Induction Of Double-strand Breaks Research Articles

6PPDQ induces cardiomyocyte senescence via AhR/ROS-mediated autophagic flux blockage

Recently, attention has been drawn to the adverse outcomes of N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6PPDQ) on human health, but its cardiac toxicity has been relatively understudied. This work aims to investigate the effects of 6PPDQ on differentiated H9c2 cardiomyocytes. Our findings demonstrated that exposure to 6PPDQ altered cellular morphology and disrupted the expression of cardiac-specific markers. Significantly, 6PPDQ exposure led to cardiomyocyte senescence, characterized by elevated β-Galactosidase activity, upregulation of cell cycle inhibitor, induction of DNA double-strand breaks, and remodeling of Lamin B1. Furthermore, 6PPDQ hindered autophagy flux by promoting the formation of autophagosomes while inhibiting the degradation of autolysosomes. Remarkably, restoration of autophagic flux using rapamycin counteracted 6PPDQ-induced cardiomyocyte senescence. Additionally, our study revealed that 6PPDQ significantly increased the ROS production. However, ROS scavenger effectively reduced the blockage of autophagic flux and cardiomyocyte senescence caused by 6PPDQ. Furthermore, we discovered that 6PPDQ activated the Aryl hydrocarbon receptor (AhR) signaling pathway. AhR antagonist was found to reverse the blockage of autophagy and alleviate cardiac senescence, while also reducing ROS levels in 6PPDQ-treated group. In conclusion, our research unveils that exposure to 6PPDQ induces ROS overproduction through AhR activation, leading to disruption of autophagy flux and ultimately contributing to cardiomyocyte senescence.

DNA double-stranded breaks, a hallmark of aging, defined at the nucleotide resolution, are increased and associated with transcription in the cardiac myocytes in LMNA-cardiomyopathy.

An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. We posit that DSBs would be more prevalent at the genomic sites that are associated with gene expression. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. We identified the genome-wide DSBs in ∼1 million cardiac myocytes per heart in three wild-type and three myocyte-specific LMNA-deficient (Myh6-Cre:LmnaF/F) mice by END-Sequencing. The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively. The END-Seq signals were enriched for 8 and 6764 DSBs in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively (q < 0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the lamin-associated domains (LADs), we defined the LADs in cardiac myocytes by a Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay (N = 5). On average there were 818 LADs per myocyte. Constitutive LADs (cLADs), defined as LADs that were shared by at least three genomes (N = 2572), comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N = 3975) were ∼16-fold lower than those at the non-LAD regions (N = ∼17 778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of-LAD regions, defined as the genomic regions in the Myh6-Cre:LmnaF/F that were juxtaposed to the LAD regions in the wild-type myocytes. To our knowledge, this is the first identification of the DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various states and aging.

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

Proton relative biological effectiveness for the induction of DNA double strand breaks based on Geant4

Uncertainties in the relative biological effectiveness (RBE) of proton remains a major barrier to the biological optimization of proton therapy. A large amount of experimental data suggest that proton RBE is variable. As an evolving Monte Carlo code toolkit, Geant4-DNA is able to simulate the initial DNA damage caused by particle beams through physical and chemical interactions at the nanometer scale over a short period of time. This contributes to evaluating the radiobiological effects induced by ionizing radiation. Based on the Geant4-DNA toolkit, this study constructed a DNA geometric model containing 6.32Gbp, simulated the relationship between radiochemical yields (G-values) and their corresponding chemical constructors, and calculated a detailed calculation of the sources of damage and the complexity of damage in DNA strand breaks. The damage model constructed in this study can simulate the relative biological effectiveness (RBE) in the proton Bragg peak region. The results indicate that: (1) When the electron energy is below 400 keV, the yield of OH· account for 18.1% to 25.3% of the total water radiolysis yields. (2) Under the influence of histone clearance function, the yield of indirect damage account for over 72.93% of the yield of DNA strand breaks (SBs). When linear energy transfer (LET) increased from 29.79 (keV/μm) to 64.29 (keV/μm), the yield of double strand breaks (DSB) increased from 17.27% to 32.65%. (3) By investigating the effect of proton Bragg peak depth on the yield of direct DSB (DSBdirect) and total DSB (DSBtotal), the RBEDSBtot and RBEDSBdir levels of cells show that the RBE value of protons reaches 2.2 in the Bragg peak region.

