60 had no impact on the formation of 53BP1 and γH2AX foci and did not further affect these end-points if combined with ATMi or DNA-PKi. Conclusions: Downregulation of Tip60 enhances the radiation sensitivity of both glioma cells and markedly elevates the radiation sensitivity when combined with DNA-PKi. Therefore, treatment with DNA-PK inhibitors represents a promising approach to augment the radiation sensitivity of glioma cell lines with deficient Tip60 activity in a synergistic manner. [ABSTRACT FROM AUTHOR]

The DNA‐dependent protein kinase (DNA‐PK) is an abundant nuclear protein that mediates DNA double‐strand break repair by nonhomologous end joining (NHEJ). As such, DNA‐PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA‐PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high‐performance liquid chromatography‐mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid–liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive‐mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1–300 ng/mL for the metabolite and proved to be both accurate (97.3%–103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography‐tandem mass spectroscopy (LC–MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics. [ABSTRACT FROM AUTHOR]

DNA-dependent protein kinase (DNA-PK) is a key effector of non-homologous end joining (NHEJ)-mediated double-strand break (DSB) repair. Since its identification, a substantial body of evidence has demonstrated that DNA-PK is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis in cancer patients. Recent studies have also uncovered novel functions of DNA-PK, shifting the paradigm of the role of DNA-PK in oncogenesis and renewing interest in targeting DNA-PK for cancer therapy. To gain genetic insight into the cellular pathways requiring DNA-PK activity, we used a CRISPR/Cas9 screen to identify genes in which defects cause hypersensitivity to DNA-PK inhibitors. We identified over one hundred genes involved in DNA replication, cell cycle regulation, and RNA processing that promoted cell survival when DNA-PK kinase activity was suppressed. This gene set will be useful for characterizing novel biological processes that require DNA-PK activity and identifying predictive biomarkers of response to DNA-PK inhibition in the clinic. We also validated several genes from this set and reported previously undescribed genes that modulate the response to DNA-PK inhibitors. In particular, we found that compromising the mRNA splicing pathway led to marked hypersensitivity to DNA-PK inhibition, providing a possible rationale for the combined use of splicing inhibitors and DNA-PK inhibitors for cancer therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA-dependent protein kinase (DNA-PK) is a key player in repairing DNA damage. Ku proteins detect DNA damage and activate DNA-PK. Blocking DNA-PK can make cancer treatments such as radiation more effective. We have developed Ku–DNA binding inhibitors (Ku-DBis) that stop DNA-PK activation, making cancer cells more sensitive to radiation. We recently discovered new Ku-DBis that enter cells better than predecessor compounds, allowing cellular and in vivo analyses. Some non-small cell lung cancer (NSCLC) cells, especially those lacking ATM, were very sensitive to these inhibitors and radiation. However, cells lacking BRCA1 were resistant, which reduced the effectiveness of radiation. In animal studies of NSCLC, Ku-DBi treatment inhibited DNA-PK and enhanced a radiation-dependent decrease in tumor cell proliferation. This is the first time a Ku-targeted inhibitor has been shown to work in living organisms, suggesting their potential application in cancer treatment. Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku–DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment. [ABSTRACT FROM AUTHOR]

Our understanding of the DNA damage responses of human cells to radiation has increased remarkably over the recent years although some notable signaling events remain to be discovered. Here we provide a brief account of the key molecular events of the responses to reflect the current understanding of the key underlying mechanisms involved. [ABSTRACT FROM AUTHOR]

Integration of the DNA copy of HIV-1 genome into the cellular genome results in series of damages, repair of which is critical for successful replication of the virus. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate the HIV-1 DNA postintegrational repair, even though integration does not result in DNA double-strand breaks. In this study, we analyzed changes in phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056), and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139), and p53 (pSer15) during the HIV-1 DNA postintegrational repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegrational DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between the double-strand DNA break repair and postintegrational repair of HIV-1 DNA. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients. [ABSTRACT FROM AUTHOR]

Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of the DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the higher localization of the DNA-PK into the nucleus. The finding that cocaine increases the nuclear localization of the DNA-PK provides further support to our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear localization of the DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study demonstrates that the cocaine-induced DNA-PK promotes the hyper-phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both the initiation and elongation phases, respectively, of HIV transcription. The cocaine-mediated enhancement of transcriptional initiation is supported by its activation of cyclin-dependent kinase 7 (CDK7). Additionally, the induction of transcriptional elongation is marked by higher LTR recruitment and the increased phosphorylation of CDK9, which indicates the stimulation of positive transcriptional elongation factor b (P-TEFb). We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observed that the phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the previously observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed the elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results unravel the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV− cases. [ABSTRACT FROM AUTHOR]

Abstract Background Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE‐13) and to investigate their underlying mechanisms. Methods Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA‐repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double‐strand break (DSB) repair were measured by western blot. Results Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation‐induced inhibition of clonogenic survival in ECA109 and TE‐13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE‐13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. Conclusions These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC.

