Topoisomerase IIβ Research Articles

Discovery of 2‐Amino‐4H‐1, 3, 4‐thiadiazine‐5(6H)‐one Derivatives and Their In Vitro Antitumor Investigation

AbstractNovel 2‐Amino‐4H‐1,3,4‐thiadiazine‐5‐(6H)‐one derivatives 3–6 were designed, synthesized, and assessed for their anticancer effects on MCF‐7 and A549 cell lines. The cytotoxicity screening found several active compounds. Meanwhile, compound 5 b exhibited the strongest cytotoxic activity compared to Doxorubicin (DOX). Furthermore, DNA flow cytometry investigation over MCF‐7 cells indicated that compound 5 b demonstrated arrest at G1/S stages of the cell cycle and induction of apoptosis by rising pre‐G1 stage. In addition, the underlying mechanistic studies of compound 5b′s proapoptotic activities revealed that they induced the elevation of P53 and the BAX/BCL‐2 ratio expression that led to a rise in active Caspase 3/7 levels and triggered apoptosis. Finally, Compound 5 b displayed a significant dual topoisomerase IIβ enzyme and β‐tubulin polymerization inhibition.

TOPBP1 regulates resistance of gastric cancer to oxaliplatin by promoting transcription of PARP1

Gastric cancer (GC) is the third leading cause of cancer-associated mortality worldwide. The platinum derivative oxaliplatin is widely applied in standard GC chemotherapy but recurrence and metastasis are common in advanced GC cases due to intrinsic or induced chemoresistance. Poly(ADP-Ribose) polymerase 1 (PARP1) is an enzyme crucial for repairing DNA damage induced by platinum compounds, which undermines the effectiveness of platinum-based chemotherapy. Data from the current study showed that topoisomerase IIβ binding protein 1 (TOPBP1), an interacting partner of topoisomerase IIβ, is highly expressed in oxaliplatin-resistant GC (OR-GC) cells and promotes PARP1 transcription through direct binding to its proximal promoter region. Furthermore, AKT-mediated phosphorylation of TOPBP1 at Ser1159 was indispensable for inducing PARP1 expression in OR-GC cells. Disruption of the TOPBP1/PARP1 regulatory pathway decreased cell viability and augmented apoptosis of OR-GC cells. The positive correlation between TOPBP1 and PARP1 was confirmed using both the TCGA database and immunohistochemical analysis of GC tissues. In GC patients receiving oxaliplatin treatment, high expression of TOPBP1 or PARP1 was associated with poor prognosis. Our finding that the TOPBP1/PARP1 pathway facilitates acquisition of oxaliplatin resistance uncovers a novel mechanism underlying platinum-based chemotherapy resistance in gastric cancer that may be utilized for developing effective therapeutic strategies.

Disease-associated mutations in topoisomerase IIβ result in defective NK cells

Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.

Synthesis and Evaluation of the Antibacterial and Antioxidant Activities of Some Novel Chloroquinoline Analogs