Abstract 3267: Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection of GBM, chemotherapy and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions, contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis, while SAMHD1 depletes the pool by hydrolyzing dNTPs. Also, previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate-limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our results show that elevating the cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single-cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM, as demonstrated by apoptosis pathway activation. Our result suggests that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutic efficacy. In conclusion, this study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3267.

Abstract 6569: KLIPP: Precision targeting of cancer with CRISPR

Abstract Current cancer therapeutic treatments are typically associated with significant collateral damage to normal tissues and the emergence of treatment resistance. Thus, there is an unmet need to develop therapies that are uniquely cancer specific and unhindered by the emergence of resistance. We have developed an RNA therapy approach based on CRISPR technology, known as “KLIPP”, that targets cancer-specific structural variant junctions (SVJs), a so far untapped cancer “Achilles heel”, that are highly abundant across cancer types. KLIPP uses a “split enzyme” system consisting of dCas9 conjugated to an endonuclease, Fok1, that requires dimerization for its activity. By designing sgRNAs that bind to sequences flanking the cancer-specific SVJs, Fok1-dCas9 dimers are formed, leading to the induction of DNA double strand breaks (DSBs) specifically at SVJs in cancer cells. We verify induction of DSBs by γH2AX foci staining in the nucleus of KLIPP-treated cells and resulting loss of clonogenic survival and induction of apoptosis, while cells lacking the targeted SVJs are unaffected. Importantly, we show elimination of orthotopic bladder cancer tumors in vivo after turning on expression of the KLIPP reagents by doxycycline in the drinking water. Finally, we show successful lipid nanoparticle delivery of KLIPP reagents to orthotopic bladder tumors in vivo using catheter-mediated delivery, leading to specific targeting of bladder cancer cells. Independently of the cancer-driving mutations, the omnipresence of cancer-specific SVJs is exploited by KLIPP to force cancer cells into apoptosis while leaving normal cells unperturbed. We believe that the successful implementation of precision KLIPP therapy could be transformative for the treatment of cancer patients. Citation Format: Mats Ljungman, Huibin Yang, Radhika Suhas Hulbatte, Natalie Gratsch, Alan Kelher, John Raupp, Yin Wang, Kristen Hong Dorsey, Troy Halseth, Anna Schwendeman, Philip Palmbos. KLIPP: Precision targeting of cancer with CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6569.

Abstract 3273: Tirapazamine sensitizes homologous recombination-proficient cancers by enhancing DNA damage andferroptosis

Abstract Despite its success in HR-deficient tumors, the application of PARP inhibitors (PARPis) in HR-proficient cancers is very limited. Since the majority of cancers are HR-proficient, there is a need for developing strategies to extend the use of PARPis to HR-proficient cancers. We investigated whether the hypoxic cytotoxin, tirapazamine (TPZ) sensitizes HR-proficient cancer cells to PARPis under normoxia and hypoxia (2% O2), and found that TPZ enhanced PARPi cell killing to a greater extent under hypoxia compared to normoxia. Mechanistically, this enhanced killing was due to the induction of DNA double-strand breaks and free radicals that lead to ferroptotic cell death. To determine the role of hypoxia in the anti-tumor effect of the combination treatment, we examined the efficacy of TPZ and PARPi treatment in hypoxic spheroids and subcutaneous xenograft models derived from a panel of HR-proficient human non-small cell lung cancer (NSCLC) cells. We found that PARPis combined with TPZ significantly inhibited the growth of the hypoxic NSCLC spheroids. In addition, the combination treatment significantly inhibited the growth of A549 and CORL105 xenografts that possess significant hypoxic fractions, but did not significantly inhibit the growth of well-oxygenated Calu3 and H3122 xenografts. Taken together, this study demonstrates that TPZ can sensitize HR-proficient tumor cells to PARPi through the introduction of DNA strand breaks and free radicals, suggesting that TPZ treatment can broaden the usage of PARPi to HR-proficient cancers. Citation Format: Yanyan Jiang, Hala Estephan, Yizhou Wang, Ftoon Aljarbou, Nuria V. Lopera, Eui Jung Moon, Ester M. Hammond, Amato J. Giaccia. Tirapazamine sensitizes homologous recombination-proficient cancers by enhancing DNA damage andferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3273.

Investigation of the Impact of Temporal Dose Delivery Patterns of Ion Irradiation with the Local Effect Model.

We present an extension of the Local Effect Model (LEM) to include time-dose relationships for predicting effects of protracted and split-dose ion irradiation at arbitrary LET. With this kinetic extension, the spatial and temporal induction and processing of DNA double strand breaks (DSB) in cellular nuclei can be simulated for a wide range of ion radiation qualities, doses and dose rates. The key concept of the extension is based on the joint spatial and temporal coexistence of initial DSB, leading to the formation of clustered DNA damage on the µm scale (as defined e.g., by the size scale of Mbp chromatin loops), which is considered to have an increased cellular lethality as compared to isolated, single DSB. By simulating the time dependent induction and repair of DSB and scoring of isolated and clustered DSB upon irradiation, the impact of dose rate and split dose on the cell survival probability can be computed. In a first part of this work, we systematically analyze the predicted impact of protraction in dependence of factors like dose, LET, ion species and radiosensitivity as characterized by the photon LQ-parameters. We establish links to common concepts that describe dose rate effects for low LET radiation. We also compare the model predictions to experimental data and find agreement with the general trends observed in the experiments. The relevant concepts of our approach are compared to other models suitable for predicting time effects. We investigate an apparent analogy between spatial and temporal concentration of radiation delivery, both leading to increased effectiveness, and discuss similarities and differences between the general dependencies of these clustering effects on their impacting factors. Finally, we conclude that the findings give additional support for the general concept of the LEM, i.e. the characterization of high LET radiation effects based on the distinction of just two classes of DSB (isolated DSB and clustered DSB).

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Abstract B020: Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection, chemotherapy, and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis while SAMHD1 depletes the pool by hydrolyzing dNTPs. Previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our result shows that elevating cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM as demonstrated by apoptosis pathway activation. Our result suggest that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutics' efficacy. This study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Maya Jenkins, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B020.

Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells.

Attaching/effacing (A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagic Escherichia coli. The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1, followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of replication protein A 32 in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.IMPORTANCEOxidative DNA lesions play causative roles in colitis-associated colon cancer. Accumulating evidence shows strong links between attaching/effacing (A/E) pathogens and colorectal cancer (CRC). EspF is one of many effector proteins exclusive to A/E pathogens with defined roles in the induction of oxidative stress, double-strand breaks (DSBs), and repair dysregulation. Here, we found that EspF promotes reactive oxygen species generation and 8-oxoguanine (8-oxoG) lesions when the repair system is activated, contributing to sustained cell survival. However, infected cells exposed to EspF presented 8-oxoG, which results in DSBs and ssDNA accumulation when the cell cycle is arrested at the G2/M phase and the repair system is defective or saturated by DNA lesions. In addition, we found that EspF could intensify the accumulation of nuclear DNA lesions through oxidative and replication stress. Overall, our work highlights the involvement of EspF in DNA lesions and DNA damage response, providing a novel avenue by which A/E pathogens may contribute to CRC.

Natural pentacyclic triterpenoid from Pristimerin sensitizes p53-deficient tumor to PARP inhibitor by ubiquitination of Chk1

Inhibition of checkpoint kinase 1 (Chk1) has shown to overcome resistance to poly (ADP-ribose) polymerase (PARP) inhibitors and expand the clinical utility of PARP inhibitors in a broad range of human cancers. Pristimerin, a naturally occurring pentacyclic triterpenoid, has been the focus of intensive studies for its anticancer potential. However, it is not yet known whether low dose of pristimerin can be combined with PARP inhibitors by targeting Chk1 signaling pathway. In this study, we investigated the efficacy, safety and molecular mechanisms of the synergistic effect produced by the combination olaparib and pristimerin in TP53-deficient and BRCA-proficient cell models. As a result, an increased expression of Chk1 was correlated with TP53 mutation, and pristimerin preferentially sensitized p53-defective cells to olaparib. The combination of olaparib and pristimerin resulted in a more pronounced abrogation of DNA synthesis and induction of DNA double-strand breaks (DSBs). Moreover, pristimerin disrupted the constitutional levels of Chk1 and DSB repair activities. Mechanistically, pristimerin promoted K48-linked polyubiquitination and proteasomal degradation of Chk1 while not affecting its kinase domain and activity. Importantly, combinatorial therapy led to a higher rate of tumor growth inhibition without apparent hematological toxicities. In addition, pristimerin suppressed olaparib-induced upregulation of Chk1 and enhanced olaparib-induced DSB marker γΗ2ΑΧ in vivo. Taken together, inhibition of Chk1 by pristimerin has been observed to induce DNA repair deficiency, which may expand the application of olaparib in BRCA-proficient cancers harboring TP53 mutations. Thus, pristimerin can be combined for PARP inhibitor-based therapy.

The Influence of Computed Tomography Contrast Agent on Radiation-Induced Gene Expression and Double-Strand Breaks.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07-0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03-0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events

BackgroundThe aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125).ResultsTargeted chondriome deletions of different sizes (236–1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11–12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring.ConclusionsBoth editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

CRISPR/Cas9 Vector Construction for Gene Knockout.

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352bp) and scaffold (83bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.

Induction of double-strand breaks with the non-steroidal androgen receptor ligand flutamide in patients on androgen suppression: a study protocol for a randomized, double-blind prospective trial

BackgroundProstate cancer remains the most prevalent malignancy and the second-leading cause of cancer-related death in men in the USA. Radiation therapy, typically with androgen suppression, remains a mainstay in the treatment of intermediate- and high-risk, potentially lethal prostate cancers. However, local recurrence and treatment failure remain common. Basic and translational research has determined the potential for using androgen receptor (AR) ligands (e.g., dihydrotestosterone and flutamide) in the context of androgen-deprived prostate cancer to induce AR- and TOP2B-mediated DNA double-strand breaks (DSBs) and thereby synergistically enhance the effect of radiation therapy (RT). The primary aim of this study is to carry out pharmacodynamic translation of these findings to humans.MethodsPatients with newly diagnosed, biopsy-confirmed localized prostatic adenocarcinoma will be recruited. Flutamide, an oral non-steroidal androgen receptor ligand, will be administered orally 6–12 h prior to prostate biopsy (performed under anesthesia prior to brachytherapy seed implantation). Key study parameters will include the assessment of DNA double-strand breaks by γH2A.x foci and AR localization to the nucleus. The initial 6 patients will be treated in a single-arm run-in phase to assess futility by establishing whether at least 2 subjects from this group develop γH2A.x foci in prostate cancer cells. If this criterion is met, the study will advance to a two-arm, randomized controlled phase in which 24 participants will be randomized 2:1 to either flutamide intervention or placebo standard-of-care (with all patients receiving definitive brachytherapy). The key pharmacodynamic endpoint will be to assess whether the extent of γH2A.x foci (proportion of cancer cells positive and number of foci per cancer cell) is greater in patients receiving flutamide versus placebo. Secondary outcomes of this study include an optional, exploratory analysis that will (a) describe cancer-specific methylation patterns of cell-free DNA in plasma and urine and (b) assess the utility of serum and urine samples as a DNA-based biomarker for tracking therapeutic response.DiscussionThis study will confirm in humans the pharmacodynamic effect of AR ligands to induce transient double-strand breaks when administered in the context of androgen deprivation as a novel therapy for prostate cancer. The findings of this study will permit the development of a larger trial evaluating flutamide pulsed-dose sequencing in association with fractionated external beam RT (+/− brachytherapy). The study is ongoing, and preliminary data collection and recruitment are underway; analysis has yet to be performed.Trial registrationClinicalTrials.gov NCT03507608. Prospectively registered on 25 April 2018.

Human RAD52 stimulates the RAD51-mediated homology search

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

Multiplex Prime Editing and PASSIGE TM for Non-Viral Generation of an Allogeneic CAR-T Cell Product

While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system. Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells. Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product. To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality. CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 + tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 + tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.

Effects of Differing Underlying Assumptions in In Silico Models on Predictions of DNA Damage and Repair.

The induction and repair of DNA double-strand breaks (DSBs) are critical factors in the treatment of cancer by radiotherapy. To investigate the relationship between incident radiation and cell death through DSB induction many in silico models have been developed. These models produce and use custom formats of data, specific to the investigative aims of the researchers, and often focus on particular pairings of damage and repair models. In this work we use a standard format for reporting DNA damage to evaluate combinations of different, independently developed, models. We demonstrate the capacity of such inter-comparison to determine the sensitivity of models to both known and implicit assumptions. Specifically, we report on the impact of differences in assumptions regarding patterns of DNA damage induction on predicted initial DSB yield, and the subsequent effects this has on derived DNA repair models. The observed differences highlight the importance of considering initial DNA damage on the scale of nanometres rather than micrometres. We show that the differences in DNA damage models result in subsequent repair models assuming significantly different rates of random DSB end diffusion to compensate. This in turn leads to disagreement on the mechanisms responsible for different biological endpoints, particularly when different damage and repair models are combined, demonstrating the importance of inter-model comparisons to explore underlying model assumptions.