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Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNAdamage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline. [ABSTRACT FROM AUTHOR]

Abstract Background Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. Methods Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. Results DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. Conclusions Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Abstract Background The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks. Methods Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage. Results Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett’s cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. Conclusions IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends.

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

Background: Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it also has roles in modulating gene expression and thus, the overall cellular response to DNA damage. Methods: Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Results: Downregulated genes were mostly involved in protein, sugar, and nucleic acid biosynthesis pathways in both genotypes, but enriched biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Finally, we have used qPCR to validate selected genes among the differentially regulated pathways to validate RNA sequence data. Conclusion: Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trials as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment., Competing Interests: Author JS was employed by the National Center for Genome Resources. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ali, Najaf-Panah, Pyper, Lujan, Sena and Ashley.)

Renal cell carcinoma (RCC) is one of the most aggressive urological malignancies and has a poor prognosis, especially in patients with metastasis. Although RCC is traditionally considered to be radioresistant, radiotherapy (RT) is still a common treatment for palliative management of metastatic RCC. Novel approaches are urgently needed to overcome radioresistance of RCC. Black phosphorus quantum dots (BPQDs) have recently received great attention due to their unique physicochemical properties and good biocompatibility. In the present study, we found that BPQDs enhance ionizing radiation (IR)-induced apoptotic cell death of RCC cells. BPQDs treatment significantly increases IR-induced DNA double-strand breaks (DSBs), as indicated by the neutral comet assay and the DSBs biomarkers γH2AX and 53BP1. Mechanistically, BPQDs can interact with purified DNA–protein kinase catalytic subunit (DNA-PKcs) and promote its kinase activity in vitro. BPQDs impair the autophosphorylation of DNA-PKcs at S2056, and this site phosphorylation is essential for efficient DNA DSBs repair and the release of DNA-PKcs from the damage sites. Consistent with this, BPQDs suppress nonhomologous end-joining (NHEJ) repair and lead to sustained high levels of autophosphorylated DNA-PKcs on the damaged sites. Moreover, animal experiments indicate that the combined approach with both BPQDs and IR displays better efficacy than monotreatment. These findings demonstrate that BPQDs have potential applications in radiosensitizing RCC cells.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3′-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18–24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes. [ABSTRACT FROM AUTHOR]

The presence of endotoxemia is strongly linked to the development of endothelial dysfunction and disruption of myocardial microvascular reactivity. These factors play a crucial role in the progression of endotoxemic cardiomyopathy. Sepsis-related multiorgan damage involves the participation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). However, whether DNA-PKcs contributes to endothelial dysfunction and myocardial microvascular dysfunction during endotoxemia remains unclear. Hence, we conducted experiments in mice subjected to lipopolysaccharide (LPS)-induced endotoxemic cardiomyopathy, as well as assays in primary mouse cardiac microvascular endothelial cells. Results showed that endothelial-cell-specific DNA-PKcs ablation markedly attenuated DNA damage, sustained microvessel perfusion, improved endothelial barrier function, inhibited capillary inflammation, restored endothelium-dependent vasodilation, and improved heart function under endotoxemic conditions. Furthermore, we show that upon LPS stress, DNA-PKcs recognizes a TQ motif in cofilin2 and consequently induces its phosphorylation at Thr 25 . Phosphorylated cofilin2 shows increased affinity for F-actin and promotes F-actin depolymerization, resulting into disruption of the endothelial barrier integrity, microvascular inflammation, and defective eNOS-dependent vasodilation. Accordingly, cofilin2-knockin mice expressing a phospho-defective (T25A) cofilin2 mutant protein showed improved endothelial integrity and myocardial microvascular function upon induction of endotoxemic cardiomyopathy. These findings highlight a novel mechanism whereby DNA-PKcs mediates cofilin2 Thr25 phosphorylation and subsequent F-actin depolymerization to contribute to endotoxemia-related cardiac microvascular dysfunction., Competing Interests: Competing interests: The authors declare no conflict of interest in the present work., (Copyright © 2024 Yingzhen Du et al.)

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a member of the phosphatidylinositol 3-kinase related kinase family, which can phosphorylate more than 700 substrates. As the core enzyme, DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to play crucial roles in cellular DNA damage response (DDR). Once DNA double strand breaks (DSBs) occur in the cells, DNA-PKcs is promptly recruited into damage sites and activated. DNA-PKcs is auto-phosphorylated and phosphorylated by Ataxia-Telangiectasia Mutated at multiple sites, and phosphorylates other targets, participating in a series of DDR and repair processes, which determine the cells’ fates: DSBs NHEJ repair and pathway choice, replication stress response, cell cycle checkpoints, telomeres length maintenance, senescence, autophagy, etc. Due to the special and multi-faceted roles of DNA-PKcs in the cellular responses to DNA damage, it is important to precisely regulate the formation and dynamic of its functional complex and activities for guarding genomic stability. On the other hand, targeting DNA-PKcs has been considered as a promising strategy of exploring novel radiosensitizers and killing agents of cancer cells. Combining DNA-PKcs inhibitors with radiotherapy can effectively enhance the efficacy of radiotherapy, offering more possibilities for cancer therapy.

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters--PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQR Xlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQR Xlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation. [ABSTRACT FROM AUTHOR]

Abstract Precise biomarkers are needed to guide better diagnostics and therapeutics for basal-like breast cancer, for which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has been recently reported by the Clinical Proteomic Tumor Analysis Consortium as the most specific biomarker. We evaluated DNA-PKcs expression in clinically-annotated breast cancer tissue microarrays and correlated results with immune biomarkers (training set: n = 300; validation set: n = 2401). Following a pre-specified study design per REMARK criteria, we found that high expression of DNA-PKcs was significantly associated with stromal and CD8 + tumor infiltrating lymphocytes. Within the basal-like subtype, tumors with low DNA-PKcs and high tumor-infiltrating lymphocytes displayed the most favourable survival. DNA-PKcs expression by immunohistochemistry identified estrogen receptor-positive cases with a basal-like gene expression subtype. Non-silent mutations in PRKDC were significantly associated with poor outcomes. Integrating DNA-PKcs expression with validated immune biomarkers could guide patient selection for DNA-PKcs targeting strategies, DNA-damaging agents, and their combination with an immune-checkpoint blockade.

Modulation of T cell activity is an effective strategy for the treatment of autoimmune diseases, immune‐related disorders and cancer. This highlights a critical need for the identification of proteins that regulate T cell function. The kinase DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) is emerging as a potent regulator of the immune system, spurring interest in its use as a therapeutic target. In murine models of immune‐related diseases including asthma and rheumatoid arthritis, treatment with small‐molecule DNA‐PKcs inhibitors decreased the disease severity. Additionally, DNA‐PKcs inhibitors reduced T cell‐mediated graft rejection in a murine allogenic skin graft model. These in vivo studies suggest the use of DNA‐PKcs inhibitors as immunotherapy for autoimmune and T cell‐mediated disorders. In this study, we sought to characterize further the effects of DNA‐PKcs inhibitors on T cells to better understand their clinical potential. We determined that inhibition of DNA‐PKcs using inhibitor NU7441 and the inhibitors currently in clinical trials for cancer therapy, M3184 and AZD7648, abrogated the activation of murine and human CD4+ and CD8+ T cells as evidenced by the reduced expression of the activation markers CD69 and CD25. Furthermore, inhibition of DNA‐PKcs impeded metabolic pathways and the proliferation of activated T cells. This reduced the ability of OTI‐CD8+ T cells to kill cancer cells and the expression of IFNγ and cytotoxic genes. These results highlight a critical role for DNA‐PKcs in T cells and validate future studies using DNA‐PKcs inhibitors as immune modulation therapy for the treatment of immune‐related diseases. [ABSTRACT FROM AUTHOR]

Abstract Radioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

Introduction: Radiation‐induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial–mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) exhibits strong repair capacity when cells undergo radiation‐induced damage; whether DNA‐PKcs regulates EMT during RIPF remains unclear. Objectives: To investigate the role and molecular mechanism of DNA‐PKcs in RIPF and provide an important theoretical basis for utilising DNA‐PKcs‐targeted drugs for preventing RIPF. Methods: DNA‐PKcs knockout (DPK−/−) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20 Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA‐PKcs‐targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR. Results: The expression of DNA‐PKcs is low in pulmonary fibrosis (PF) patients. DNA‐PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA‐PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA‐PKcs and Twist1. High Twist1 expression and EMT‐associated changes caused by DNA‐PKcs deletion were blocked by insulin‐like growth factor‐1 (IGF‐1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR‐induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA‐PKcs. Conclusion: Our study clarified the critical role and mechanism of DNA‐PKcs in RIPF and showed that it could be a potential target for preventing RIPF. [ABSTRACT FROM AUTHOR]

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.

Abstract Radioresistance is the main obstacle in the clinical management of nasopharyngeal carcinoma (NPC). linc00312 is deregulated in a number of human cancers, including NPC. However, the detailed functions and underlying mechanisms of linc00312 in regulating radiosensitivity of NPC remains unknown. In this study, cox regression analysis was used to assess the association between linc00312 and NPC patients’ survival after radiotherapy. Our results reveal that linc00312 is significantly down-regulated in NPC tissues and patients with higher expression of linc00312 are significantly associated with longer overall survival and better short-term radiotherapy efficacy. Overexpression of linc00312 could increase the sensitivity of NPC cells to ionizing radiation, as indicated by clonogenic survival assay, comet assay, and flow cytometry. Mechanistically, RNA pull down and RNA immunoprecipitation were performed to investigate the binding proteins of linc00312. linc00312 directly binds to DNA-PKcs, hinders the recruitment of DNA-PKcs to Ku80, and inhibits phosphorylation of AKT–DNA–PKcs axis, therefore inhibiting the DNA damage signal sensation and transduction in the NHEJ repair pathway. In addition, linc00312 impairs DNA repair and cell cycle control by suppressing MRN–ATM–CHK2 signal and ATR–CHK1 signal. In summary, we identified DNA-PKcs as the binding protein of linc00312 and revealed a novel mechanism of linc00312 in the DNA damage response, providing evidence for a potential therapeutic strategy in NPC.

Abstract The activation of DNA‐dependent kinase (DNA‐PKcs) upon DNA damage contains a cascade of reactions, covering acetylation by TIP60, binding with Ku70/80, and autophosphorylation. However, how cells regulate TIP60‐mediated acetylation of DNA‐PKcs and the following DNA‐PKcs activation upon DNA damage remains obscure. This present study reported that TIP60 is hyper‐SUMOylated in normal conditions, but upon irradiation‐induced DNA damage, small ubiquitin‐like modifier (SUMO)‐specific protease 3 (SENP3)‐mediated deSUMOylation of TIP60 promoted its interaction with DNA‐PKcs to form the TIP60‐DNA‐PKcs complex. We show that TIP60 SUMOylation is reduced quickly in response to DNA damage and the deSUMOylation of TIP60 by SENP3 is required for DNA‐PKcs acetylation and its autophosphorylation. Comet and γH2AX immunofluorescence assay showed that knockdown of SENP3 impaired DNA damage repair. Using the NHEJ report system, we found that knockdown of SENP3 affected the efficiency of NHEJ. Further exploration using clonogenic survival assay, cell viability assay and cytoflow assay suggested that leaking SENP3 increased the sensitivity of tumour cells to serval DNA damage treatment. Overall, our findings revealed a previously unidentified role of SENP3 in regulating DNA‐PKcs activity and DNA damage repair.