Quinoline heterocycle is a useful scaffold to develop bioactive molecules used as anticancer, antimalaria, and antimicrobials. Inspired by their numerous biological activities, an attempt was made to synthesize a series of novel 7-chloroquinoline derivatives, including 2,7-dichloroquinoline-3-carbonitrile (5), 2,7-dichloroquinoline-3-carboxamide (6), 7-chloro-2-methoxyquinoline-3-carbaldehyde (7), 7-chloro-2-ethoxyquinoline-3-carbaldehyde (8), and 2-chloroquinoline-3-carbonitrile (12) by the application of Vilsmeier–Haack reaction and aromatic nucleophilic substitution of 2,7-dichloroquinoline-3-carbaldehyde. The carbaldehyde functional group was transformed into nitriles using POCl3 and NaN3, which was subsequently converted to amide using CH3CO2H and H2SO4. The compounds synthesized were screened for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes. Compounds 6 and 8 showed good activity against E. coli with an inhibition zone of 11.00 ± 0.04 and 12.00 ± 0.00 mm, respectively. Compound 5 had good activity against S. aureus and P. aeruginosa with an inhibition zone of 11.00 ± 0.03 mm relative to standard amoxicillin (18 ± 0.00 mm). Compound 7 displayed good activity against S. pyogenes with an inhibition zone of 11.00 ± 0.02 mm. The radical scavenging activity of these compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), and compounds 5 and 6 displayed the strongest antioxidant activity with IC50 of 2.17 and 0.31 µg/mL relative to ascorbic acid (2.41 µg/mL), respectively. The molecular docking study of the synthesized compounds was conducted to investigate their binding pattern with topoisomerase IIβ and E. coli DNA gyrase B. Compounds 6 (−6.4 kcal/mol) and 8 (−6.6 kcal/mol) exhibited better binding affinity in their in silico molecular docking against E. coli DNA gyrase. The synthesized compounds were also found to have minimum binding energy ranging from −6.9 to −7.3 kcal/mol against topoisomerase IIβ. The SwissADME predicted results showed that the synthesized compounds 5–8 and 12 satisfy Lipinski’s rule of five with zero violations. The ProTox-II predicted organ toxicity results revealed that all the synthesized compounds were inactive in hepatotoxicity, immunotoxicity, mutagenicity, and cytotoxicity. The findings of the in vitro antibacterial and molecular docking analysis suggested that compound 8 might be considered a hit compound for further analysis as antibacterial and anticancer drug. The radical scavenging activity displayed by compounds 5 and 6 suggests these compounds as a radical scavenger.

Design, Synthesis, Cytotoxic Screening and Molecular Docking Studies of Novel Hybrid Thiosemicarbazone Derivatives as Anticancer Agents.

Thiosemicarbazones have been the focus of scientists owing to their broad clinical anticancer range. Herein, A Series of new thiosemicarbazone derivatives 5-9 were synthesized and confirmed through the use of different spectroscopic techniques along with elemental analysis. The in vitro cytotoxic activity of compounds 5-9 against MCF-7 and A549 cell lines and normal breast cells were assessed. Several compounds were found to be active. The most active compound 7 caused MCF-7 cell cycle arrest at G1/ S phases; and induced apoptosis at the pre-G1 phase. The apoptosis-inducing activity of compound 7 was proofed by the elevation of caspase 3/7 activity and also by up-regulation of the expression of Bax and p53 proteins together with the down-regulation of the expression of the Bcl-2 protein. It also had a strong inhibitory effect topoisomerase IIβ enzyme. Molecular Docking study revealed that the synthesized compounds had good docking scores compared to the standard drug Etoposide towards the topoisomerase IIβ protein (3QX3). Overall, these findings confirmed that the new thiosemicarbazone derivatives could aid in the development of promising cancer drug candidates.

Jadomycins: A potential chemotherapy for multi-drug resistant metastatic breast cancer.

Breast cancer causes the most cancer fatalities in women worldwide. Approximately one‐third of breast cancers metastasize, or spread from primary tumors to other tissues, and have a 70% 5‐year mortality rate. Current breast cancer treatments like doxorubicin and paclitaxel become ineffective when breast cancer cells develop multi‐drug resistance and overexpress ATP‐binding cassette transporters, as the transporters cause a substantial efflux of the chemotherapies. Jadomycins, a group of molecules isolated from Streptomyces venezuelae ISP5230, are shown to be cytotoxic against a variety of cancers, especially breast cancer. Furthermore, jadomycins retain their cytotoxic properties in multi‐drug resistant breast cancer cells, as they are not expelled through ATP‐binding cassette transporters. Here, we describe the research that supports the potential use of jadomycins as a novel chemotherapy in the treatment of multi‐drug resistant, metastatic breast cancer. We present the supportive findings, as well as the mechanisms of action investigated thus far. These include copper‐mediated reactive oxygen species generation, aurora B kinase inhibition, and topoisomerase IIα and IIβ inhibition. We also suggest future directions of jadomycin research, which will help to determine if jadomycins can be used as a breast cancer chemotherapy in clinical practice.

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Evaluation of Synthetic 2,4-Disubstituted-benzo[g]quinoxaline Derivatives as Potential Anticancer Agents.

