Formation Of Strand Breaks Research Articles

Kinetic Features of Degradation of R-Loops by RNase H1 from Escherichia coli

R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants Kd were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1.

Effect of Ultrahigh Dose Rate on Biomolecular Radiation Damage.

Dose rate is one of the important parameters in radiation-induced biomolecular damage. The effects of dose rate have been known to modify radiation toxicity in biological systems. The rate and extent of sublethal DNA damage (e.g., base damage and single-strand breaks) repair and those of cell proliferation have been manifested by dose rate. However, the recent preclinical application of ultrahigh dose rate [(UHDR) ca. 40 Gy/s and higher] radiation modalities have been shown to lower the type and extent of radiation damage to biological systems. At these UHDR, radiation-induced physicochemical and chemical processes are expected to differ from those observed after irradiation at conventional dose rates (CONV). It is unclear whether these UHDR conditions can affect the quality (type) and quantity (extent) of biomolecular damage such as DNA lesions. Here, we comparatively study the influence of indirect effects of CONV and UHDR on the formation of DNA strand breaks and clustered damage including densely accumulated lesions in an aerated and an anoxic dilute aqueous solution of a plasmid DNA model under low and high hydroxyl radical (•OH) scavenging conditions. Aqueous solutions of purified supercoiled plasmid DNA (pUC19) were prepared in either air- or nitrogen-saturated conditions, with Tris buffer added as the radiation-produced •OH scavenger at low and high scavenging capacities. These DNA samples were irradiated using kV X-ray systems at CONV (0.1 Gy/s) and high dose rate (HDR, 25 Gy/s) as well as UHDR (55 and 125 Gy/s) under different scavenging and environmental conditions. DNA lesions including strand breaks and clustered damage including densely accumulated lesions were quantified by gel electrophoresis and the yields of these lesions were calculated from the dose-response curve. Non-DSB clustered damage including densely accumulated lesions were evaluated by treating DNAs using bacterial endonuclease enzymes (Fpg and Nth) prior to gel electrophoresis. UHDR of 55 and 125 Gy/s induced lower amounts of both isolated strand breaks and clustered DNA damage including densely accumulated lesions at doses >40 Gy in the presence of oxygen, compared to the abundance of these lesions induced by 0.1 and 25 Gy/s irradiation under the same dose conditions. Overall, the strand break and clustered damage including densely accumulated lesions yields decreased by factors of 1.3-3.5 after UHDR. We did not observe these differences either via •OH scavenging or by removing oxygen from the solution. In addition, our results point out that the inter-track recombination reactions did not contribute to the observed dose-rate effects on DNA damage. The effects of dose rate on DNA damage are highly dependent on the total dose, as expected, but also on the •OH scavenging capacity that is employed in the aqueous DNA solutions. These important variables may be relevant in biological systems as well. On a practical level, our in vitro plasmid DNA model, which permits to precisely vary the scavenging capacity and gassing conditions (air saturated vs. N2 saturated) can help to differentiate dose-rate effects on biomolecular damage. Our results indicate that the radical-radical reactions are important in understanding the dose-rate effect on DNA damage.

Genotoxicity of beauvericin

Abstract The European Commission (EC) asked EFSA to assess the genotoxicity of beauvericin (BEA). Relevant information, including that which has become available since the 2014 Scientific Opinion on the risks to human and animal health related to the presence of BEA and enniatins in food and feed, was reviewed. In the previous Opinion the Panel concluded that in vitro genotoxicity data were equivocal and there were no in vivo genotoxicity data available. New in vitro studies in mammalian cell lines provided no convincing evidence for induction of chromosomal damage by BEA as measured by micronucleus and chromosome aberration tests or an increase of DNA strand breaks as assessed by the Comet assay. In these studies, no concentration‐dependent effects or potential for interference from associated cytotoxicity were observed. In addition, DNA double‐strand breaks as measured by γ‐H2AX analysis were only observed following exposure to highly cytotoxic BEA concentrations. In vivo studies (Comet and Pig‐a assays, micronucleus test) with BEA were negative. In vitro gene expression studies showed no indication of a DNA damage response and (quantitative) structure activity relationship analysis was also not indicative of genotoxic potential. Some effects of BEA might play an indirect role in the formation of DNA strand breaks. These include increased reactive oxygen species, induction of cell cycle arrest and apoptosis, associated with interference in mitochondrial function and cell signalling. There was no compelling evidence of inflammatory and immunosuppressive effects. Taken together, the available data indicate that BEA is devoid of genotoxic potential.

Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR). The natural imbalance creates conditions where single strand breaks (SSBs) can convert to double strand breaks (DSBs) and reversibly switch between states in response to oxidation both in vivo and in vitro. Our data suggest that, in a normal background of repair, SSBs and DSBs are in an equilibrium which is pushed or pulled by metabolic state. Interconversion of SSB to DSBs provides a physiological check point, which would allow the formation of unrepaired DSBs for productive functions, but would also restrict them from exceeding tolerable limits.

Specificity of DNA damage formation induced by femtosecond near-infrared laser filamentation in water

We investigated the deoxyribonucleic acid (DNA) damage induced by laser filamentation, which was generated by focusing femtosecond near-infrared Ti:Sapphire laser light in water at several repetition rates ranging from 1000 Hz to 10 Hz. Using plasmid DNA (pUC19), the single-strand break, double-strand break, nucleobase lesions, and the fragmented DNA were analyzed and quantified by agarose gel electrophoresis. Additionally, the H2O2 concentration after irradiation was determined. We observed that (1) the DNA damage per laser shot and (2) the enzyme-sensitive base lesions per total DNA damage decreased as the laser repetition rate increased. Furthermore, (3) extraordinarily short DNA fragments were likely to be produced, compared with those produced using X-rays, and (4) most OH radicals could be eliminated by recombination to generate H2O2, preventing them from damaging the DNA. The Monte-Carlo simulation of the strand break formation implies that the observed dependency of strand break efficiency on the laser repetition rate is mainly due to diffusion of DNA molecules. These findings quantitatively and qualitatively revealed that an intense laser pulse induces a specific DNA damage profile that is not induced by X-rays, a sparsely ionizing radiation source.

The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

Multiple Genomic Landscapes of Recombination and Genomic Divergence in Wild Populations of House Mice-The Role of Chromosomal Fusions and Prdm9.

Chromosomal fusions represent one of the most common types of chromosomal rearrangements found in nature. Yet, their role in shaping the genomic landscape of recombination and hence genome evolution remains largely unexplored. Here, we take advantage of wild mice populations with chromosomal fusions to evaluate the effect of this type of structural variant on genomic landscapes of recombination and divergence. To this aim, we combined cytological analysis of meiotic crossovers in primary spermatocytes with inferred analysis of recombination rates based on linkage disequilibrium using single nucleotide polymorphisms. Our results suggest the presence of a combined effect of Robertsonian fusions and Prdm9 allelic background, a gene involved in the formation of meiotic double strand breaks and postzygotic reproductive isolation, in reshaping genomic landscapes of recombination. We detected a chromosomal redistribution of meiotic recombination toward telomeric regions in metacentric chromosomes in mice with Robertsonian fusions when compared to nonfused mice. This repatterning was accompanied by increased levels of crossover interference and reduced levels of estimated recombination rates between populations, together with high levels of genomic divergence. Interestingly, we detected that Prdm9 allelic background was a major determinant of recombination rates at the population level, whereas Robertsonian fusions showed limited effects, restricted to centromeric regions of fused chromosomes. Altogether, our results provide new insights into the effect of Robertsonian fusions and Prdm9 background on meiotic recombination.

Targeting HUWE1 in Multiple Myeloma Leads to Impaired Replicative Stress Response and Enhanced Sensitivity to Bortezomib

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.

Dissociative Electron Attachment to 5-Iodo-4-thio-2'-deoxyuridine: A Potential Radiosensitizer of Hypoxic Cells.

In the search for effective radiosensitizers for tumor cells, halogenated uracils have attracted more attention due to their large cross section for dissociation upon the attachment of low-energy electrons. In this study, we investigated dissociative electron attachment (DEA) to 5-iodo-4-thio-2'-deoxyuridine, a potential radiosensitizer using a crossed electron-molecule beam experiment coupled with quadrupole mass spectrometry. The experimental results were supported by calculations on the threshold energies of formed anions and transition state calculations. We show that low-energy electrons with kinetic energies near 0 eV may effectively decompose the molecule upon DEA. The by far most abundant anion observed corresponds to the iodine anion (I-). Due to the associated bond cleavage, a radical site is formed at the C5 position, which may initiate strand break formation if the molecule is incorporated into a DNA strand. Our results reflect the conclusion from previous radiolysis studies with the title compound, suggesting its potential as a radiosensitizer.

