Search Results (33)

Glioblastoma is the most common primary brain tumor in adults. Our previous studies revealed a functional interplay of myelin transcription factor 1-like (MYT1L) with the DNA-dependent protein kinase (DNA-PK) in the regulation of p21 transcription. However, the contributing role of this functional interplay in glioblastoma remains largely unknown. Here, we used cell lines with normal DNA-PK (HEK293 and M059K) or deficient DNA-PK (M059J) as a model system to demonstrate the importance of the DNA-PK-dependent activation of MYT1L in controlling the transcription of CXC chemokine receptor 1 (CXCR1) in a positive-feedback proliferative signaling loop in glioblastoma with numerous conventional techniques. In normal DNA-PK cells, MYT1L acted as an oncogene by promoting cell proliferation, inhibiting apoptosis, and shortening a cell cycle S phase. However, in DNA-PK-deficient cells, MYT1L functioned as a tumor suppressor by inhibiting cell proliferation and inducing a G1 arrest. The enforced expression of MYT1L promoted CXCR1 transcription in DNA-PK-normal cells but attenuated transcription in DNA-PK-deficient cells. Bioinformatics analysis predicted a MYT1L-binding sequence at the CXCR1 promoter. The functional dependence of MYT1L on DNA-PK in CXCR1 transcription was validated by luciferase assay. Although the expression of CXCR1 was lower in M059J cells as compared to M059K cells, it was higher than in normal brain tissue. The CXCR1 ligands interleukin 8 (IL-8) and GRO protein alpha (GROα) expressed in M059J and M059K cells may signal through the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway that can be blocked by CXCR1 siRNA. Our findings demonstrate the existence of a positive feedback DNA-PK/MYT1L-CXCR1-ERK1/2 proliferation loop in glioblastoma cells that may represent a pharmacological target loop for therapeutic intervention. Full article

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. Full article

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. Full article

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. Full article

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor. Full article

Ataxia-telangiectasia mutated gene (ATM) is a key component of the DNA damage response (DDR) and double-strand break repair pathway. The functional loss of ATM (ATM deficiency) is hypothesised to enhance sensitivity to DDR inhibitors (DDRi). Whole-exome sequencing (WES), immunohistochemistry (IHC), and Western blotting (WB) were used to characterise the baseline ATM status across a panel of ATM mutated patient-derived xenograft (PDX) models from a range of tumour types. Antitumour efficacy was assessed with poly(ADP-ribose)polymerase (PARP, olaparib), ataxia- telangiectasia and rad3-related protein (ATR, AZD6738), and DNA-dependent protein kinase (DNA-PK, AZD7648) inhibitors as a monotherapy or in combination to associate responses with ATM status. Biallelic truncation/frameshift ATM mutations were linked to ATM protein loss while monoallelic or missense mutations, including the clinically relevant recurrent R3008H mutation, did not confer ATM protein loss by IHC. DDRi agents showed a mixed response across the PDX’s but with a general trend toward greater activity, particularly in combination in models with biallelic ATM mutation and protein loss. A PDX with an ATM splice-site mutation, 2127T > C, with a high relative baseline ATM expression and KAP1 phosphorylation responded to all DDRi treatments. These data highlight the heterogeneity and complexity in describing targetable ATM-deficiencies and the fact that current patient selection biomarker methods remain imperfect; although, complete ATM loss was best able to enrich for DDRi sensitivity. Full article

Minute Virus of Mice (MVM) is an autonomous parvovirus of the Parvoviridae family that replicates in mouse cells and transformed human cells. MVM genomes localize to cellular sites of DNA damage with the help of their essential non-structural phosphoprotein NS1 to establish viral replication centers. MVM replication induces a cellular DNA damage response that is mediated by signaling through the ATM kinase pathway, while inhibiting induction of the ATR kinase signaling pathway. However, the cellular signals regulating virus localization to cellular DNA damage response sites has remained unknown. Using chemical inhibitors to DNA damage response proteins, we have discovered that NS1 localization to cellular DDR sites is independent of ATM or DNA-PK signaling but is dependent on ATR signaling. Pulsing cells with an ATR inhibitor after S-phase entry leads to attenuated MVM replication. These observations suggest that the initial localization of MVM to cellular DDR sites depends on ATR signaling before it is inactivated by vigorous virus replication. Full article

In this work, we describe the synthesis of new macrocycles derived from 3-phenyl-1,2,4-triazole-5-thione 1 in a heterogeneous medium using liquid–solid phase transfer catalysis (PTC) conditions. The structures of the two compounds (3 and 4) isolated were elucidated based on spectral data (¹H-NMR, ¹³C-NMR) and confirmed in the case of 3-phenyl-1,2,4-triazolo [3,4-h]-13,4--thiaza-11-crown-4 (3) by a single-crystal X-ray diffraction analysis. Furthermore, the experimental spectral and the X-ray geometrical parameters were compared with their corresponding predicted ones obtained at the B3LYP/6-311++G(d,p) level of theory. The intercontacts between crystal units were investigated through Hirshfeld surface analysis. The drug-like macrocycles were predicted using ADMET and drug-likeness properties, which showed that 3 may act as an inhibitor of DNA-dependent protein kinase (DNA-PK). This assumption was confirmed by the well-binding fitting of 3 into the binding site of DNA-PK and the formation of a stable 3-DNA-PK complex with a binding energy of −7 kcal-mol⁻¹. Finally, the anticancer activity of 3 was assessed by an MTT assay against A549 cells, which showed that 3 has moderate anticancer activity compared to that of the doxorubicin reference drug. Full article

Lung cancer is considered the most commonly diagnosed cancer and one of the leading causes of death globally. Despite the responses from small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) patients to conventional chemo- and radiotherapies, the current outcomes are not satisfactory. Recently, novel advances in DNA sequencing technologies have started to take off which have provided promising tools for studying different tumors for systematic mutation discovery. To date, a limited number of DDR inhibition trials have been conducted for the treatment of SCLC and NSCLC patients. However, strategies to test different DDR inhibitor combinations or to target multiple pathways are yet to be explored. With the various biomarkers that have either been recently discovered or are the subject of ongoing investigations, it is hoped that future trials would be designed to allow for studying targeted treatments in a biomarker-enriched population, which is defensible for the improvement of prognosis for SCLC and NSCLC patients. This review article sheds light on the different DNA repair pathways and some of the inhibitors targeting the proteins involved in the DNA damage response (DDR) machinery, such as ataxia telangiectasia and Rad3-related protein (ATR), DNA-dependent protein kinase (DNA-PK), and poly-ADP-ribose polymerase (PARP). In addition, the current status of DDR inhibitors in clinical settings and future perspectives are discussed. Full article

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling. Full article

DNA double-strand break (DSB) is considered the most deleterious type of DNA damage, which is generated by ionizing radiation (IR) and a subset of anticancer drugs. DNA-dependent protein kinase (DNA-PK), which is composed of a DNA-PK catalytic subunit (DNA-PKcs) and Ku80-Ku70 heterodimer, acts as the molecular sensor for DSB and plays a pivotal role in DSB repair through non-homologous end joining (NHEJ). Cells deficient for DNA-PKcs show hypersensitivity to IR and several DNA-damaging agents. Cellular sensitivity to IR and DNA-damaging agents can be augmented by the inhibition of DNA-PK. A number of small molecules that inhibit DNA-PK have been developed. Here, the development and evolution of inhibitors targeting DNA-PK for cancer therapy is reviewed. Significant parts of the inhibitors were developed based on the structural similarity of DNA-PK to phosphatidylinositol 3-kinases (PI3Ks) and PI3K-related kinases (PIKKs), including Ataxia-telangiectasia mutated (ATM). Some of DNA-PK inhibitors, e.g., NU7026 and NU7441, have been used extensively in the studies for cellular function of DNA-PK. Recently developed inhibitors, e.g., M3814 and AZD7648, are in clinical trials and on the way to be utilized in cancer therapy in combination with radiotherapy and chemotherapy. Full article

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

The DNA-dependent protein kinase (DNA-PK) is composed of a DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/Ku80 heterodimer. DNA-PK is thought to act as the “sensor” for DNA double-stranded breaks (DSB), which are considered the most deleterious type of DNA damage. In particular, DNA-PKcs and Ku are shown to be essential for DSB repair through nonhomologous end joining (NHEJ). The phenotypes of animals and human individuals with defective DNA-PKcs or Ku functions indicate their essential roles in these developments, especially in neuronal and immune systems. DNA-PKcs are structurally related to Ataxia–telangiectasia mutated (ATM), which is also implicated in the cellular responses to DSBs. DNA-PKcs and ATM constitute the phosphatidylinositol 3-kinase-like kinases (PIKKs) family with several other molecules. Here, we review the accumulated knowledge on the functions of DNA-PKcs, mainly based on the phenotypes of DNA-PKcs-deficient cells in animals and human individuals, and also discuss its relationship with ATM in the maintenance of genomic stability. Full article

Metastatic cancers resistant to immunotherapy require novel management strategies. DNA damage response (DDR) proteins, including ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), have been promising therapeutic targets for decades. Specific, potent DDR inhibitors (DDRi) recently entered clinical trials. Surprisingly, preclinical studies have now indicated that DDRi may stimulate anti-tumor immunity to augment immunotherapy. The mechanisms governing how DDRi could promote anti-tumor immunity are not well understood; however, early evidence suggests that they can potentiate immunogenic cell death to recruit and activate antigen-presenting cells to prime an adaptive immune response. Merkel cell carcinoma (MCC) is well suited to test these concepts. It is inherently immunogenic as ~50% of patients with advanced MCC persistently benefit from immunotherapy, making MCC one of the most responsive solid tumors. As is typical of neuroendocrine cancers, dysfunction of p53 and Rb with upregulation of Myc leads to the very rapid growth of MCC. This suggests high replication stress and susceptibility to DDRi and DNA-damaging agents. Indeed, MCC tumors are particularly radiosensitive. Given its inherent immunogenicity, cell cycle checkpoint deficiencies and sensitivity to DNA damage, MCC may be ideal for testing whether targeting the intersection of the DDR checkpoint and the immune checkpoint could help patients with immunotherapy-refractory cancers. Full article

CC-115 is a dual inhibitor of the mechanistic target of rapamycin (mTOR) kinase and the DNA-dependent protein kinase (DNA-PK) that is currently being studied in phase I/II clinical trials. DNA-PK is essential for the repair of DNA-double strand breaks (DSB). Radiotherapy is frequently used in the palliative treatment of metastatic melanoma patients and induces DSBs. Melanoma cell lines and healthy-donor skin fibroblast cell lines were treated with CC-115 and ionizing irradiation (IR). Apoptosis, necrosis, and cell cycle distribution were analyzed. Colony forming assays were conducted to study radiosensitizing effects. Immunofluorescence microscopy was performed to determine the activity of homologous recombination (HR). In most of the malign cell lines, an increasing concentration of CC-115 resulted in increased cell death. Furthermore, strong cytotoxic effects were only observed in malignant cell lines. Regarding clonogenicity, all cell lines displayed decreased survival fractions during combined inhibitor and IR treatment and supra-additive effects of the combination were observable in 5 out of 9 melanoma cell lines. CC-115 showed radiosensitizing potential in 7 out of 9 melanoma cell lines, but not in healthy skin fibroblasts. Based on our data CC-115 treatment could be a promising approach for patients with metastatic melanoma, particularly in the combination with radiotherapy. Full article