A new series of 2,4-disubstituted benzo[g]quinoxaline molecules have been synthesized, using naphthalene-2,3-diamine and 1,4-dibromonaphthalene-2,3-diamine as the key starting materials. The structures of the new compounds were confirmed by spectral data along with elemental microanalyses. The cytotoxic activity of all synthesized benzo[g]quinoxaline derivatives was assessed in vitro against the breast MCF-7 cancer cell line. The tested molecules revealed good cytotoxicity toward the breast MCF-7 cancer cell line, especially compound 3. The results of topoisomerase IIβ inhibition assay revealed that compound 3 exhibits potent inhibitory activity in submicromolar concentration. Additionally, compound 3 was found to cause pre-G1 apoptosis, and slightly increase the cell population at G1 and S phases of the cell cycle profile in MCF-7 cells. Finally, compound 3 induces apoptosis via Bax activation and downregulation of Bcl2, as revealed by ELISA assay.

Prodrug of ICRF-193 provides promising protective effects against chronic anthracycline cardiotoxicity in a rabbit model in vivo.

The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane (DEX) has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented tumor suppressor p53 (p53)-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIβ (TOP2B) inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.

Monascin and monascinol, azaphilonoid pigments from Mortierella polycephala AM1: in silico and in vitro targeting of the angiogenic VEGFR2 kinase

The fungus, Mortierella polycephala is one of the most productive sources of anticancer bioactive compounds namely those of pigment nature. During our investigation of the produced bioactive metabolites by the terrestrial M. polycephala AM1 isolated from Egyptian poultry feather waste, two main azaphilonoid pigments, monascin (1) and monascinol (2) were obtained as major products; their structures were identified by 1D (1H&13C) and 2D (1H-1H COSY, HMBC) NMR and HRESI-MS spectroscopic data. Biologically, cytotoxic activities of these compounds were broadly studied compared with the fungal extract. To predict the biological target for the presumed antitumor activity, an in silico study was run toward three proteins, topoisomerase IIα, topoisomerase IIβ, and VEGFR2 kinase. Monascinol (2) was expected to be moderately active against VEGFR2 kinase without any anticipated inhibition toward topo II isoforms. The in vitro study confirmed the docked investigation consistently and introduced monascinol (2) rather than its counterpart (1) as a potent inhibitor to the tested VEGFR2 kinase. Taxonomically, the fungus was identified using morphological and genetic assessments.

Synthesis and cytotoxicity screening of some synthesized hybrid nitrogen molecules as anticancer agents

A Series of hybrid organic nitrogen compounds 4, 6, and 8 were synthesized and confirmed through the use of different spectroscopic techniques (IR, NMR, and MS spectrometry) along with elemental analysis. The in vitro antitumor activity of the compounds (4b–d), (6b,c), and (8b,c) against A549 and MCF-7 cell lines and normal breast and lung cells were assessed. Several compounds were found to be potent antitumor agents. Further, in vitro cell cycle study of compounds, 4d and 8c revealed A549 and MCF-7 cell cycle arrest at S and G2/ M phases, respectively; and induce apoptosis at the pre-G1 phase. The apoptosis-inducing activity of synthesized compounds was proofed by the elevation of caspase 3/7 activity and also by up-regulation of the expression of Bax and p53 proteins together with the down-regulation of the expression of the Bcl-2 protein. They also had a strong inhibitory effect against β-tubulin polymerization and topoisomerase IIβ enzyme.

BRCA1‐BARD1 complex regulates the stability of topoisomerase IIβ in transcription of human immediate early genes

Recent studies indicated that activated transcription of serum-inducible genes in humans requires topoisomerase IIβ (TOP2B)-mediated DNA strand break (DSB) and DNA damage response (DDR) signaling. However, the mechanisms by which TOP2B is regulated in this process are unknown. Here, we identified a novel role of BRCA1-BARD1 complex in transcription of these genes by regulating TOP2B binding to the transcription start site (TSS). Our genomic and in vitro biochemical analyses demonstrated that BRCA1-BARD1 complex and TOP2B colocalize in a large number of genes and physically interact for TOP2B ubiquitination. Upon transcriptional activation, BRCA1 is phosphorylated at S1524 by ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). Intriguingly, unphosphorylatable BRCA1 (S1524A)-BARD1 complex ubiquitinates TOP2B, but not WT or S1524D BRCA1. Using the TSS of a representative serum-inducible gene, EGR1, we found that ubiquitinated TOP2B binds to the DNA with much more stability than deubiquitinated one does. Together, our findings suggest that BRCA1-BARD1 complex stabilizes TOP2B association with serum-inducible genes through its E3 ligase function in transcription. Transcriptional activation induces TOP2B-mediated DSB and DDR signaling to activate ATM/ATR that phosphorylates BRCA1 S1524. Phosphorylated BRCA1 is ineffective in ubiquitinating TOP2B, which loosens it from the DNA. We propose that this loosening may contribute to either mobility or removal of TOP2B.