DNA-dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional serine‑threonine protein kinase that plays roles in non-homologous end joining of DNA repair in cells. NU7441 is a specific DNA-PKcs inhibitor. We investigated the effects of NU7441 on the transcriptome of BT549 triple negative breast cancer cells. Total RNA extracted from NU7441-treated or control BT549 cells was processed for preparation of sequencing libraries. Assessment of read quality was performed using fastqc tool. Trimming and filtering low-quality reads were performed using fastp. Reads were aligned by hisat2. SAM files were converted to BAM files using Samtools. The gene differential expression analysis, Gene Ontology (GO) analysis and KEGG pathway analysis were performed. After NU7441 treatment, total number of 2045 differential genes were selected according to |log2(FoldChange)| >= 1 & padj

Background: Due to the high demand for novel approaches for leukemia-targeted therapy, this study investigates the impact of DNA-PK inhibitor NU7441 on the sensitivity of pre-B ALL cells to the telomerase inhibitor MST-312. Methods: The study involved NALM-6 cells treated with MST-312 and NU7441, assessing their viability and metabolic activity using trypan blue and MTT assays. The study also evaluated apoptosis, gene expression changes, and DNA damage using flow cytometry, qRT-PCR, and micronucleus assays. The binding energy of MST-312 in the active site of telomerase was calculated using molecular docking. Results: The study's findings revealed a synergistic decline in both cell viability and metabolic activity in NALM-6 cells when exposed to the combined treatment of MST-312 and NU7441, and this decrease occurred without any adverse effects on healthy PBMC cells. Furthermore, the combination treatment exhibited a significantly higher induction of apoptosis than treatment with MST-312 alone, as observed through flow cytometry assay. qRT-PCR analysis revealed that this enhanced apoptosis was associated with a notable downregulation of Bcl-2 expression and an upregulation of Bax gene expression. Moreover, the combination therapy decreased expression levels of hTERT and c-Myc genes. The micronucleus assay indicated that the combination treatment increased DNA damage in NALM-6 cells. Also, a good conformation between MST-312 and the active site of telomerase was revealed by docking data. Conclusions: The study suggests that simultaneous inhibition of telomerase and DNA-PK in pre-B ALL presents a novel targeted therapy approach. Highlights: MST-312/NU7441 exerts a synergistic cytotoxic effect against NALM-6 cells. NU7441 accentuates MST-312-induced apoptosis of NALM-6 cells. MST-312/NU7441-induced apoptosis progresses through upregulation of the Bax/Bcl-2 ratio. A combination of MST-312/NU7441 downregulates c-Myc and hTERT gene expression. NU7441 increases MST-312-induced DNA damage in NALM-6 cells. [ABSTRACT FROM AUTHOR]

The proteins and protein assemblies involved in DNA repair have been the focus of a multitude of structural studies for the past few decades. Historically, the structures of these protein complexes have been resolved by X-ray crystallography. However, more recently with the advancements in cryo-electron microscopy (cryo-EM) ranging from optimising the methodology for sample preparation to the development of improved electron detectors, the focus has shifted from X-ray crystallography to cryo-EM. This methodological transition has allowed for the structural determination of larger, more complex protein assemblies involved in DNA repair pathways and has subsequently led to a deeper understanding of the mechanisms utilised by these fascinating molecular machines. Here, we review some of the key structural advancements that have been gained in the study of non-homologous end joining (NHEJ) by the use of cryo-EM, with a focus on assemblies composed of DNA-PKcs and Ku70/80 (Ku) and the various methodologies utilised to obtain these structures. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focuses on finding improved treatment strategies for advanced or metastatic synovial sarcoma, which is a rare and aggressive type of soft tissue sarcoma. The researchers tested a combination of two drugs, peposertib and doxorubicin, to see if they could work together to kill synovial sarcoma cells more efficiently. The experiments conducted in cultured cancer cells and mouse models of synovial sarcoma demonstrated that when these drugs were used together, they had a significantly stronger effect against cancer cells compared to using either drug alone. It is noteworthy that the combination could successfully overcome resistance to doxorubicin monotherapy in a patient-derived tumor model. This study also shed light on the underlying molecular mechanisms of this combination effect. Overall, the findings suggest that combining peposertib with doxorubicin could be a promising treatment option for synovial sarcoma patients in the future. Synovial sarcoma is a rare and highly aggressive subtype of soft tissue sarcoma. The clinical challenge posed by advanced or metastatic synovial sarcoma, marked by limited treatment options and suboptimal outcomes, necessitates innovative approaches. The topoisomerase II (Topo II) inhibitor doxorubicin has remained the cornerstone systemic treatment for decades, and there is pressing need for improved therapeutic strategies for these patients. This study highlights the potential to enhance the cytotoxic effects of doxorubicin within well-characterized synovial sarcoma cell lines using the potent and selective DNA-PK inhibitor, peposertib. In vitro investigations unveil a p53-mediated synergistic anti-tumor effect when combining doxorubicin with peposertib. The in vitro findings were substantiated by pronounced anti-tumor effects in mice bearing subcutaneously implanted tumors. A well-tolerated regimen for the combined application was established using both pegylated liposomal doxorubicin (PLD) and unmodified doxorubicin. Notably, the combination of PLD and peposertib displayed enhanced anti-tumor efficacy compared to unmodified doxorubicin at equivalent doses, suggesting an improved therapeutic window—a critical consideration for clinical translation. Efficacy studies in two patient-derived xenograft models of synovial sarcoma, accurately reflecting human metastatic disease, further validate the potential of this combined therapy. These findings align with previous evidence showcasing the synergy between DNA-PK inhibition and Topo II inhibitors in diverse tumor models, including breast and ovarian cancers. Our study extends the potential utility of combined therapy to synovial sarcoma. [ABSTRACT FROM AUTHOR]

Simple Summary: Localized prostate cancer has a favorable prognosis and can be effectively treated with radiotherapy, as well as therapies that target hormone production and signaling. However, if the disease progresses past these treatments, treatment options are limited, and the disease often becomes fatal. In this review, we discuss the changes in treatment as prostate cancer develops, as well as the genetic mutations that accompany advanced prostate cancer. We highlight how these mutations can provide clinical opportunities to manipulate the DNA damage response for therapeutic gain using cancer-specific alterations in the genome and new therapeutic agents that are under development or entering clinical trials. Prostate cancers that progress despite androgen deprivation develop into castration-resistant prostate cancer, a fatal disease with few treatment options. In this review, we discuss the current understanding of prostate cancer subtypes and alterations in the DNA damage response (DDR) that can predispose to the development of prostate cancer and affect its progression. We identify barriers to conventional treatments, such as radiotherapy, and discuss the development of new therapies, many of which target the DDR or take advantage of recurring genetic alterations in the DDR. We place this in the context of advances in understanding the genetic variation and immune landscape of CRPC that could help guide their use in future treatment strategies. Finally, we discuss several new and emerging agents that may advance the treatment of lethal disease, highlighting selected clinical trials. [ABSTRACT FROM AUTHOR]

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins. [ABSTRACT FROM AUTHOR]

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

Background: Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE‐13) and to investigate their underlying mechanisms. Methods: Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA‐repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double‐strand break (DSB) repair were measured by western blot. Results: Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation‐induced inhibition of clonogenic survival in ECA109 and TE‐13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE‐13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. Conclusions: These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC. [ABSTRACT FROM AUTHOR]

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.

Abstract Background DNA‐dependent protein kinase (DNA‐PK) is engaged in DNA damage repair and is significantly expressed in triple negative breast cancer (TNBC). Inhibiting DNA‐PK to reduce DNA damage repair provides a possibility of tumor treatment. NU7441, a DNA‐PK inhibitor, can regulate the function and differentiation of CD4+T cells and effectively enhance immunogenicity of monocyte‐derived dendritic cells. However, the effect of NU7441 on the tumor progression activity of immunosuppressive myeloid‐derived suppressor cells (MDSCs) in TNBC remains unclear. Results In this study, we found that NU7441 alone significantly increased tumor growth in 4 T1 (a mouse TNBC cell line) tumor‐bearing mice. Bioinformatics analysis showed that DNA‐PK and functional markers of MDSCs (iNOS, Arg1, and IDO) tended to coexist in breast cancer patients. The mutations of these genes were significantly correlated with lower survival in breast cancer patients. Moreover, NU7441 significantly decreased the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs), spleen and tumor, but enhanced the immunosuppressive function of splenic MDSCs. Furthermore, NU7441 increased MDSCs' DNA‐PK and pDNA‐PK protein levels in PBMCs and in the spleen and increased DNA‐PK mRNA expression and expression of MDSCs functional markers in splenic MDSCs from tumor‐bearing mice. NU7441 combined with gemcitabine reduced tumor volume, which may be because gemcitabine eliminated the remaining MDSCs with enhanced immunosuppressive ability. Conclusions These findings highlight that the regulation of DNA‐PK activity by NU7441 promotes TNBC progression via enhancing the immunosuppressive function of MDSCs. Moreover, NU7441 combined with gemcitabine offers an efficient therapeutic approach for TNBC and merits deeper investigation.

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. [ABSTRACT FROM AUTHOR]

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy. [ABSTRACT FROM AUTHOR]

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV− cases.

DNA double-strand breaks (DSBs) are a serious form of DNA damage that can cause genetic mutation. On the induction of DSBs, histone H2AX becomes phosphorylated by kinases, including ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). Phosphorylated H2AX (γ-H2AX) can be a platform to recruit DNA repair machinery. Here, we analyzed the immediate early kinetics of γ-H2AX upon laser-induced DNA damage in ATM-proficient and -deficient living cells by using fluorescently labeled antigen-binding fragments specific for γ-H2AX. The accumulation kinetics of γ-H2AX were similar in both ATM-proficient and -deficient cells. However, γ-H2AX accumulation was delayed when the cells were treated with a DNA-PK inhibitor, suggesting that DNA-PK rapidly phosphorylates H2AX at DSB sites. Ku80 (also known as XRCC5), a DNA-PK subunit, diffuses freely in the nucleus without DNA damage, whereas ATM repeatedly binds to and dissociates from chromatin. The accumulation of ATM at damage sites was regulated by the histone H4K16 acetyltransferase MOF (also known as KAT8 in mammals), but its accumulation was not necessarily reflected in the γ-H2AX level. These results suggest distinct actions of ATM and DNA-PK in immediate γ-H2AX accumulation. [ABSTRACT FROM AUTHOR]

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP‐AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro‐inflammatory signaling. We show that the DNA‐PK DNA repair complex (i) drives cGAS‐independent IRF3‐mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS‐dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA‐PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS‐dependent signaling is acquired during tumorigenesis and that cGAS and DNA‐PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity. Synopsis: Upon cytosolic DNA detection, the DNA damage repair kinase DNA‐PK promotes optimal type I Interferon and chemokine production by regulating IRF3 activation and enabling cGAS‐dependent cGAMP production. This cooperation fuels cancer‐mediated inflammation, affecting both tumour initiation and patient prognosis.DNA‐PK promotes STING‐independent IRF3‐dependent type I Interferon responses.DNA‐PK enhances genotoxic stress‐dependent type I Interferon signaling in the absence of cGAS.DNA‐PK and cGAS cooperate for optimal type I Interferon responses and chemokine/cytokine production.DNA‐PK enables cGAS‐dependent cGAMP production.cGAS expression compromises early tumorigenesis but promotes cancer‐associated chronic inflammation. [ABSTRACT FROM AUTHOR]

Background: DNA‐dependent protein kinase (DNA‐PK) is engaged in DNA damage repair and is significantly expressed in triple negative breast cancer (TNBC). Inhibiting DNA‐PK to reduce DNA damage repair provides a possibility of tumor treatment. NU7441, a DNA‐PK inhibitor, can regulate the function and differentiation of CD4+T cells and effectively enhance immunogenicity of monocyte‐derived dendritic cells. However, the effect of NU7441 on the tumor progression activity of immunosuppressive myeloid‐derived suppressor cells (MDSCs) in TNBC remains unclear. Results: In this study, we found that NU7441 alone significantly increased tumor growth in 4 T1 (a mouse TNBC cell line) tumor‐bearing mice. Bioinformatics analysis showed that DNA‐PK and functional markers of MDSCs (iNOS, Arg1, and IDO) tended to coexist in breast cancer patients. The mutations of these genes were significantly correlated with lower survival in breast cancer patients. Moreover, NU7441 significantly decreased the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs), spleen and tumor, but enhanced the immunosuppressive function of splenic MDSCs. Furthermore, NU7441 increased MDSCs' DNA‐PK and pDNA‐PK protein levels in PBMCs and in the spleen and increased DNA‐PK mRNA expression and expression of MDSCs functional markers in splenic MDSCs from tumor‐bearing mice. NU7441 combined with gemcitabine reduced tumor volume, which may be because gemcitabine eliminated the remaining MDSCs with enhanced immunosuppressive ability. Conclusions: These findings highlight that the regulation of DNA‐PK activity by NU7441 promotes TNBC progression via enhancing the immunosuppressive function of MDSCs. Moreover, NU7441 combined with gemcitabine offers an efficient therapeutic approach for TNBC and merits deeper investigation. [ABSTRACT FROM AUTHOR]

The integration of a DNA copy of an HIV-1 RNA genome into the host genome, carried out by the viral enzyme integrase, results in the formation of single-stranded gaps in cellular DNA that must be repaired. Here, we have analyzed the involvement of the PI3K kinases, ATM, ATR, and DNA-PKcs, which are important players in the DNA damage response (DDR) in HIV-1 post-integrational DNA repair (PIR). The participation of the DNA-PK complex in HIV-1 PIR has been previously shown, and the formation of a complex between the viral integrase and the DNA-PK subunit, Ku70, has been found to be crucial for efficient PIR. Now, we have shown that the inhibition of both DNA-PKcs and ATM, but not ATR, significantly reduces PIR efficiency. The activation of both kinases is a sequential process, where one kinase, being activated, activates the other, and it occurs simultaneously with the integration of viral DNA. This fact suggests that the activation of both kinases triggers PIR. Most interestingly, the activation of not only DNA-PKcs, but also ATM depends on the complex formation between integrase and Ku70. The elucidation of the interactions between viruses and DDR is important both for understanding the modulation of host cell functions by these pathogens and for developing new approaches to combat viral infections. [ABSTRACT FROM AUTHOR]

Here we report that simultaneous inhibition of the three primary DNA damage recognition PI3 kinase-like kinases (PIKKs) —ATM, ATR, and DNA-PK— induces severe combinatorial synthetic lethality in mammalian cells. Utilizing Chinese hamster cell lines CHO and V79 and their respective PIKK mutants, we evaluated effects of inhibiting these three kinases on cell viability, DNA damage response, and chromosomal integrity. Our results demonstrate that while single or dual kinase inhibition increased cytotoxicity, inhibition of all three PIKKs results in significantly higher synergistic lethality, chromosomal aberrations, and DNA double-strand break (DSB) induction as calculated by their synergy scores. These findings suggest that the overlapping redundancy of ATM, ATR, and DNA-PK functions is critical for cell survival, and their combined inhibition greatly disrupts DNA damage signaling and repair processes, leading to cell death. This study provides insights into the potential of multi-targeted DDR kinase inhibition as an effective anticancer strategy, necessitating further research to elucidate underlying mechanisms and therapeutic applications. • Simultaneous inhibition of ATM, ATR, and DNA-PK induces synergistic lethality stronger than dual inhibitions of any of them. • Cellular lethality is associated with chromosome aberration induction rather than SCE. • This study suggests that overlapping redundancy of ATM, ATR, and DNA-PK functions in DNA damage responses. [ABSTRACT FROM AUTHOR]

Treatment of locally advanced rectal cancer includes chemoradiation and surgery, but patient response to treatment is variable. Patients who have a complete response have improved outcomes; therefore, there is a critical need to identify mechanisms of resistance to circumvent them. DNA-PK is involved in the repair of DNA double-strand breaks caused by radiation, which we found to be increased in rectal cancer after treatment. We hypothesized that inhibiting this complex with a DNA-PK inhibitor, Peposertib (M3814), would improve treatment response.We assessed pDNA-PK in a rectal cancer cell line and mouse model utilizing western blotting, viability assays, γH2AX staining, and treatment response. The three treatment groups were: standard of care (SOC) (5-fluorouracil (5FU) with radiation), M3814 with radiation, and M3814 with SOC.SOC treatment of rectal cancer cells increased pDNA-PK protein and increased γH2AX foci, but this was abrogated by the addition of M3814. Mice with CT26 tumors treated with M3814 with SOC did not differ in average tumor size but individual tumor response varied. The clinical complete response rate improved significantly with the addition of M3814 but pathological complete response did not. We investigated alterations in DNA repair and found that Kap1 and pATM are increased after M3814 addition suggesting this may mediate resistance.When the DNA-PK inhibitor, M3814, is combined with SOC treatment, response improved in some rectal cancer models but an increase in other repair mechanisms likely diminishes the effect. A clinical trial is ongoing to further explore the role of DNA-PK inhibition in rectal cancer treatment.

Integrin a6b4 is highly expressed in triple negative breast cancer (TNBC) and drives its most aggressive traits; however, its impact on chemotherapeutic efficacy remains untested. We found that integrin a6b4 signaling promoted sensitivity to cisplatin and carboplatin but not to other chemotherapies tested. Mechanistic investigations revealed that integrin a6b4 stimulated the activation of ATM, p53, and 53BP1, which required the integrin b4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin a6b4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we found that in response to cisplatin-induced DNA double strand break (DSB), integrin a6b4 suppressed the homologous recombination (HR) activity and enhanced non-homologous end joining (NHEJ) repair activity. Finally, we discovered that integrin a6b4 preferentially activated DNA-PK, facilitated DNA-PK-p53 and p53-53BP1 complex formation in response to cisplatin and required DNA-PK to enhance ATM, 53BP1 and p53 activation as well as cisplatin sensitivity. In summary, we discovered a novel function of integrin a6b4 in promoting cisplatin sensitivity in TNBC through DNA damage response pathway. [ABSTRACT FROM AUTHOR]

The human body reacts to tissue damage by generating damage-associated molecular patterns (DAMPs) that activate sterile immune responses. To date, little is known about how DAMPs are removed to avoid excessive immune responses. Here, we show that proteasomal dysfunction induces the release of mitochondrial DNA (mtDNA) as a DAMP that activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) pathway and is subsequently degraded through the N-degron pathway. In the resolution phase of sterile immune responses, DNA-dependent protein kinase (DNA-PK) senses cytosolic mtDNA and activates N-terminal (Nt-) arginylation by ATE1 R-transferases. The substrates of Nt-arginylation include the molecular chaperone BiP/GRP78 retrotranslocated from the endoplasmic reticulum (ER). R-BiP, the Nt-arginylated species of BiP, is associated with cytosolic mtDNA to accelerate its targeting to autophagic membranes for lysosomal degradation. Thus, cytosolic mtDNA activates the N-degron pathway to facilitate its own degradation and form a negative feedback loop, by which the cell can turn off sterile immune responses at the right time., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract 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.

DNA damage response inhibitors are widely used anti-cancer agents that have potent activity against tumor cells with deficiencies in various DNA damage response proteins such as BRCA1/2. Inhibition of other proteins in this pathway including PARP, DNA-PK, WEE1, CHK1/2, ATR, or ATM can sensitize cancer cells to radiotherapy and chemotherapy, and such combinations are currently being tested in clinical trials for treatment of many malignancies including breast, ovarian, rectal, and lung cancer. Unrepaired DNA damage induced by DNA damage response inhibitors alone or in combination with radio- or chemotherapy has a direct cytotoxic effect on cancer cells and can also engage anti-cancer innate and adaptive immune responses. DNA damage-induced immune stimulation occurs by a variety of mechanisms including by the cGAS/STING pathway, STAT1 and downstream TRAIL pathway activation, and direct immune cell activation. Whether or not the relative contribution of these mechanisms varies after treatment with different DNA damage response inhibitors or across cancers with different genetic aberrations in DNA damage response enzymes is not well-characterized, limiting the design of optimal combinations with radio- and chemotherapy. Here, we review how the inhibition of key DNA damage response enzymes including PARP, DNA-PK, WEE1, CHK1/2, ATR, and ATM induces innate and adaptive immune responses alone or in combination with radiotherapy, chemotherapy, and/or immunotherapy. We also discuss current progress in the clinical translation of immunostimulatory DNAdamaging treatment regimens and necessary future directions to optimize the immune-sensitizing potential of DNA damage response inhibitors. [ABSTRACT FROM AUTHOR]

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with a Ku70/Ku80 heterodimer constitutes the intact DNA-PK kinase, which is an upstream component of the DNA repair machinery that signals the DNA damage, orchestrates the DNA repair, and serves to maintain genome integrity. Beyond its role in DNA damage repair, the DNA-PK kinase is also implicated in transcriptional regulation and RNA metabolism, with an illuminated impact on tumor progression and therapeutic responses. However, the efforts to identify DNA-PK regulated transcriptomes are limited by short-read sequencing to resolve the full complexity of the transcriptome. Therefore, we leveraged the PacBio Single Molecule, Real-Time (SMRT) Sequencing platform to study the transcriptome after DNA-PK inactivation to further underscore the importance of its role in diseases. Our analysis revealed additional novel transcriptome and complex gene structures in the DNA-PK inactivated cells, identifying 8,355 high-confidence new isoforms from 3,197 annotated genes and 523 novel genes. Among them, 380 lncRNAs were identified. We validated these findings using computational approaches and confirmatory transcript quantification with short-read sequencing. Several novel isoforms representing distinct splicing events have been validated through PCR experiments. Our analyses provide novel insights into DNA-PK function in transcriptome regulation and RNA metabolism. [ABSTRACT FROM AUTHOR]

DNA-dependent protein kinase (DNA-PK) is a key player in the NHEJ repair pathway. DNA-PK and its subunits, Ku70, Ku80, and catalytic subunit (DNA-PKcs), also participate in other cellular processes; however, there are still no systemic data on the effect of depletion of Ku70, Ku80 and DNA-PKcs on cell functions in the same cell line. Here, we analyzed transcriptome changes in HEK 293T cells after depletion of each DNA-PK subunit. Depletion of various DNA-PK subunits resulted in dramatic differences in the number of differentially expressed genes: only 7 genes changed more than 2-fold in DNA-PKcs-deficient cells, 29 genes in Ku80-deficient, 219 genes in Ku70-deficient. All DNA-PKcs-dependent genes were stress-related and depended on both Ku70 and Ku80. Two-thirds of Ku80-dependent genes were also differentially expressed in the Ku70-deficient line. Most Ku70-dependent genes were altered exclusively in Ku70-depleted cells, indicating that Ku70 is involved in the regulation of more processes than Ku80. GO enrichment analysis showed the effect of Ku70 knockdown on cell adhesion and matrix organization, protein degradation, cell proliferation, and differentiation. Depletion of Ku70, but not Ku80, provided greater cell motility and disassembly of cell-cell contacts. These data clearly indicate that Ku70 is more functionally important for the cell life than DNA-PKcs and even Ku80. • Transcriptome analysis of cells with depletion of each of the DNA-PK subunits is done. • Depletion of Ku70 changes expression of more genes than depletion of Ku80. • Depletion of Ku70 increases cell motility and disassembly of cell-cell contacts. [ABSTRACT FROM AUTHOR]

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

DNA damage response inhibitors are widely used anti-cancer agents that have potent activity against tumor cells with deficiencies in various DNA damage response proteins such as BRCA1/2. Inhibition of other proteins in this pathway including PARP, DNA-PK, WEE1, CHK1/2, ATR, or ATM can sensitize cancer cells to radiotherapy and chemotherapy, and such combinations are currently being tested in clinical trials for treatment of many malignancies including breast, ovarian, rectal, and lung cancer. Unrepaired DNA damage induced by DNA damage response inhibitors alone or in combination with radio- or chemotherapy has a direct cytotoxic effect on cancer cells and can also engage anti-cancer innate and adaptive immune responses. DNA damage-induced immune stimulation occurs by a variety of mechanisms including by the cGAS/STING pathway, STAT1 and downstream TRAIL pathway activation, and direct immune cell activation. Whether or not the relative contribution of these mechanisms varies after treatment with different DNA damage response inhibitors or across cancers with different genetic aberrations in DNA damage response enzymes is not well-characterized, limiting the design of optimal combinations with radio- and chemotherapy. Here, we review how the inhibition of key DNA damage response enzymes including PARP, DNA-PK, WEE1, CHK1/2, ATR, and ATM induces innate and adaptive immune responses alone or in combination with radiotherapy, chemotherapy, and/or immunotherapy. We also discuss current progress in the clinical translation of immunostimulatory DNA-damaging treatment regimens and necessary future directions to optimize the immune-sensitizing potential of DNA damage response inhibitors.

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not. Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK's function in repair. • DNA-PK activation occurs in two ways and is controlled by how DNA-PK's DNA End Blocking helix (DEB) interacts with DNA ends. • Different DNA ends promote DNA-PK dimers with distinct functions. • A Ku-80 mediated dimer promotes nucleolytic end-processing. • An XLF-dependent dimer promotes fill-in end processing and the ligation competent NHEJ complex derives from this dimer. [ABSTRACT FROM AUTHOR]

[Display omitted] • The structure and the NHEJ role of DNA-PK were elaborated. • The development progress of DNA-PK inhibitors and their SARs based on different scaffolds were analyzed. • The DNA-PK inhibitors under clinical trials were listed. The deployment of DNA damage response (DDR) combats various forms of DNA damage, ensuring genomic stability. Cancer cells' propensity for genomic instability offers therapeutic opportunities to selectively kill cancer cells by suppressing the DDR pathway. DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is crucial for the non-homologous end joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs). Therefore, targeting DNA-PK is a promising cancer treatment strategy. This review elaborates on the structures of DNA-PK and its related large protein, as well as the development process of DNA-PK inhibitors, and recent advancements in their clinical application. We emphasize our analysis of the development process and structure-activity relationships (SARs) of DNA-PK inhibitors based on different scaffolds. We hope this review will provide practical information for researchers seeking to develop novel DNA-PK inhibitors in the future. [ABSTRACT FROM AUTHOR]

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1–2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. [Display omitted] • DNA-PKcs structurally blocks Ku from sliding into chromatin in human and Xenopus • A neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin • In S phase, ATM/CtIP overcomes Ku accumulation • Without DNA-PKcs, transcription at the DNA end vicinity is inhibited in a Ku-dependent manner The DNA end binding protein Ku can slide onto naked DNA, but this is limited in cells. Using human cells and Xenopus egg extracts, Bossaert, Moreno, et al. identify DNA-PKcs as the main structural barrier to Ku entry into chromatin, along with two active mechanisms that limit Ku accumulation in the absence of DNA-PKcs. [ABSTRACT FROM AUTHOR]

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role. • Mutation of the KBM-binding domain in KU80 increases sensitivity to DNA replication stress • CHO cells expressing KU80 with a mutated KBM-binding domain accumulate high levels of pan-nuclear γH2AX in S/G2 phase • This phenotype is also observed in CHO cells in which the KBM-containing protein WRN is depleted. • These data implicate the KBM-binding domain in KU80 in the response to replication stress, and suggest that this role is facilitated via WRN. [ABSTRACT FROM AUTHOR]

The intra-S-phase checkpoint was among the first reported cell cycle checkpoints in mammalian cells. It transiently slows down the rate of DNA replication after DNA damage to facilitate repair and thus prevents genomic instability. The ionizing radiation (IR)-induced intra-S-phase checkpoint in mammalian cells is thought to be mainly dependent upon the kinase activity of ATM. Defects in the intra-S-phase checkpoint result in radio-resistant DNA synthesis (RDS), which promotes genomic instability. ATM belongs to the PI3K kinase family along with ATR and DNA-PKcs. ATR has been shown to be the key kinase for intra-S-phase checkpoint signaling in yeast and has also been implicated in this checkpoint in higher eukaryotes. Recently, contributions of DNA-PKcs to IR-induced G2-checkpoint could also be established. Whether and how ATR and DNA-PKcs are involved in the IR-induced intra-S-phase checkpoint in mammalian cells is incompletely characterized. Here, we investigated the contributions of ATM, ATR, and DNA-PKcs to intra-S-phase checkpoint activation after exposure to IR of human and hamster cells. The results suggest that the activities of both ATM and ATR are essential for efficient intra-S-phase checkpoint activation. Indeed, in a wild-type genetic background, ATR inhibition generates stronger checkpoint defects than ATM inhibition. Similar to G2 checkpoint, DNA-PKcs contributes to the recovery from the intra-S-phase checkpoint. DNA-PKcs–deficient cells show persistent, mainly ATR-dependent intra-S-phase checkpoints. A correlation between the degree of DSB end resection and the strength of the intra-S-phase checkpoint is observed, which again compares well to the G2 checkpoint response. We conclude that the organization of the intra-S-phase checkpoint has a similar mechanistic organization to that of the G2 checkpoint in cells irradiated in the G2 phase. [ABSTRACT FROM AUTHOR]

Simple Summary: We performed a CRISPR-Cas9 synthetic lethality screen in order to identify molecular targets whose inhibition would synergistically enhance the effect of everolimus in uveal melanoma cells. IGF1R and PRKDC, among others, were identified as hits. We verified these hits effects genetically: we treated the uveal melanoma cell lines depleted of PRKDC or IGF1R with everolimus and, in case of IGF1R, observed a synergistic effect. Additionally, we found synergistic growth inhibition with the inhibitors targeting DNA-PKcs or IGF1R in combination with everolimus. Moreover, we investigated the combination of targeted inhibitors of DNA-PKcs and IGF1R with everolimus on uveal melanoma in an in vivo model. The dual DNA-PKcs/mTOR inhibitor CC-115 demonstrated activity in vivo. Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model. [ABSTRACT FROM AUTHOR]

This study aims at analyzing the impact of the pharmacological inhibition of DNA damage response (DDR) targets (DNA-PK and ATR) on radiosensitization of bladder cancer cell lines of different molecular/histological subtypes. Applying DNA-PK (AZD7648) and ATR (Ceralasertib) inhibitors on SCaBER, J82 and VMCUB-1 bladder cancer cell lines, we revealed sensitization upon ionizing radiation (IR), i.e., the IC50 for each drug shifted to a lower drug concentration with increased IR doses. In line with this, drug exposure retarded DNA repair after IR-induced DNA damage visualized by a neutral comet assay. Western blot analyses confirmed specific inhibition of targeted DDR pathways in the analyzed bladder cancer cell lines, i.e., drugs blocked DNA-PK phosphorylation at Ser2056 and the ATR downstream mediator CHK1 at Ser317. Interestingly, clonogenic survival assays indicated a cell-line-dependent synergism of combined DDR inhibition upon IR. Calculating combined index (CI) values, with and without IR, according to the Chou–Talalay method, confirmed drug- and IR-dose-specific synergistic CI values. Thus, we provide functional evidence that DNA-PK and ATR inhibitors specifically target corresponding DDR pathways retarding the DNA repair process at nano-molar concentrations. This, in turn, leads to a strong radiosensitizing effect and impairs the survival of bladder cancer cells. [ABSTRACT FROM AUTHOR]

High fructose consumption is a significant risking factor for glomerular podocyte injury. However, the causes of high fructose-induced glomerular podocyte injury are still unclear. In this study, we reported a novel mechanism by which high fructose induced ferroptosis, a newly form of programmed cell death, in glomerular podocyte injury. We performed quantitative proteomic analysis in glomeruli of high fructose-fed rats to identify key regulating proteins involved in glomerular injury, and found that mitochondrial single-strand DNA-binding protein 1 (SSBP1) was markedly upregulated. Depletion of SSBP1 could alleviate high fructose-induced ferroptotic cell death in podocytes. Subsequently, we found that SSBP1 positively regulated a transcription factor p53 by interacting with DNA-dependent protein kinase (DNA-PK) and p53 to drive ferroptosis in high fructose-induced podocyte injury. Mechanically, SSBP1 activated DNA-PK to induce p53 phosphorylation at serine 15 (S15) to promote the nuclear accumulation of p53, and thereby inhibited expression of ferroptosis regulator solute carrier family 7 member 11 (SLC7A11) in high fructose-exposed podocytes. Natural antioxidant pterostilbene was showed to downregulate SSBP1 and then inhibit DNA-PK/p53 pathway in its alleviation of high fructose-induced glomerular podocyte ferroptosis and injury. This study identified SSBP1 as a novel intervention target against high fructose-induced podocyte ferroptosis and suggested that the suppression of SSBP1 by pterostilbene may be a potential therapy for the treatment of podocyte ferroptosis in glomerular injury.

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.