Class I HDAC inhibition reduces DNA damage repair capacity of MYC-amplified medulloblastoma cells

PurposeMYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations.MethodsMYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry.ResultsEntinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment.ConclusionOur study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

SCFRMF mediates degradation of the meiosis-specific recombinase DMC1

Meiotic recombination requires the specific RecA homolog DMC1 recombinase to stabilize strand exchange intermediates in most eukaryotes. Normal DMC1 levels are crucial for its function, yet the regulatory mechanisms of DMC1 stability are unknown in any organism. Here, we show that the degradation of Arabidopsis DMC1 by the 26S proteasome depends on F-box proteins RMF1/2-mediated ubiquitination. Furthermore, RMF1/2 interact with the Skp1 ortholog ASK1 to form the ubiquitin ligase complex SCFRMF1/2. Genetic analyses demonstrate that RMF1/2, ASK1 and DMC1 act in the same pathway downstream of SPO11-1 dependent meiotic DNA double strand break formation and that the proper removal of DMC1 is crucial for meiotic crossover formation. Moreover, six DMC1 lysine residues were identified as important for its ubiquitination but not its interaction with RMF1/2. Our results reveal mechanistic insights into how the stability of a key meiotic recombinase that is broadly conserved in eukaryotes is regulated.

Efficacy of a HER2-Targeted Thorium-227 Conjugate in a HER2-Positive Breast Cancer Bone Metastasis Model.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-30% of breast cancers but has low expression in normal tissue, making it attractive for targeted alpha therapy (TAT). HER2-positive breast cancer typically metastasizes to bone, resulting in incurable disease and significant morbidity and mortality. Therefore, new strategies for HER2-targeting therapy are needed. Here, we present the preclinical in vitro and in vivo characterization of the HER2-targeted thorium-227 conjugate (HER2-TTC) TAT in various HER2-positive cancer models. In vitro, HER2-TTC showed potent cytotoxicity in various HER2-expressing cancer cell lines and increased DNA double strand break formation and the induction of cell cycle arrest in BT-474 cells. In vivo, HER2-TTC demonstrated dose-dependent antitumor efficacy in subcutaneous xenograft models. Notably, HER2-TTC also inhibited intratibial tumor growth and tumor-induced abnormal bone formation in an intratibial BT-474 mouse model that mimics breast cancer metastasized to bone. Furthermore, a match in HER2 expression levels between primary breast tumor and matched bone metastases samples from breast cancer patients was observed. These results demonstrate proof-of-concept for TAT in the treatment of patients with HER2-positive breast cancer, including cases where the tumor has metastasized to bone.

Alkylation of nucleobases by 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) sensitizes PARP1-deficient tumors.

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is amonofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

Sensitive MALDI-TOF MS and ‘turn-on’ fluorescent genosensor for the determination of DNA damage induced by CNS acting drugs

The genotoxic and carcinogenic adverse effects of various drugs should be considered for assessing drug benefit/risk ratio. On that account, the scope of this study is to examine the kinetics of DNA damage triggered by three CNS acting drugs; carbamazepine, quetiapine and desvenlafaxine. Two precise, simple and green approaches were proposed for probing drug induced DNA impairment; MALDI-TOF MS and terbium (Tb3+) fluorescent genosensor. The results revealed that all the studied drugs induced DNA damage manifested by the MALDI-TOF MS analysis as a significant disappearance of the DNA molecular ion peak with the appearance of other peaks at smaller m/z indicating the formation of DNA strand breaks. Moreover, significant enhancement of Tb3+ fluorescence occurred, proportional to the amount of DNA damage, upon incubation of each drug with dsDNA. Furthermore, the DNA damage mechanism is examined. The proposed Tb3+ fluorescent genosensor showed superior selectivity and sensitivity and is significantly simpler and less expensive than other methods reported for the detection of DNA damage. Moreover, the DNA damaging potency of these drugs was studied using calf thymus DNA in order to clarify the potential safety hazards associated with the studied drugs on natural DNA.

Abstract 2723: Treatment of gastric cancer cells with tumor treating fields (TTFields) and concomitant FOLFOX

Abstract Background: Gastric cancer, one of the most common types of cancers, is mainly treated with the FOLFOX chemotherapy regimen (oxaliplatin, fluorouracil [5-FU], and leucovorin); yet long-term survival remains poor. Tumor Treating Fields (TTFields) are electrical fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. In several tumor types, the mechanism of action of TTFields included an antimitotic effect and DNA damage induction with impaired DNA damage repair. The current study aimed to examine the efficacy of TTFields for treating gastric cancer cells, and the potential application of TTFields concomitant with FOLFOX. Methods: Human gastric cancer cell lines, AGS and KATO III, were treated with TTFields (intensity of 1.1 and 1.7 V/cm, respectively; frequency of 150 kHz) using the inovitro system. Cell count and colony formation were examined following treatment, and the overall effect calculated by multiplication of the two. RNA sequencing was also performed on samples from control and TTFields-treated cells. Mitotic spindle defects, chromosome miss-localization, and DNA double strand breaks (DSB) formation were examined by fluorescent staining for α-tubulin, phospho-histone 3 (PH3), and phospho-histone H2AX (γH2AX), respectively. Expression of DNA repair proteins was measured using Western Blot. The impact of TTFields with FOLFOX and its individual therapeutic components, oxaliplatin and 5-FU on cell count and clonogenicity was tested. Results: TTFields reduced cell count and the colony forming ability of the cells. Cells treated with TTFields displayed abnormal mitotic figures and increased levels of DNA damage. Expression of DNA repair proteins was downregulated following treatment with TTFields, as evident from RNA and protein levels. Furthermore, TTFields augmented the cytotoxic and clonogenic effects of FOLFOX and its individual therapeutic components, oxaliplatin and 5-FU. Conclusions: TTFields show potential as an effective gastric cancer treatment, with a mechanism of action involving an anti-mitotic effect and DNA damage repair impairment. TTFields may be applied to enhance the efficacy of gastric cancer standard-of-care. Citation Format: Naama Flint-Brodsly, Einav Zeevi, Kerem Wainer-Katsir, Hila Fishman, Antonia Martinez-Conde, Eyal Dor-On, Mijal Munster, Yaara Porat, Tali Voloshin, Shiri Davidi, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Treatment of gastric cancer cells with tumor treating fields (TTFields) and concomitant FOLFOX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2723.

A shared 'vulnerability code' underpins varying sources of DNA damage throughout paternal germline transmission in mouse.

During mammalian spermatogenesis, the paternal genome is extensively remodelled via replacement of histones with protamines forming the highly compact mature sperm nucleus. Compaction occurs in post-meiotic spermatids and is accompanied by extensive double strand break (DSB) formation. We investigate the epigenomic and genomic context of mouse spermatid DSBs, identifying primary sequence motifs, secondary DNA structures and chromatin contexts associated with this damage. Consistent with previously published results we find spermatid DSBs positively associated with short tandem repeats and LINE elements. We further show spermatid DSBs preferentially occur in association with (CA)n, (NA)n and (RY)n repeats, in predicted Z-DNA, are not associated with G-quadruplexes, are preferentially found in regions of low histone mark coverage and engage the remodelling/NHEJ factor BRD4. Locations incurring DSBs in spermatids also show distinct epigenetic profiles throughout later developmental stages: regions retaining histones in mature sperm, regions susceptible to oxidative damage in mature sperm, and fragile two-cell like embryonic stem cell regions bound by ZSCAN4 all co-localise with spermatid DSBs and with each other. Our results point to a common 'vulnerability code' unifying several types of DNA damage occurring on the paternal genome during reproduction, potentially underpinned by torsional changes during sperm chromatin remodelling.

Anti-tumour and radiosensitising effects of PARP inhibitor on cervical cancer xenografts

This study evaluated the radiosensitising effect of niraparib; a poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor on HeLa cervical cancer cells in nude mice and explored its possible mechanism. Twenty-four 3–5-week-old female BALB/c nude mice, inoculated with HeLa cells into the right hind leg, were randomly assigned into eight groups with three mice per group and treated. The tumour volume was significantly reduced under niraparib + radiotherapy combination as compared to monotherapy and untreated mice. The tumour growth was significantly delayed by 23.33–39 days when treated with combination therapy (p<.05). Further, univariate analysis revealed prolonged time for tumour growth when radiotherapy was followed by niraparib (I.G.) rather than niraparib (I.P.) (p=.003). Combination therapy reduced levels of PARP-1 precursor, PARP-1 splicer, PAR and RAD51 protein with high expression of γ-H2AX/CC3 and low expression of Ki-67. Niraparib in combination with radiotherapy can enhance the formation of DNA double strand breaks in HeLa cells and up regulate the expression of γ-H2AX/CC3. IMPACT STATEMENT What is already known on this subject? Asia has the highest incidence of cervical cancer (58.2%). Poly(adenosine diphosphate-ribose) polymerases (PARPs) are family of enzymes involved in single-strand break (SSB) and double-strand break (DSB) repair pathways. Niraparib is an effective inhibitor of both PARP-1 and PARP-2 and has the ability to cross the blood–brain barrier. What the results of this study add? Our study demonstrated that the combination of niraparib and radiotherapy can significantly enhance the cytotoxicity induced by radiotherapy. The inhibition effect of radiotherapy combined with niraparib on the tumour growth of mice was prominent, thereby establishing the radio-sensitisation activity of niraparib. What are the implications of these findings for clinical practice and/or further research? Niraparib can improve the cytotoxic effect of radiotherapy by increasing the formation of DSBs and up regulating the expression of apoptotic protein in HeLa cells.

ATR inhibition overcomes platinum tolerance associated with ERCC1- and p53-deficiency by inducing replication catastrophe.

ERCC1/XPF is a heterodimeric DNA endonuclease critical for repair of certain chemotherapeutic agents. We recently identified that ERCC1- and p53-deficient lung cancer cells are tolerant to platinum-based chemotherapy. ATR inhibition synergistically re-stored platinum sensitivity to platinum tolerant ERCC1-deficient cells. Mechanistically we show this effect is reliant upon several functions of ATR including replication fork protection and altered cell cycle checkpoints. Utilizing an inhibitor of replication protein A (RPA), we further demonstrate that replication fork protection and RPA availability are critical for platinum-based drug tolerance. Dual treatment led to increased formation of DNA double strand breaks and was associated with chromosome pulverization. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATR inhibition may also enhance response to immunotherapy in ERCC1-deficient tumors. In vivo studies demonstrate a significant effect on tumor growth delay with combination therapy compared with single agent treatment. Results of this study have led to the identification of a feasible therapeutic strategy combining ATR inhibition with platinum and potentially immune checkpoint blockade inhibitors to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.

MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

Tumor Treating Fields (TTFields) Concomitant with PARP Inhibitors for Treatment of Ovarian Cancer Cell Lines

<h3>Purpose/Objective(s)</h3> TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Recently, TTFields have also been shown to induce a state of BRCAness in various cancer types. PARP inhibitors (PARPi) demonstrate clinical efficacy in patients with solid tumors and deleterious BRCA mutations; however, 3 of 4 patients with ovarian cancer do not harbor BRCA mutations, and thus may experience limited benefit from PARP inhibition. TTFields have shown in vitro and in vivo efficacy in ovarian cancer models. The objective of this study was to examine the effect of TTFields concomitant with PARPi on ovarian cancer cell lines. <h3>Materials/Methods</h3> A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 deep deletion) ovarian carcinoma cells were treated for 72 h with TTFields at an intensity of 1 V/cm RMS and frequency of 200 kHz, alone or with concomitant application of the PARP inhibitors niraparib or olaparib. Efficacy was examined via measurements of cell survival, colony forming ability, and induction of apoptosis. Formation of DNA double strand breaks was examined by fluorescence staining for ɣH2AX. <h3>Results</h3> Application of TTFields or PARPi to A2780 or OVCAR3 cells resulted in cytotoxicity, anti-clonogenic effect, and induction of apoptosis. Concomitant application of TTFields with PARPi displayed synergistic interaction in the BRCA wild type A2780 cells and additively in the BRCA mutant OVCAR-3 cells. Co-application of TTFields and PARPi also resulted in increased levels of DNA double strand breaks in both cell lines relative to each modality alone. <h3>Conclusion</h3> The results support that TTFields induce a state of BRCAness, which together with PARP inhibition can possibly lead to synthetic lethality. Collectively, the data suggest potential benefits for TTFields concomitant application with PARP inhibition for treatment of ovarian cancer even in the absence of background BRCA mutations.