DNA supercoil geometry differentially affects the stability of DNA cleavage complexes formed by different type II topoisomerases

Type II topoisomerases are enzymes that regulate DNA supercoiling and remove knots and tangles from the genome. Humans encode two isoforms of the type II enzyme, topoisomerases IIɑ and IIβ. Type II topoisomerases function by passing an intact double helix through a transient double-stranded break that they generate in a second DNA segment. This strand passage mechanism generates a requisite covalent enzyme-cleaved DNA intermediate, known as the cleavage complex. Under normal circumstances, the cleavage complex exists in a rapid cleavage/religation reaction equilibrium. However, when DNA tracking systems attempt to traverse topoisomerase-cleaved DNA complexes, the double-stranded DNA breaks cannot be rejoined by the enzyme and require DNA recombination/repair mechanisms to reestablish the integrity of the genetic material. In sufficient numbers, these strand breaks can overwhelm the repair processes and result in genotoxic effects including chromosomal translocations and cell death. Chemicals that stabilize cleavage complexes and increase genome fragmentation by converting type II topoisomerases into DNA-damaging agents and are known as topoisomerase poisons. Previous work has shown that levels of DNA cleavage mediated by type II topoisomerase are differentially affected by the handedness of DNA supercoil geometry. Human topoisomerase IIɑ and topoisomerase IIβ maintain higher levels of DNA cleavage complexes with negatively-supercoiled [(-)SC] DNA than with positively-supercoiled [(+)SC] substrates. However, it is not known whether increased DNA cleavage correlates with increased stability of cleavage complexes. We hypothesized that the stability of cleavage complexes may also vary by DNA supercoil geometry and enzyme type. To test our hypotheses, we assessed cleavage complex stability by measuring persistence of DNA cleavage in the presence or absence of human type II topoisomerase poisons. We determined that human topoisomerase IIɑ-mediated cleavage with (-)SC DNA persisted longer than with (+)SC DNA in the presence of etoposide and its derivative F14512, or amsacrine. The etoposide derivative TOP-53 generated a less pronounced difference in the persistence of topoisomerase IIɑ-mediated DNA cleavage. In contrast to the IIɑ isoform, the stability of IIβ-mediated DNA cleavage complexes across all poisons tested did not appear to vary by DNA supercoil handedness. We conclude that increased levels of DNA cleavage do not always correlate with greater stability of cleavage complexes. Both topoisomerases IIɑ and IIβ discriminate supercoil handedness during the DNA cleavage events, but how long enzyme-DNA cleavage complexes remain stable appears to be dependent on isoform. Although the IIɑ isoform both recognizes and prefers (-)SC over (+)SC DNA when maintaining cleavage complex stability, the IIβ isoform does not appear to discriminate. We are currently extending our experiments to bacteria with the Escherichia coli type II topoisomerases, gyrase and topoisomerase IV.

A Four-Component Domino Reaction: An Eco-Compatible and Highly Efficient Construction of 1,8-Naphthyridine Derivatives, Their In Silico Molecular Docking, Drug Likeness, ADME, and Toxicity Studies

A multicomponent domino reaction of enaminone, malononitrile, and o-phthalaldehyde has been established, providing direct access to novel highly functionalized pentacyclic cyclopenta [b] indeno [1, 2, 3-de] [1,8] naphthyridine derivatives. The simplicity of execution, readily available substrates, high yields, excellent functional group tolerance, scalability, and good scores of environmental parameters make this synthetic strategy more sustainable and worthy of further attention. This one-pot transformation, which involved multiple steps and did not require the use of a catalyst, constructed four new C-C bonds, two new C-N bonds, and three new rings, with efficient use of all reactants. Furthermore, we performed in silico molecular docking analysis for prediction of anticancer (against human topoisomerase IIβ protein) and antimicrobial (against E.coli. DNA gyrase B protein) activities. Drug likeness and ADMET studies were also predicted. Overall investigation indicates that compound 6i may serve as a candidate that could be developed as potential anticancer and antimicrobial agent among all.

