DNA Single-strand Breaks Research Articles

Abstract 4644: Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or a PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and a USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alexandre Andre B. A. Da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David Martigneti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Golbahar Sadatrezai, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey Shapiro, Kalindi Parmar, Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity in ovarian cancer patient-derived organoid models [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 4644.

Genome-wide profiles of DNA damage represent highly accurate predictors of mammalian age.

The identification of novel age-related biomarkers represents an area of intense research interest. Despite multiple studies associating DNA damage with aging, there is a glaring paucity of DNA damage-based biomarkers of age, mainly due to the lack of precise methods for genome-wide surveys of different types of DNA damage. Recently, we developed two techniques for genome-wide mapping of the most prevalent types of DNA damage, single-strand breaks and abasic sites, with nucleotide-level resolution. Herein, we explored the potential of genomic patterns of DNA damage identified by these methods as a source of novel age-related biomarkers using mice as a model system. Strikingly, we found that models based on genomic patterns of either DNA lesion could accurately predict age with higher precision than the commonly used transcriptome analysis. Interestingly, the informative patterns were limited to relatively few genes and the DNA damage levels were positively or negatively correlated with age. These findings show that previously unexplored high-resolution genomic patterns of DNA damage contain useful information that can contribute significantly to both practical applications and basic science.

Podocyte Ercc1 is indispensable for glomerular integrity

As life expectancy continues to increase, age-related kidney diseases are becoming more prevalent. Chronic kidney disease (CKD) is not only a consequence of aging but also a potential accelerator of aging process. Here we report the pivotal role of podocyte ERCC1, a DNA repair factor, in maintaining glomerular integrity and a potential effect on multiple organs. Podocyte-specific ERCC1-knockout mice developed severe proteinuria, glomerulosclerosis, and renal failure, accompanied by a significant increase in glomerular DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). ERCC1 gene transfer experiment in the knockout mice attenuated proteinuria and glomerulosclerosis with reduced DNA damage. Notably, CD44+CD8+ memory T cells, indicative of T-cell senescence, were already elevated in the peripheral blood of knockout mice at 10 weeks old. Additionally, levels of senescence-associated secretory phenotype (SASP) factors were significantly increased in both the circulation and multiple organs of the knockout mice. In older mice and human patients, we observed an accumulation of DSBs and an even greater buildup of SSBs in glomeruli, despite no significant reduction in ERCC1 expression with age in mice. Collectively, our findings highlight the crucial role of ERCC1 in repairing podocyte DNA damage, with potential implications for inflammation in various organs.

A-T neurodegeneration and DNA damage-induced transcriptional stress

Loss of the ATM protein kinase in humans results in Ataxia-telangiectasia, a disorder characterized by childhood-onset neurodegeneration of the cerebellum as well as cancer predisposition and immunodeficiency. Although many aspects of ATM function are well-understood, the mechanistic basis of the progressive cerebellar ataxia that occurs in patients is not. Here we review recent progress related to the role of ATM in neurons and the cerebellum that comes from many sources: animal models, post-mortem brain tissue samples, and human neurons in culture. These observations have revealed new insights into the consequences of ATM loss on DNA damage, gene expression, and immune signaling in the brain. Many results point to the importance of reactive oxygen species as well as single-strand DNA breaks in the progression of molecular events leading to neuronal dysfunction. In addition, innate immunity signaling pathways appear to play a critical role in ATM functions in microglia, responding to various forms of nucleic acid sensors and regulating survival of neurons and other cell types. Overall, the results lead to an updated view of transcriptional stress and DNA damage resulting from ATM loss that results in changes in gene expression as well as neuroinflammation that contribute to the cerebellar neurodegeneration observed in patients.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling.

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays)or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.

APOBEC3A induces DNA gaps through PRIMPOL and confers gap-associated therapeutic vulnerability.

Mutation signatures associated with apolipoprotein B mRNA editing catalytic polypeptide-like 3A/B (APOBEC3A/B) cytidine deaminases are prevalent across cancers, implying their roles as mutagenic drivers during tumorigenesis and tumor evolution. APOBEC3A (A3A) expression induces DNA replication stress and increases the cellular dependency on the ataxia telangiectasia and Rad3-related (ATR) kinase for survival. Nonetheless, how A3A induces DNA replication stress remains unclear. We show that A3A induces replication stress without slowing replication forks. We find that A3A induces single-stranded DNA (ssDNA) gaps through PrimPol-mediated repriming. A3A-induced ssDNA gaps are repaired by multiple pathways involving ATR, RAD51, and translesion synthesis. Both ATR inhibition and trapping of poly(ADP-ribose) polymerase (PARP) on DNA by PARP inhibitor impair the repair of A3A-induced gaps, preferentially killing A3A-expressing cells. When used in combination, PARP and ATR inhibitors selectively kill A3A-expressing cells synergistically in a manner dependent on PrimPol-generated gaps. Thus, A3A-induced replication stress arises from PrimPol-generated ssDNA gaps, which confer a therapeutic vulnerability to gap-targeted DNA repair inhibitors.

Abstract B020: Targeting the DNA damage response sensor Replication Protein A for first in class cancer therapy

Abstract The DNA Damage Response (DDR) is a DNA repair and cell signaling pathway crucial in maintaining genomic stability. The most successful DDR targeted therapies inhibit the DNA damage sensor, PARP, a protein that recognizes single strand DNA (ssDNA) breaks as well as other DNA secondary structures induced by single strand gaps that can initiate chromosome instability and cell death. Building on the initial clinical success of PARP inhibitors, development of DDR targeted therapeutics has become increasingly popular with the majority of new agents targeting protein kinases downstream of the DNA damage sensors. The clinical outcomes with these therapeutics have not met expectations and thus a new approach to targeting the DDR pathway is needed. NERx Biosciences has developed a novel strategy of targeting the DDR by intervening upstream of the DDR kinases and targeting specific DDR sensors. The human ssDNA binding protein, replication protein A (RPA), is a critical sensor for the DDR, serving to sense ssDNA at replication forks that arise from replication stress (RS) and is a novel target for cancer therapy. We have discovered, developed, and characterized a novel small molecule RPA inhibitor (RPAi) NERx-329 that blocks the RPA-DNA interaction and elicits a state of chemical RPA exhaustion that results in in vivo anticancer activity. Chemical optimization enabled in vitro and in vivo analyses to elucidate mechanism of action, cellular engagement, and therapeutic activity of RPA-targeted agents. Biochemical reconstitution of the ATR signaling pathway shows that NERx-329 disrupts ATR kinase activity, further suggesting a novel mechanism of action driven by RPA inhibition induced replication catastrophe. NERx-329 elicits single agent in vitro anticancer activity across a broad spectrum of cancers and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. In vivo analysis reveals activity in a series of lung and ovarian PDX models of human cancer. Single agent activity results in a tumor growth delay, which can be enhanced in combination therapy with DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Data demonstrate that tumor growth observed in vivo is a function of NERx329 bioavailability and not development of a specific resistance mechanism. In addition, specific genetic predictors of RPAi sensitivity have been identified and results demonstrate specific genetic alterations increase RPAi activity in vitro and in vivo. NERx-329 represents the first clinically viable agent to target the DDR pathway by disrupting the RPA-DNA interaction and holds potential for significant impact in cancer treatment. This is supported by data from a retrospective clinical assessment of RPA expression to assess treatment outcomes in lung cancer patients, revealing that high RPA expression is a negative prognostic biomarker correlating with worse overall patient survival. NERx 329 offers a novel therapeutic approach for these cancers that are not effectively treated with existing therapeutics. Citation Format: Katherine Pawelczak, Pamela VanderVere-Carozza, Matthew Jordon, Navnath Gavande, John Turchi. Targeting the DNA damage response sensor Replication Protein A for first in class cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B020.

Abstract B032: USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells

Abstract Targeting BRCA-deficient tumors with PARP inhibitors has improved the progression-free survival of ovarian and breast cancer patients in the clinic. However, the majority of patients develop acquired resistance. Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulating single-stranded DNA (ssDNA) gaps at replication forks. We have previously shown that USP1 deficiency is synthetic lethal with BRCA1 deficiency, suggesting USP1 is a potential therapeutic target for overcoming PARPi resistance (Lim et al., Mol Cell, 2018). Accordingly, USP1 inhibitors (USPi) are now undergoing clinical development. Here, we show that USP1i also promote ssDNA gap accumulation during replication in BRCA1-deficient cells. USP1 inhibition results in the accumulation of monoubiquitinated PCNA at the replication fork and increased levels of non-cleaved USP1 and RAD18 at the replication fork. Accumulation of ssDNA gaps is dependent on RAD18 activity and monoubiquitinated PCNA levels. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, resulted in reduced PCNA-Ub levels, resistance to the USP1i, and suppression of ssDNA gaps. Moreover, the accumulation of ssDNA gaps is associated with increased S-phase PAR levels and is independent of PRIMPOL, suggesting defects on the lagging strand of the replication fork. Interestingly, the induction of ssDNA gaps was detected in USP1i-sensitive but not USP1i-resistant BRCA1-deficient cells, suggesting this is the mechanism of cytotoxicity by USP1 inhibition. Indeed, USP1i also sensitized BRCA1-deficient cancer cells to other ssDNA gap-inducing agents such as POLQ and PARP inhibitors. Notably, USP1i exhibited monotherapy activity in ovarian and breast cancer cell lines with acquired PARPi resistance and re-sensitized them to PARP inhibition. PARPi-resistant BRCA1-mutated patient-derived ovarian cancer organoids were also sensitized by USP1 inhibition. Similar to the cell lines, the ssDNA gap accumulation induced by USP1i in organoids strongly correlated with the drug sensitivity of both PARPi-sensitive and PARPi-resistant models. Finally, USPi overcame PARPi resistance in a BRCA1-mutant PARPi-resistant PDX model. In conclusion, USPi exhibits monotherapy activity in both PARPi-sensitive. Citation Format: Alexandre A. B. A. da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David B. Martignetti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey I. Shapiro, Kalindi Parmar, Alan D. D’Andrea. USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B032.

Abstract IA001: Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity

Abstract Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA gaps (ssDNA gaps) at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells. USP1 inhibition increased monoubiquitinated PCNA at replication forks and resulted in accumulation of ssDNA gaps in BRCA1-deficient cancer cells. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, caused USP1 inhibitor resistance and suppression of ssDNA gaps. ssDNA gap accumulation induced by USP1 inhibition correlates with drug sensitivity of BRCA1-mutated cancer cells and overcomes PARP inhibitor resistance in cell line and xenograft models. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. A set of patient-derived ovarian cancer organoids (PDOs) were used to confirm the sensitivity of BRCA1-deficient cells to USP1 inhibition. Five PDOs were derived from patients with high grade-serous ovarian cancer (HGSOC). Among the five PDOs, two of them were from tumors harboring germline BRCA1 pathogenic mutations. BRCA1-mutant PDOs, but not BRCA1-WT PDOs, were sensitive to a USP1 inhibitor and to PARP inhibitor monotherapies. The accumulation of ssDNA gaps after treatment with a USP1 inhibitor or PARP inhibitor correlated with the sensitivity to these drugs in all the models tested. Moreover, the combination of a PARP inhibitor and USP1 inhibitor showed synergy in a BRCA1-WT model, which was resistant to both monotherapies. Interestingly, ssDNA gaps accumulated with the combination treatment and not with monotherapy in this model. Ovarian cancer PDOs therefore provide a powerful tool for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to USP1 inhibition as monotherapy or in combination in ongoing clinical trials. Citation Format: Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for USP1 inhibitor sensitivity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA001.

USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

DNA replication stress-induced fork arrest represents a significant threat to genomic integrity. One major mechanism of replication restart involves repriming downstream of the arrested fork by PRIMPOL, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of nascent strand ssDNA gaps has emerged as a possible determinant of the cellular hypersensitivity to genotoxic agents in certain genetic backgrounds such as BRCA deficiency, but how gaps are converted into cytotoxic structures is still unclear. Here, we investigate the processing of PRIMPOL-dependent ssDNA gaps upon replication stress induced by hydroxyurea and cisplatin. We show that gaps generated in PRIMPOL-overexpressing cells are expanded in the 3'-5' direction by the MRE11 exonuclease, and in the 5'-3' direction by the EXO1 exonuclease. This bidirectional exonucleolytic gap expansion ultimately promotes their conversion into DSBs. We moreover identify the de-ubiquitinating enzyme USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps. USP1 promotes gap accumulation during S-phase, and their expansion by the MRE11 and EXO1 nucleases. This activity of USP1 is linked to its role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination prevents gap accumulation during replication. Finally, we show that USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions.

Putative Role of the Futile Repair Initiated by Human Thymine-DNA Glycosylase in Formation of Programmed Strand Breaks in Neuronal Enhancers

Enhancers are regulatory DNA elements that play a crucial role in controlling gene expression in specific cell types, including neurons. Enhancer activity is tightly regulated and involves the recruitment of various proteins and enzymes to facilitate the opening of chromatin and the activation of target genes. Given the importance of enhancers in neuronal function, the presence of single-strand DNA breaks (SSBs) in these regions raises intriguing questions about their potential impact on gene regulation and neuronal activity. Single-strand DNA breaks (SSBs) have been identified as important lesions in the genome, with the potential to influence gene expression and genomic stability. By understanding the role of SSB repair and human mono-functional Thymine-DNA glycosylase (TDG) catalyzed futile excision of regular bases in enhancer regions, we may gain insights into the molecular mechanisms underlying neurological disorders and potentially identify new therapeutic targets for intervention.

Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.

Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.

Osnovni principi otkrivanja oštećenja DNK pomoću alkalnog komet testa

The Comet assay is a highly sensitive and rapid method for detecting DNA damage at the level of individual cells. It measures single-stranded and double-stranded DNA breaks, alkali-labile, incomplete excision repair and cross-linking sites. A major advantage of the Comet assay is its applicability to both proliferating and non-proliferating cells, and it can be performed on all types of eukaryotic cells. The basic principle of the alkaline Comet assay is to lyse the biological membranes to release proteins bound to the DNA, followed by a short-term electrophoresis at a pH above 13. Through this process, the DNA fragments migrate and form a comet-like shape, which is visualized by staining with a DNA-binding fluorescent dye. The extent of electrophoretic migration is proportional to the amount of DNA damage. Key advantages of the Comet assay include sensitivity-the assay can detect low levels of DNA damage, versatility-it can be used with a small amount of test substance, speed-results are usually available within 24 to 48 hours, and broad applicability-the method is suitable for numerous purposes, including molecular epidemiology, occupational exposure studies, environmental biomonitoring, antigenotoxicity assessments and clinical research. Overall, the Comet assay provides valuable data for a range of scientific and clinical fields, making it a versatile and powerful tool for assessing DNA damage.

Combined strategies with PARP inhibitors for the treatment of BRCA wide type cancer.

Genomic instability stands out as a pivotal hallmark of cancer, and PARP inhibitors (PARPi) emerging as a groundbreaking class of targeted therapy drugs meticulously crafted to inhibit the repair of DNA single-strand breaks(SSB) in tumor cells. Currently, PARPi have been approved for the treatment of ovarian cancer, pancreatic cancer, breast cancer, and prostate cancer characterized by homologous recombination(HR) repair deficiencies due to mutations in BRCA1/2 or other DNA repair associated genes and acquiring the designation of breakthrough therapy. Nonetheless, PARPi exhibit limited efficacy in the majority of HR-proficient BRCA1/2 wild-type cancers. At present, the synergistic approach of combining PARPi with agents that induce HR defects, or with chemotherapy and radiotherapy to induce substantial DNA damage, significantly enhances the efficacy of PARPi in BRCA wild-type or HR-proficient patients, supporting extension the use of PARPi in HR proficient patients. Therefore, we have summarized the effects and mechanisms of the combined use of drugs with PARPi, including the combination of PARPi with HR defect-inducing drugs such as ATRi, CHKi, HR indirectly inducing drugs like VEGFRi, CDKi, immune checkpoint inhibitors and drugs instigating DNA damage such as chemotherapy or radiotherapy. In addition, this review discusses several ongoing clinical trials aimed at analyzing the clinical application potential of these combined treatment strategies.

Improving olaparib exposure to optimize adverse effects management.

Olaparib is an inhibitor of the human poly-(ADP-ribose)-polymerase enzymes (PARP1/2) needed to repair single-strand DNA breaks. It is used in breast, ovarian, prostate and pancreatic cancer. This work aimed to describe the pharmacokinetics/pharmacodynamics (PK/PD) relationship between olaparib plasma concentrations and common adverse effects (i.e. anaemia and hypercreatininaemia), in a real-life setting, to propose a target concentration for therapeutic drug monitoring. Two PK/PD models describing the evolution of haemoglobinaemia and creatininaemia as a function of time were developed, based on data from, respectively, 38 and 37 patients receiving olaparib. The final model estimates were used to calculate the incidence of anaemia and creatinine increase according to plasma trough concentrations for 1000 virtual subjects to define target exposure. The final models correctly described the temporal evolution of haemoglobinaemia and creatininaemia for all patients. The haemoglobinaemia PK/PD model is inspired by Friberg's model, and the creatininaemia PK/PD model is an indirect response model. Model parameters were in agreement with physiological values and close to literature values for similar models. The mean (population) plasma haemoglobin concentration at treatment initiation, as estimated by the model, was 11.62 g/dL, while creatinine concentration was 71.91 µmol/L. Using simulations, we have identified a target trough concentration of 3500-4000 ng/mL, above which more than 20% of patients would report grade ≥3 anaemia. Based on real-world data, we were able to properly describe the time course of haemoglobinaemia and plasma creatininaemia during olaparib treatment.

The protective effects of empagliflozin on DNA oxidative changes in a model of vascular endothelial and smooth muscle cells damaged by oxidized cholesterol

BackgroundDiabetes patients often suffer chronic vascular complications resulting from endothelial dysfunction, smooth muscle cell (SMC) proliferation, inflammation and disturbed oxidative balance. Empagliflozin is one of three approved sodium-glucose cotransporter 2 (SGLT2) inhibitors for type 2 diabetes mellitus. The aim of this studywas to determine the protective and repairing effect of EMPA in a model of vascular endothelial and SMC damage with 25-hydroxycholesterol (25-OHC). MethodsHuman umbilical vascular endothelial cells (HUVECs) and SMCs were treated with compounds which induce DNA single-strand breaks (SSBs) and subjected to comet assay. Oxidative DNA damage was detected using endonuclease III (Nth) or human 8 oxoguanine DNA glycosylase (hOOG1). Reactive oxygen species (ROS) formation was determined by the fluorescence of a 6-carboxy-2′,7′-dichlorodihydrofluoresce probe in diacetate (H2DCFDA). Results25-OHC-stimulated SMCs showed greater resistance to ROS generation and DNA damage compared to HUVECs. In both experimental models, EMPA treatment was associated with lower ROS production and DNA damage, including oxidative damage to purines and pyrimidines. This effect was not dose-dependent. EMPA was found to counteract this DNA damage by inhibiting ROS production. ConclusionsIt appears that the EMPA induced indirect repair of DNA by inhibiting ROS production.

Short-term Effects of Non-invasive Physical Plasma Treatment on Genomic Stability.

The application of non-invasive physical plasma (NIPP) generates reactive oxygen species. These can lead to chemical oxidation of cellular molecules including DNA. On the other hand, NIPP can induce therapeutically intended apoptosis, which also leads to DNA fragmentation in the late phase. Therefore, to assess unwanted genotoxic effects, the formation of DNA damage was investigated in this study in discrimination from apoptotic processes. Mutation events after NIPP application were analyzed in CCL-93 fibroblast cells using the hypoxanthine phosphoribosyl transferase assay. Additionally, DNA single-strand breaks (SSB) and double-strand breaks (DSB) were quantified by performing the alkaline comet assay, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. DSBs were quantified by phospho-histone 2AX-p53-binding protein 1 co-localization DSB focus assay. The data were compared with cell death quantification by the caspase-3/7 apoptosis assay. Treatment with NIPP led to exceedingly rapid damage to genomic DNA and the appearance of DNA SSBs and DSBs in the initial 4 h. However, damage decreased again within the first 4-8 h, then the late phase began, characterized by DNA DSB and increasing caspase-3/7 activation. Although NIPP treatment leads to extremely rapid damage to genomic DNA, this damage is reversed very quickly by efficient DNA-repair processes. As a consequence, only those cells whose genome damage can be repaired actually survive and proliferate. Persistent genotoxic effects were not observed in the cell system used.

Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia–telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study’s data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

The DNA repair enzyme, aprataxin, plays a role in innate immune signaling.

Ataxia with oculomotor apraxia type 1 (AOA1) is a progressive neurodegenerative disorder characterized by a gradual loss of coordination of hand movements, speech, and eye movements. AOA1 is caused by an inactivation mutation in the APTX gene. APTX resolves abortive DNA ligation intermediates. APTX deficiency may lead to the accumulation of 5'-AMP termini, especially in the mitochondrial genome. The consequences of APTX deficiency includes impaired mitochondrial function, increased DNA single-strand breaks, elevated reactive oxygen species production, and altered mitochondrial morphology. All of these processes can cause misplacement of nuclear and mitochondrial DNA, which can activate innate immune sensors to elicit an inflammatory response. This study explores the impact of APTX knockout in microglial cells, the immune cells of the brain. RNA-seq analysis revealed significant differences in the transcriptomes of wild-type and APTX knockout cells, especially in response to viral infections and innate immune pathways. Specifically, genes and proteins involved in the cGAS-STING and RIG-I/MAVS pathways were downregulated in APTX knockout cells, which suggests an impaired immune response to cytosolic DNA and RNA. The clinical relevance of these findings was supported by analyzing publicly available RNA-seq data from AOA1 patient cell lines. Comparisons between APTX-deficient patient cells and healthy control cells also revealed altered immune responses and dysregulated DNA- and RNA-sensing pathways in the patient cells. Overall, this study highlights the critical role of APTX in regulating innate immunity, particularly in DNA- and RNA-sensing pathways. Our findings contribute to a better understanding of the underlying molecular mechanisms of AOA1 pathology and highlights potential therapeutic targets for this disease.