1,2-Naphthoquinone as a Poison of Human Type II Topoisomerases.

1,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic properties. Because many quinones have been shown to act as topoisomerase II poisons, the effects of this compound on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined. The compound increased the levels of double-stranded DNA breaks generated by both enzyme isoforms and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphthoquinone was a more efficacious poison against topoisomerase IIα than IIβ. Topoisomerase II poisons can be classified as interfacial (which interact noncovalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation) or covalent (which adduct the protein and increase DNA cleavage by closing the N-terminal gate of the enzyme). Therefore, experiments were performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Moreover, the compound inhibited cleavage activity when incubated with the enzyme prior to the addition of DNA and induced virtually no cleavage with the catalytic core of the enzyme. It also induced stable covalent topoisomerase IIα-DNA cleavage complexes and was a partial inhibitor of DNA ligation. Findings were also consistent with 1,2-naphthoquinone acting as a covalent poison of topoisomerase IIβ; however, mechanistic studies with this isoform were less conclusive. Whereas the activity of 1,2-naphthoquinone was blocked in the presence of a sulfhydryl reagent, it was much less sensitive to the presence of a reducing agent. Furthermore, the reduced form of 1,2-naphthoquinone, 1,2-dihydroxynaphthalene, displayed high activity against the β isoform. Taken together, results suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission (at least in part) by acting as a covalent poison of the human type II enzymes.

Structure-Activity Relationship Study of Dexrazoxane Analogues Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against Anthracycline Toxicity to Cardiomyocytes Due to Its Strong Topoisomerase IIβ Interactions.

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIβ (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.

Topoisomerase IIβ immunoreactivity (IR) co-localizes with neuronal marker-IR but not glial fibrillary acidic protein-IR in GLI3-positive medulloblastomas: an immunohistochemical analysis of 124 medulloblastomas from the Japan Children's Cancer Group.

We previously reported observing GLI3 in medulloblastomas expressing neuronal markers (NM) and/or glial fibrillary acidic protein (GFAP). Furthermore, patients with medulloblastomas expressing NM or GFAP tended to show favorable or poor prognosis, respectively. In the present study, we focused on the role of topoisomerase IIβ (TOP2β) as a possible regulator for neuronal differentiation in medulloblastomas and examined the pathological roles of GLI3, NM, GFAP, and TOP2β expressions in a larger population. We divided 124 medulloblastomas into three groups (NM-/GFAP-, NM +/GFAP-, and GFAP +) based on their immunoreactivity (IR) against NM and GFAP. The relationship among GLI3, NM, GFAP, and TOP2β was evaluated using fluorescent immunostaining and a publicly available single-cell RNA sequencing dataset. In total, 87, 30, and 7 medulloblastomas were classified as NM-/GFAP-, NM + /GFAP-, and GFAP +, and showed intermediate, good, and poor prognoses, respectively. GLI3-IR was frequently observed in NM +/GFAP- and GFAP + , and TOP2β-IR was frequently observed only in NM +/GFAP- medulloblastomas. In fluorescent immunostaining, TOP2β-IR was mostly co-localized with NeuN-IR but not with GFAP-IR. In single-cell RNA sequencing, TOP2β expression was elevated in CMAS/DCX-positive, but not in GFAP-positive, cells. NM-IR and GFAP-IR are important for estimating the prognosis of patients with medulloblastoma; hence they should be assessed in clinical practice.

On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response.

NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIβ, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases.

Genome-wide prediction of topoisomerase IIβ binding by architectural factors and chromatin accessibility.

DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.

A switch in mechanism of action prevents doxorubicin-mediated cardiac damage

Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIβ poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate).Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways.Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIβ poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours.