bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒07‒26
forty-two papers selected by
Sean Rudd
Karolinska Institutet
  1. Endogenous toxic metabolites and implications in cancer therapy.
  2. NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.
  3. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism.
  4. Development of a chemical probe against NUDT15.
  5. NME3 Regulates Mitochondria to Reduce ROS-Mediated Genome Instability.
  6. GTP Metabolic Reprogramming by IMPDH2: Unlocking Cancer Cells' Fueling Mechanism.
  7. BRG1 loss predisposes lung cancers to replicative stress and ATR dependency.
  8. Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase.
  9. Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53.
  10. Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models.
  11. Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks.
  12. How to fix DNA-protein crosslinks.
  13. Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.
  14. Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.
  15. Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses.
  16. Role of H3K9 demethylases in DNA double-strand break repair.
  17. Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin.
  18. DNA Damage Response and Metabolic Reprogramming in Health and Disease.
  19. Detection of base analogs incorporated during DNA replication by nanopore sequencing.
  20. Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.
  21. Design, Synthesis and Evaluation of New Quinazolinone Derivatives that Inhibit Bloom's Syndrome Protein (BLM) Helicase, Trigger DNA damage at the Telomere Region and Synergize with PARP Inhibitors.
  22. Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal.
  23. Inhibition of c-MET increases the antitumour activity of PARP inhibitors in gastric cancer models.
  24. Ralaniten Sensitizes Enzalutamide-Resistant Prostate Cancer to Ionizing Radiation in Prostate Cancer Cells that Express Androgen Receptor Splice Variants.
  25. The Effects of Genetic and Epigenetic Alterations of BARD1 on the Development of Non-Breast and Non-Gynecological Cancers.
  26. Targeting DNA repair in combination with radiotherapy in pancreatic cancer: A systematic review of preclinical studies.
  27. Recent Advances in DNA Repair Pathway and Its Application in Personalized Care of Metastatic Castration-Resistant Prostate Cancer (mCRPC).
  28. Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells.
  29. Targeting the PLK1-FOXO1 pathway as a novel therapeutic approach for treating advanced prostate cancer.
  30. Control of the chromatin response to DNA damage: Histone proteins pull the strings.
  31. Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication.
  32. CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.
  33. Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction.
  34. CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.
  35. Ethanol exposure increases mutation rate through error-prone polymerases.
  36. SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase.
  37. A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.
  38. Stabilization of an abasic site paired against an unnatural triazolyl nitrobenzene nucleoside.
  39. Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia.
  40. NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase.
  41. Partial Purine Nucleoside Phosphorylase Deficiency Helps Determine Minimal Activity Required for Immune and Neurological Development.
  42. Fused and Substituted Pyrimidine Derivatives as a Profound Anticancer Agent.
  1. Oncogene. 2020 Jul 24.
    Endogenous toxic metabolites and implications in cancer therapy.
    Namgyu Lee, Meghan E Spears, Anne E Carlisle, Dohoon Kim.
      It is well recognized that many metabolic enzymes play essential roles in cancer cells in producing building blocks such as nucleotides, which are required in greater amounts due to their increased proliferation. On the other hand, the significance of enzymes in preventing the accumulation of their substrates is less recognized. Here, we outline the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, such as metabolites containing reactive groups (e.g., methylglyoxal, 4-hydroxynonenal, and glutaconyl-CoA), or metabolites that act as competitive analogs against other metabolites (e.g., deoxyuridine triphosphate and l-2-hydroxyglutarate). Thus, if a metabolic pathway contains a toxic intermediate, then we may be able to induce accumulation and poison a cancer cell by targeting the downstream enzyme. Furthermore, this poisoning may be cancer cell selective if this pathway is overactive in a cancer cell relative to a nontransformed cell. We describe this concept as illustrated in selenocysteine metabolism and other pathways and discuss future directions in exploiting toxic metabolites to kill cancer cells.
    DOI:  https://doi.org/10.1038/s41388-020-01395-9
  2. Nat Metab. 2020 Apr;2(4): 318-334
    NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.
    Douglas B Fox, Nina Marie G Garcia, Brock J McKinney, Ryan Lupo, Laura C Noteware, Rachel Newcomb, Juan Liu, Jason W Locasale, Matthew D Hirschey, James V Alvarez.
      The survival and recurrence of dormant tumour cells following therapy is a leading cause of death in cancer patients. The metabolic properties of these cells are likely distinct from those of rapidly growing tumours. Here we show that Her2 down-regulation in breast cancer cells promotes changes in cellular metabolism, culminating in oxidative stress and compensatory upregulation of the antioxidant transcription factor, NRF2. NRF2 is activated during dormancy and in recurrent tumours in animal models and breast cancer patients with poor prognosis. Constitutive activation of NRF2 accelerates recurrence, while suppression of NRF2 impairs it. In recurrent tumours, NRF2 signalling induces a transcriptional metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. The NRF2-driven metabolic state renders recurrent tumour cells sensitive to glutaminase inhibition, which prevents reactivation of dormant tumour cells in vitro, suggesting that NRF2-high dormant and recurrent tumours may be targeted. These data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer.
    Keywords:  Breast cancer recurrence; Her2; NRF2; ROS; Residual disease; Tumor metabolism
    DOI:  https://doi.org/10.1038/s42255-020-0191-z
  3. Nat Metab. 2019 Mar;1(3): 404-415
    NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism.
    Li Chen, Zhaoyue Zhang, Atsushi Hoshino, Henry D Zheng, Michael Morley, Zoltan Arany, Joshua D Rabinowitz.
      NADPH donates high-energy electrons for antioxidant defence and reductive biosynthesis. Cytosolic NADP is recycled to NADPH by the oxidative pentose-phosphate pathway (oxPPP), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). Here we show that any one of these routes can support cell growth, but the oxPPP is uniquely required to maintain a normal NADPH/NADP ratio, mammalian dihydrofolate reductase (DHFR) activity and folate metabolism. These findings are based on CRISPR deletions of glucose-6-phosphate dehydrogenase (G6PD, the committed oxPPP enzyme), ME1, IDH1 and combinations thereof in HCT116 colon cancer cells. Loss of G6PD results in high NADP, which induces compensatory increases in ME1 and IDH1 flux. But the high NADP inhibits DHFR, resulting in impaired folate-mediated biosynthesis, which is reversed by recombinant expression of Escherichia coli DHFR. Across different cancer cell lines, G6PD deletion produced consistent changes in folate-related metabolites, suggesting a general requirement for the oxPPP to support folate metabolism.
    DOI:  https://doi.org/10.1038/s42255-019-0043-x
  4. Nat Chem Biol. 2020 Jul 20.
    Development of a chemical probe against NUDT15.
    Si Min Zhang, Matthieu Desroses, Anna Hagenkort, Nicholas C K Valerie, Daniel Rehling, Megan Carter, Olov Wallner, Tobias Koolmeister, Adam Throup, Ann-Sofie Jemth, Ingrid Almlöf, Olga Loseva, Thomas Lundbäck, Hanna Axelsson, Shruti Regmi, Antonio Sarno, Andreas Krämer, Linda Pudelko, Lars Bräutigam, Azita Rasti, Mona Göttmann, Elisée Wiita, Juliane Kutzner, Torsten Schaller, Christina Kalderén, Armando Cázares-Körner, Brent D G Page, Rosa Krimpenfort, Saeed Eshtad, Mikael Altun, Sean G Rudd, Stefan Knapp, Martin Scobie, Evert J Homan, Ulrika Warpman Berglund, Pål Stenmark, Thomas Helleday.
      The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
    DOI:  https://doi.org/10.1038/s41589-020-0592-z
  5. Int J Mol Sci. 2020 Jul 17. pii: E5048. [Epub ahead of print]21(14):
    NME3 Regulates Mitochondria to Reduce ROS-Mediated Genome Instability.
    Chih-Wei Chen, Ning Tsao, Wei Zhang, Zee-Fen Chang.
      NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.
    Keywords:  DNA damage; NME3; mitochondrial morphology; oxidative stress
    DOI:  https://doi.org/10.3390/ijms21145048
  6. J Biochem. 2020 Jul 23. pii: mvaa085. [Epub ahead of print]
    GTP Metabolic Reprogramming by IMPDH2: Unlocking Cancer Cells' Fueling Mechanism.
    Satoshi Kofuji, Atsuo T Sasaki.
      Growing cells increase multiple biosynthetic processes in response to the high metabolic demands needed to sustain proliferation. The even higher metabolic requirements in the setting of cancer provoke proportionately greater biosynthesis. Underappreciated key aspects of this increased metabolic demand are guanine nucleotides and adaptive mechanisms to regulate their concentration. Using the malignant brain tumor, glioblastoma, as a model, we have demonstrated that one of the rate-limiting enzymes for guanosine triphosphate (GTP) synthesis, inosine monophosphate dehydrogenase-2 (IMPDH2), is increased and IMPDH2 expression is necessary for the activation of de novo GTP biosynthesis. Moreover, increased IMPDH2 enhances RNA polymerase I and III transcription directly linking GTP metabolism to both anabolic capacity as well as nucleolar enlargement historically observed as associated with cancer. In this review, we will review in detail the basis of these new discoveries and, more generally, summarize the current knowledge on the role of GTP metabolism in cancer.
    Keywords:  GTP; IMPDH; glioblastoma; nucleolus; ribosome
    DOI:  https://doi.org/10.1093/jb/mvaa085
  7. Cancer Res. 2020 Jul 20. pii: canres.1744.2020. [Epub ahead of print]
    BRG1 loss predisposes lung cancers to replicative stress and ATR dependency.
    Manav Gupta, Carla P Concepcion, Caroline G Fahey, Hasmik Keshishian, Arjun Bhutkar, Christine F Brainson, Francisco J Sanchez-Rivera, Patrizia Pessina, Jonathan Y Kim, Antoine Simoneau, Margherita Paschini, Mary C Beytagh, Caroline R Stanclift, Monica Schenone, D R Mani, Chendi Li, Audris Oh, Fei Li, Hai Hu, Angeliki Karatza, Roderick T Bronson, Alice T Shaw, Aaron N Hata, Kwok-Kin Wong, Lee Zou, Steven A Carr, Tyler Jacks, Carla F Kim.
      Inactivation of SMARCA4/BRG1, the core ATPase subunit of mammalian SWI/SNF complexes, occurs at very high frequencies in non-small cell lung cancers. There are no targeted therapies for this subset of lung cancers, nor is it known how mutations in BRG1 contribute to lung cancer progression. Using a combination of gain- and loss-of-function approaches, we demonstrate that deletion of BRG1 in lung cancer leads to activation of replication stress responses. Single-molecule assessment of replication fork dynamics in BRG1-deficient cells revealed increased origin firing mediated by the pre-licensing protein CDC6. Quantitative mass spectrometry and co-immunoprecipitation assays showed that BRG1-containing SWI/SNF complexes interact with RPA complexes. Lastly, BRG1-deficient lung cancers were sensitive to pharmacological inhibition of ATR. These findings provide novel mechanistic insight into BRG1-mutant lung cancers and suggest that their dependency on ATR can be leveraged therapeutically and potentially expanded to BRG1-mutant cancers in other tissues.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1744
  8. Nat Commun. 2020 Jul 24. 11(1): 3713
    Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase.
    Hazal B Kose, Sherry Xie, George Cameron, Melania S Strycharska, Hasan Yardimci.
      A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.
    DOI:  https://doi.org/10.1038/s41467-020-17443-7
  9. Elife. 2020 Jul 23. pii: e58571. [Epub ahead of print]9
    Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53.
    Syafiq Abd Wahab, Dirk Remus.
      Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.
    Keywords:  S. cerevisiae; biochemistry; chemical biology; chromosomes; gene expression
    DOI:  https://doi.org/10.7554/eLife.58571
  10. Nat Commun. 2020 Jul 24. 11(1): 3726
    Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models.
    Hyoung Kim, Haineng Xu, Erin George, Dorothy Hallberg, Sushil Kumar, Veena Jagannathan, Sergey Medvedev, Yasuto Kinose, Kyle Devins, Priyanka Verma, Kevin Ly, Yifan Wang, Roger A Greenberg, Lauren Schwartz, Neil Johnson, Robert B Scharpf, Gordon B Mills, Rugang Zhang, Victor E Velculescu, Eric J Brown, Fiona Simpkins.
      Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.
    DOI:  https://doi.org/10.1038/s41467-020-17127-2
  11. Cells. 2020 Jul 16. pii: E1699. [Epub ahead of print]9(7):
    Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks.
    Lanni Aquila, Boyko S Atanassov.
      Eukaryotic cells are constantly exposed to both endogenous and exogenous stressors that promote the induction of DNA damage. Of this damage, double strand breaks (DSBs) are the most lethal and must be efficiently repaired in order to maintain genomic integrity. Repair of DSBs occurs primarily through one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these pathways is in part regulated by histone post-translational modifications (PTMs) including ubiquitination. Ubiquitinated histones not only influence transcription and chromatin architecture at sites neighboring DSBs but serve as critical recruitment platforms for repair machinery as well. The reversal of these modifications by deubiquitinating enzymes (DUBs) is increasingly being recognized in a number of cellular processes including DSB repair. In this context, DUBs ensure proper levels of ubiquitin, regulate recruitment of downstream effectors, dictate repair pathway choice, and facilitate appropriate termination of the repair response. This review outlines the current understanding of histone ubiquitination in response to DSBs, followed by a comprehensive overview of the DUBs that catalyze the removal of these marks.
    Keywords:  DNA damage response; DSBs; DUBs; deubiquitinases; deubiquitinating enzymes; double strand break repair; histones; ubiquitination
    DOI:  https://doi.org/10.3390/cells9071699
  12. DNA Repair (Amst). 2020 Jul 09. pii: S1568-7864(20)30173-7. [Epub ahead of print]94 102924
    How to fix DNA-protein crosslinks.
    Ulrike Kühbacher, Julien P Duxin.
      Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks (DPCs). DPCs are generated by different chemotherapeutics, environmental or endogenous sources of crosslinking agents, or by lesions on DNA that stall the catalytic cycle of certain DNA processing enzymes. These bulky adducts impair processes on DNA such as DNA replication or transcription, and therefore pose a serious threat to genome integrity. The large diversity of DPCs suggests that there is more than one canonical mechanism to repair them. Indeed, many different enzymes have been shown to act on DPCs by either processing the protein, the DNA or the crosslink itself. In addition, the cell cycle stage or cell type are likely to dictate pathway choice. In recent years, a detailed understanding of DPC repair during S phase has started to emerge. Here, we review the current knowledge on the mechanisms of replication-coupled DPC repair, and describe and also speculate on possible pathways that remove DPCs outside of S phase. Moreover, we highlight a recent paradigm shifting finding that indicates that DPCs are not always detrimental, but can also play a protective role, preserving the genome from more deleterious forms of DNA damage.
    Keywords:  DNA repair; DNA replication; DNA-protein crosslinks (DPCs)
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102924
  13. Nat Metab. 2019 Oct;1(10): 958-965
    Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.
    Riikka H Hämäläinen, Juan C Landoni, Kati J Ahlqvist, Steffi Goffart, Sanna Ryytty, M Obaidur Rahman, Virginia Brilhante, Katherine Icay, Sampsa Hautaniemi, Liya Wang, Marikki Laiho, Anu Suomalainen.
      Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5'-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.
    DOI:  https://doi.org/10.1038/s42255-019-0120-1
  14. PLoS One. 2020 ;15(7): e0236291
    Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.
    Chen Yang, Yifan Zhang, Yi Chen, Franziska Ragaller, Mingzhi Liu, Sara Corvigno, Hanna Dahlstrand, Joseph Carlson, Zihua Chen, Anders Näsman, Ahmed Waraky, Yingbo Lin, Olle Larsson, Felix Haglund.
      Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried.
    DOI:  https://doi.org/10.1371/journal.pone.0236291
  15. Mem Inst Oswaldo Cruz. 2020 ;pii: S0074-02762020000100329. [Epub ahead of print]115 e200019
    Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses.
    Chantal Reigada, Melisa Sayé, Fabio Di Girolamo, Edward A Valera-Vera, Claudio A Pereira, Mariana R Miranda.
      BACKGROUND: NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro.OBJECTIVES: In the present study we explored the role of TcNDPK1 in DNA damage responses.
    METHODS: TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated.
    FINDINGS: Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair.
    MAIN CONCLUSION: These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.
    DOI:  https://doi.org/10.1590/0074-02760200019
  16. J Cancer Biol. 2020 ;1(1): 10-15
    Role of H3K9 demethylases in DNA double-strand break repair.
    Hee-Young Jeon, Arif Hussain, Jianfei Qi.
      H3K9 demethylases can remove the repressive H3K9 methylation marks on histones to alter chromatin structure, gene transcription and epigenetic state of cells. By counteracting the function of H3K9 methyltransferases, H3K9 demethylases have been shown to play an important role in numerous biological processes, including diseases such as cancer. Recent evidence points to a key role for some H3K9 demethylases in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) and/or non-homologous end joining (NHEJ) pathways. Mechanistically, H3K9 demethylases can upregulate the expression of DNA repair factors. They can also be recruited to the DNA damage sites and regulate the recruitment or function of DNA repair factors. Here, we will discuss the role and mechanisms of H3K9 demethylases in the regulation of DSB repair.
    Keywords:  DNA damage repair; H3K9; cancer; demethylation; double-strand breaks; epigenetic; histone demethylase
    DOI:  https://doi.org/10.46439/cancerbiology.1.003
  17. Proc Natl Acad Sci U S A. 2020 Jul 20. pii: 202003868. [Epub ahead of print]
    Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin.
    Mingrui Duan, Kathiresan Selvam, John J Wyrick, Peng Mao.
      Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.
    Keywords:  Cockayne syndrome; DNA repair; Spt4/Spt5; TFIIH; nucleosome
    DOI:  https://doi.org/10.1073/pnas.2003868117
  18. Trends Genet. 2020 Jul 16. pii: S0168-9525(20)30166-9. [Epub ahead of print]
    DNA Damage Response and Metabolic Reprogramming in Health and Disease.
    Ourania Chatzidoukaki, Evi Goulielmaki, Björn Schumacher, George A Garinis.
      Nuclear DNA damage contributes to cellular malfunction and the premature onset of age-related diseases, including cancer. Until recently, the canonical DNA damage response (DDR) was thought to represent a collection of nuclear processes that detect, signal and repair damaged DNA. However, recent evidence suggests that beyond nuclear events, the DDR rewires an intricate network of metabolic circuits, fine-tunes protein synthesis, trafficking, and secretion as well as balances growth with defense strategies in response to genotoxic insults. In this review, we discuss how the active DDR signaling mobilizes extranuclear and systemic responses to promote cellular homeostasis and organismal survival in health and disease.
    Keywords:  DNA damage; aging; cancer; disease; metabolism; stress response
    DOI:  https://doi.org/10.1016/j.tig.2020.06.018
  19. Nucleic Acids Res. 2020 Jul 25. pii: gkaa517. [Epub ahead of print]
    Detection of base analogs incorporated during DNA replication by nanopore sequencing.
    Daniela Georgieva, Qian Liu, Kai Wang, Dieter Egli.
      DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.
    DOI:  https://doi.org/10.1093/nar/gkaa517
  20. Cancers (Basel). 2020 Jul 17. pii: E1939. [Epub ahead of print]12(7):
    Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.
    Alice Bradbury, Rachel O'Donnell, Yvette Drew, Nicola J Curtin, Sweta Sharma Saha.
      In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response.
    Keywords:  ATR; PARP; homologous recombination repair; non-homologous end-joining; ovarian cancer; platinum
    DOI:  https://doi.org/10.3390/cancers12071939
  21. J Med Chem. 2020 Jul 22.
    Design, Synthesis and Evaluation of New Quinazolinone Derivatives that Inhibit Bloom's Syndrome Protein (BLM) Helicase, Trigger DNA damage at the Telomere Region and Synergize with PARP Inhibitors.
    Chen-Xi Wang, Zi-Lin Zhang, Qi-Kun Yin, Jia-Li Tu, Jia-En Wang, Yao-Hao Xu, Yong Rao, Tian-Miao Ou, Shi-Liang Huang, Ding Li, Honggen Wang, Qingjiang Li, Jia-Heng Tan, Shuo-Bin Chen, Zhi-Shu Huang.
      DNA damage response (DDR) pathways are crucial for the survival of cancer cells and are attractive targets for cancer therapy. Bloom syndrome protein (BLM) is a DNA helicase that performs important roles in DDR pathways. Our previous study discovered an effective new BLM inhibitor with a quinazolinone scaffold by a screening assay. Herein, to better understand the structure-activity relationship (SAR) and biological roles of the BLM inhibitor, a series of new derivatives were designed, synthesized and evaluated based on this scaffold. Among them, compound 9h exhibited nanomolar inhibitory activity and binding affinity for BLM. 9h could effectively disrupt BLM recruitment to DNA in cells. Furthermore, 9h inhibited the proliferation of the colorectal cell line HCT116 by significantly triggering DNA damage in the telomere region and inducing apoptosis, especially in combination with a PARP inhibitor. This result suggested a synthetic lethal effect between the BLM and PARP inhibitors in DDR pathways.
    DOI:  https://doi.org/10.1021/acs.jmedchem.0c00917
  22. Nucleic Acids Res. 2020 Jul 25. pii: gkaa611. [Epub ahead of print]
    Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal.
    Emilie Lebraud, Guillaume Pinna, Capucine Siberchicot, Jordane Depagne, Didier Busso, Damiano Fantini, Lamya Irbah, Elena Robeska, Gueorgui Kratassiouk, Jean-Luc Ravanat, Bernd Epe, J Pablo Radicella, Anna Campalans.
      One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.
    DOI:  https://doi.org/10.1093/nar/gkaa611
  23. J Cell Mol Med. 2020 Jul 20.
    Inhibition of c-MET increases the antitumour activity of PARP inhibitors in gastric cancer models.
    Evangelos Koustas, Michalis V Karamouzis, Panagiotis Sarantis, Dimitrios Schizas, Athanasios G Papavassiliou.
      Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Activation of c-MET increases tumour cell survival through the initiation of the DNA damage repair pathway. PARP is an essential key in the DNA damage repair pathway. The primary role of PARP is to detect and initiate an immediate cellular response to single-strand DNA breaks. Tumours suppressor genes such as BRCA1/2 are closely associated with the DNA repair pathway. In BRCA1/2 mutations or deficiency status, cells are more likely to develop additional genetic alterations and chromosomal instability and can lead to cancer. In this study, we investigate the role of c-MET and PARP inhibition in a gastric cancer model. We exploited functional in vitro and in vivo experiments to assess the antitumour potential of co-inhibition of c-MET (SU11274) and PARP (NU1025). This leads to a reduction of gastric cancer cells viability, especially after knockdown of BRCA1/2 through apoptosis and induction of γ-Η2ΑΧ. Moreover, in AGS xenograft models, the combinatorial treatment of NU1025 plus SU11274 reduced tumour growth and triggers apoptosis. Collectively, our data may represent a new therapeutic approach for GC thought co-inhibition of c-MET and PARP, especially for patients with BRCA1/2 deficiency tumours.
    Keywords:  BRCA1; BRCA2; PARP inhibitor; c-Met inhibitor; gastric cancer
    DOI:  https://doi.org/10.1111/jcmm.15655
  24. Cancers (Basel). 2020 Jul 21. pii: E1991. [Epub ahead of print]12(7):
    Ralaniten Sensitizes Enzalutamide-Resistant Prostate Cancer to Ionizing Radiation in Prostate Cancer Cells that Express Androgen Receptor Splice Variants.
    Carmen A Banuelos, Yusuke Ito, Jon K Obst, Nasrin R Mawji, Jun Wang, Yukiyoshi Hirayama, Jacky K Leung, Teresa Tam, Amy H Tien, Raymond J Andersen, Marianne D Sadar.
      Blocking androgen receptor (AR) transcriptional activity by androgen deprivation therapy (ADT) improves the response to radiotherapy for intermediate and high risk prostate cancer. Unfortunately, ADT, antiandrogens, and abiraterone increase expression of constitutively active splice variants of AR (AR-Vs) which regulate DNA damage repair leading to resistance to radiotherapy. Here we investigate whether blocking the transcriptional activities of full-length AR and AR-Vs with ralaniten leads to enhanced sensitivity to radiotherapy. Combination therapies using ralaniten with ionizing radiation were evaluated for effects on proliferation, colony formation, cell cycle, DNA damage, and Western blot analyses in human prostate cancer cells that express both full-length AR and AR-Vs. Ralaniten and a potent next-generation analog (EPI-7170) decreased expression of DNA repair genes whereas enzalutamide had no effect. FACS analysis revealed a dose-dependent decrease of BrdU incorporation with increased accumulation of γH2AX with a combination of ionizing radiation with ralaniten. An additive inhibitory effect on proliferation of enzalutamide-resistant cells was achieved with a combination of ralaniten compounds with ionizing radiation. Ralaniten and EPI-7170 sensitized prostate cancer cells that express full-length AR and AR-Vs to radiotherapy whereas enzalutamide had no added benefit.
    Keywords:  AR-V7; DNA repair; EPI-002; enzalutamide; prostate cancer; radiotherapy; ralaniten
    DOI:  https://doi.org/10.3390/cancers12071991
  25. Genes (Basel). 2020 Jul 21. pii: E829. [Epub ahead of print]11(7):
    The Effects of Genetic and Epigenetic Alterations of BARD1 on the Development of Non-Breast and Non-Gynecological Cancers.
    Andrea K Watters, Emily S Seltzer, Danny MacKenzie, Melody Young, Jonathan Muratori, Rama Hussein, Andrej M Sodoma, Julie To, Manrose Singh, Dong Zhang.
      Breast Cancer 1 (BRCA1) gene is a well-characterized tumor suppressor gene, mutations of which are primarily found in women with breast and ovarian cancers. BRCA1-associated RING domain 1 (BARD1) gene has also been identified as an important tumor suppressor gene in breast, ovarian, and uterine cancers. Underscoring the functional significance of the BRCA1 and BARD1 interactions, prevalent mutations in the BRCA1 gene are found in its RING domain, through which it binds the RING domain of BARD1. BARD1-BRCA1 heterodimer plays a crucial role in a variety of DNA damage response (DDR) pathways, including DNA damage checkpoint and homologous recombination (HR). However, many mutations in both BARD1 and BRCA1 also exist in other domains that significantly affect their biological functions. Intriguingly, recent genome-wide studies have identified various single nucleotide polymorphisms (SNPs), genetic alterations, and epigenetic modifications in or near the BARD1 gene that manifested profound effects on tumorigenesis in a variety of non-breast and non-gynecological cancers. In this review, we will briefly discuss the molecular functions of BARD1, including its BRCA1-dependent as well as BRCA1-independent functions. We will then focus on evaluating the common BARD1 related SNPs as well as genetic and epigenetic changes that occur in the non-BRCA1-dominant cancers, including neuroblastoma, lung, and gastrointestinal cancers. Furthermore, the pro- and anti-tumorigenic functions of different SNPs and BARD1 variants will also be discussed.
    Keywords:  BARD1; BARD1 isoforms; cancers; single nucleotide polymorphism (SNP)
    DOI:  https://doi.org/10.3390/genes11070829
  26. Crit Rev Oncol Hematol. 2020 Jul 11. pii: S1040-8428(20)30196-7. [Epub ahead of print]153 103060
    Targeting DNA repair in combination with radiotherapy in pancreatic cancer: A systematic review of preclinical studies.
    Waisse Waissi, Adrien Paix, Anaïs Nicol, Georges Noël, Hélène Burckel.
      BACKGROUND: Current research that combines radiation with targeted therapy may dramatically improve prognosis of pancreatic ductal adenocarcinoma (PDAC). We investigated preclinical outcomes of DNA repair inhibitor targeted therapy associated with radiotherapy.METHODS: We searched Pubmed database to identify publications assessing DNA damage targeted therapies in preclinical models of PDACin vitro and in vivo. Standard enhancement ratio, median survival and growth delay were extracted.
    RESULTS: We identified fourteen publications using DNA repair targeted therapies in preclinical models of PDAC. Ten publications comprising twenty-eight experiments evaluated radiosensitization with different DNA repair inhibitors in vitro and displayed cell killing by a factor of 1.35 ± 0.047. Moreover, 86 % (24/28) of in vitro experiments showed radiosensitization with DNA damage response inhibitor. However, only 60 % (9/15) of the in vivo experiments presented radiosensitization effects.
    CONCLUSION: DNA repair targeted therapies use promising radiosensitizers for PDAC and could successfully be translated into clinical trials.
    Keywords:  DNA repair; Inhibitors; Pancreatic cancer; Radiosensitization; Radiotherapy; Targeted therapy
    DOI:  https://doi.org/10.1016/j.critrevonc.2020.103060
  27. Methods Mol Biol. 2020 ;2204 75-89
    Recent Advances in DNA Repair Pathway and Its Application in Personalized Care of Metastatic Castration-Resistant Prostate Cancer (mCRPC).
    Chenyang Xu, Shanhua Mao, Haowen Jiang.
      Prostate cancer (PCa) is one of the common malignancies in male adults. In the era of precision medicine, many other novel agents targeting advanced prostate cancer, especially metastatic castration-resistant prostate cancer (mCRPC), are currently being evaluated. Among all these candidate therapies, poly-ADP ribose polymerase (PARP) inhibitors targeting DNA damage response (DDR) pathway has proven improving survival outcomes in clinical trials. In this review, we focus on recent advances in biology and clinical implication of DDR pathway and aim to discuss the latest results in advanced prostate cancer, especially mCRPC.
    Keywords:  Androgen-deprived treatment; DNA damage response; Homologous recombination deficiency; Metastatic castration-resistant prostate cancer; Prostate-specific antigen
    DOI:  https://doi.org/10.1007/978-1-0716-0904-0_7
  28. Cell Death Dis. 2020 Jul 24. 11(7): 577
    Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells.
    Beibei Zhai, Yue Li, Sudha Sravanti Kotapalli, Jeffrey Bacha, Dennis Brown, Anne Steinø, Mads Daugaard.
      1,2:5,6-Dianhydrogalactitol (DAG) is a bi-functional DNA-targeting agent currently in phase II clinical trial for treatment of temozolomide-resistant glioblastoma (GBM). In the present study, we investigated the cytotoxic activity of DAG alone or in combination with common chemotherapy agents in GBM and prostate cancer (PCa) cells, and determined the impact of DNA repair pathways on DAG-induced cytotoxicity. We found that DAG produced replication-dependent DNA lesions decorated with RPA32, RAD51, and γH2AX foci. DAG-induced cytotoxicity was unaffected by MLH1, MSH2, and DNA-PK expression, but was enhanced by knockdown of BRCA1. Acting in S phase, DAG displayed selective synergy with topoisomerase I (camptothecin and irinotecan) and topoisomerase II (etoposide) poisons in GBM, PCa, and lung cancer cells with no synergy observed for docetaxel. Importantly, DAG combined with irinotecan treatment enhanced tumor responses and prolonged survival of tumor-bearing mice. This work provides mechanistic insight into DAG cytotoxicity in GBM and PCa cells and offers a rational for exploring combination regimens with topoisomerase I/II poisons in future clinical trials.
    DOI:  https://doi.org/10.1038/s41419-020-02780-8
  29. Sci Rep. 2020 Jul 23. 10(1): 12327
    Targeting the PLK1-FOXO1 pathway as a novel therapeutic approach for treating advanced prostate cancer.
    Lilia Gheghiani, Shengzhe Shang, Zheng Fu.
      The forkhead box protein O1 (FOXO1) is considered to be a key tumor suppressor due to its involvement in a broad range of cancer-related functions, including cellular differentiation, apoptosis, cell cycle arrest, and DNA damage. Given that inactivation of FOXO1 has been reported in many types of human cancer, we sought to investigate whether restoration of the pro-apoptotic activity of FOXO1 may be used as a new promising strategy for cancer treatment. Our previous study revealed that Polo-like kinase 1 (PLK1), a serine/threonine kinase that is essential for cell cycle progression, is a novel and major regulator of FOXO1 in the late phases of the cell cycle. Here, we provided evidence that PLK1-dependent phosphorylation of FOXO1 induces its nuclear exclusion and negatively regulates FOXO1's transcriptional activity in prostate cancer (PCa). Blocking the PLK1-dependant phosphorylation of FOXO1 restored the pro-apoptotic function of FOXO1 in PCa. Combining PLK1 inhibition with nocodazole (to induce mitotic arrest) had synergistic antitumor effects in vitro, with minimal effect on normal prostate epithelial cells. These findings shed light on a novel approach to reactivate apoptotic pathways in advanced PCa and support targeting PLK1-FOXO1 pathways as a novel approach for treating advanced PCa.
    DOI:  https://doi.org/10.1038/s41598-020-69338-8
  30. Semin Cell Dev Biol. 2020 Jul 17. pii: S1084-9521(19)30096-5. [Epub ahead of print]
    Control of the chromatin response to DNA damage: Histone proteins pull the strings.
    Juliette Ferrand, Alexandre Plessier, Sophie E Polo.
      DNA damage challenges both genome integrity and its organization with histone proteins into chromatin, with prominent alterations in histone variant dynamics and histone modifications. While these alterations jeopardize epigenome stability, they are also instrumental for an efficient and timely response to DNA damage. Here, we review recent findings illustrating how histone variants and post-translational modifications actively contribute to and control the DNA damage response. We present accumulating evidence that histone protein changes help relieve the chromatin barrier to DNA repair by regulating chromatin compaction and mobility. We also highlight how histone modifications and variants control transcriptional silencing at damage sites, and we describe both pre-existing and DNA damage-induced chromatin features that govern DNA damage signaling and guide DNA repair pathway choice. We discuss how histone dynamics ultimately participate to the restoration of epigenome integrity and present our current knowledge of key molecular players involved in these critical processes.
    Keywords:  Chromatin dynamics; DNA damage repair; Histone post-translational modifications; Histone variants; Transcriptional regulation
    DOI:  https://doi.org/10.1016/j.semcdb.2020.07.002
  31. EMBO J. 2020 Jul 23. e104185
    Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication.
    Leticia K Lerner, Sandro Holzer, Mairi L Kilkenny, Saša Šviković, Pierre Murat, Davide Schiavone, Cara B Eldridge, Alice Bittleston, Joseph D Maman, Dana Branzei, Katherine Stott, Luca Pellegrini, Julian E Sale.
      Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis.
    Keywords:  DNA replication; G-quadruplex; fork protection complex; replisome; timeless
    DOI:  https://doi.org/10.15252/embj.2019104185
  32. PLoS Genet. 2020 Jul 21. 16(7): e1008933
    CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.
    Jia-Min Zhang, Jin-Xin Zheng, Yue-He Ding, Xiao-Ran Zhang, Fang Suo, Jing-Yi Ren, Meng-Qiu Dong, Li-Lin Du.
      Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.
    DOI:  https://doi.org/10.1371/journal.pgen.1008933
  33. Nat Metab. 2020 Jul;2(7): 635-647
    Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction.
    Tingting Wang, J N Rashida Gnanaprakasam, Xuyong Chen, Siwen Kang, Xuequn Xu, Hua Sun, Lingling Liu, Hayley Rodgers, Ethan Miller, Teresa A Cassel, Qiushi Sun, Sara Vicente-Muñoz, Marc O Warmoes, Penghui Lin, Zayda Lizbeth Piedra-Quintero, Mireia Guerau-de-Arellano, Kevin A Cassady, Song Guo Zheng, Jun Yang, Andrew N Lane, Xiaotong Song, Teresa W-M Fan, Ruoning Wang.
      T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.
    DOI:  https://doi.org/10.1038/s42255-020-0219-4
  34. Nucleic Acids Res. 2020 Jul 20. pii: gkaa603. [Epub ahead of print]
    CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.
    Jonathan M Geisinger, Tim Stearns.
      While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53-/- cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.
    DOI:  https://doi.org/10.1093/nar/gkaa603
  35. Nat Commun. 2020 Jul 21. 11(1): 3664
    Ethanol exposure increases mutation rate through error-prone polymerases.
    Karin Voordeckers, Camilla Colding, Lavinia Grasso, Benjamin Pardo, Lore Hoes, Jacek Kominek, Kim Gielens, Kaat Dekoster, Jonathan Gordon, Elisa Van der Zande, Peter Bircham, Toon Swings, Jan Michiels, Peter Van Loo, Sandra Nuyts, Philippe Pasero, Michael Lisby, Kevin J Verstrepen.
      Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.
    DOI:  https://doi.org/10.1038/s41467-020-17447-3
  36. Cell Death Dis. 2020 Jul 18. 11(7): 548
    SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase.
    Fanghua Li, Emil Mladenov, Sharif Mortoga, George Iliakis.
      The cell cycle-dependent engagement of DNA-end resection at DSBs is regulated by phosphorylation of CTIP by CDKs, the central regulators of cell cycle transitions. Cell cycle transitions are also intimately regulated by protein degradation via two E3 ubiquitin ligases: SCFSKP2 and APC/CCDH1 complex. Although APC/CCDH1 regulates CTIP in G1- and G2-phase, contributions by SCFSKP2 have not been reported. We demonstrate that SCFSKP2 is a strong positive regulator of resection. Knockdown of SKP2, fully suppresses resection in several cell lines. Notably, this suppression is G2-phase specific and is not observed in S-phase or G1-phase cells. Knockdown of SKP2 inactivates SCFSKP2 causing APC/CCDH1 activation, which degrades CTIP. The stabilizing function of SCFSKP2 on CTIP promotes resection and supports gene conversion (GC), alternative end joining (alt-EJ) and cell survival. We propose that CDKs and SCFSKP2-APC/CCDH1 cooperate to regulate resection and repair pathway choice at DSBs in G2-phase.
    DOI:  https://doi.org/10.1038/s41419-020-02755-9
  37. JCI Insight. 2020 Jul 23. pii: 138829. [Epub ahead of print]5(14):
    A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.
    Hao-Dong Li, Changzheng Lu, He Zhang, Qing Hu, Junqiu Zhang, Ileana C Cuevas, Subhransu S Sahoo, Mitzi Aguilar, Elizabeth G Maurais, Shanrong Zhang, Xiaojing Wang, Esra A Akbay, Guo-Min Li, Bo Li, Prasad Koduru, Peter Ly, Yang-Xin Fu, Diego H Castrillon.
      Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.
    Keywords:  Cancer immunotherapy; DNA repair; Genetics; Mouse models; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.138829
  38. Biophys Chem. 2020 Jul 11. pii: S0301-4622(20)30136-8. [Epub ahead of print]264 106428
    Stabilization of an abasic site paired against an unnatural triazolyl nitrobenzene nucleoside.
    Subhendu Sekhar Bag, Sayantan Sinha, Hiranya Gogoi, Subhashis Datta, Rajen Kundu, Sangita Talukdar.
      An abasic site is the most frequently observed among the various forms of DNA lesions in genomic DNA. If left unrepaired, an abasic site might turn out to be a principle cause for deleterious mutations and can be threat to cellular survival. Thus, to keep cellular integrity and measure the extent of DNA damage, recognition and stabilization of the abasic sites (apurinic/apyrimidinic site = Ap) are essential. Further, it is crucial to detect and stabilise the abasic site for towards the development of new diagnostics and chemotherapeutics. Herein, we report the stabilization of an abasic DNA duplex wherein the abasic site paired against a novel unnatural nucleoside, triazolylnitrobenzene (TNBBAc). This nucleoside is bulky and exhibits, high polarizability and good stacking propensity. Robust hetero-pair stabilization is another feature of it. Therefore, it is interesting to study the stabilization of an abasic DNA containing a synthesized triazolylnitrobenzene nucleoside TNBBAc We planned to study the thermal as well as the thermodynamic origin of abasic DNA stabilization by our synthesized oligonucleotide probe containing TNBBAc nucleoside. We observed that the nucleoside TNBBAc offered good thermal stabilization of a TNBBAc-Φ duplex via strong intercalative stacking interaction alongside an abasic site. The UV-visible spectroscopic study supported the intercalative stacking interaction. The stabilization though is marginal, but it would shed light on the design of bases of significant volume to stabilise abasic DNA to a greater extent.
    Keywords:  Abasic DNA stabilization; Pairing selectivity; Thermal melting/thermodynamic stability; Triazolylnitrobenzene ((TNB)B(Ac)) nucleoside; Unnatural DNA probe; Unnatural nucleoside
    DOI:  https://doi.org/10.1016/j.bpc.2020.106428
  39. Int J Mol Sci. 2020 Jul 18. pii: E5084. [Epub ahead of print]21(14):
    Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia.
    Fabian Freisleben, Lena Behrmann, Vanessa Thaden, Jana Muschhammer, Carsten Bokemeyer, Walter Fiedler, Jasmin Wellbrock.
      Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.
    Keywords:  ABCC11; AML; Ara-C; CDA; GLI3; HH; SAMHD1; cytarabine; resistance
    DOI:  https://doi.org/10.3390/ijms21145084
  40. Nat Metab. 2020 Apr;2(4): 364-379
    NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase.
    Yue Yang, Ning Zhang, Guoan Zhang, Anthony A Sauve.
      Dihydronicotinamide riboside (NRH) has been suggested to act as a precursor for the synthesis of NAD+, but the biochemical pathway converting it has been unknown. Here, we show that NRH can be converted into NAD+ via a salvage pathway in which adenosine kinase (ADK, also known as AK) acts as an NRH kinase. Using isotope-labelling approaches, we demonstrate that NRH is fully incorporated into NAD+, with NMNH acting as an intermediate. We further show that AK is enriched in fractions from cell lysates with NRH kinase activity, and that AK can convert NRH into NAD+. In cultured cells and mouse liver, pharmacological or genetic inhibition of AK blocks formation of reduced nicotinamide mononucleotide (NMNH) and inhibits NRH-stimulated NAD+ biosynthesis. Finally, we confirm the presence of endogenous NRH in the liver with metabolomics. Our findings establish NRH as a natural precursor of NAD+ and reveal a new route for NAD+ biosynthesis via an NRH salvage pathway involving AK.
    DOI:  https://doi.org/10.1038/s42255-020-0194-9
  41. Front Immunol. 2020 ;11 1257
    Partial Purine Nucleoside Phosphorylase Deficiency Helps Determine Minimal Activity Required for Immune and Neurological Development.
    Eyal Grunebaum, Nicholas Campbell, Matilde Leon-Ponte, Xiaobai Xu, Hugo Chapdelaine.
      Introduction: Complete or near complete absence of the purine nucleoside phosphorylase (PNP) enzyme causes a profound T cell immunodeficiency and neurological abnormalities that are often lethal in infancy and early childhood. We hypothesized that patients with partial PNP deficiency, characterized by a late and mild phenotype due to residual PNP enzyme, would provide important information about the minimal PNP activity needed for normal development. Methods: Three siblings with a homozygous PNP gene mutation (c.769C>G, p.His257Asp) resulting in partial PNP deficiency were investigated. PNP activity was semi-quantitively assayed by the conversion of [14C]inosine in hemolysates, mononuclear cells, and lymphoblastoid B cells. PNP protein expression was determined by Western Blotting in lymphoblastoid B cells. DNA repair was quantified by measuring viability of lymphoblastoid B cells following ionizing irradiation. Results: A 21-year-old female was referred for recurrent sino-pulmonary infections while her older male siblings, aged 25- and 28- years, did not suffer from significant infections. Two of the siblings had moderately reduced numbers of T, B, and NK cells, while the other had near normal lymphocyte subset numbers. T cell proliferations were normal in the two siblings tested. Hypogammaglobulinemia was noted in two siblings, including one that required immunoglobulin replacement. All siblings had typical (normal) neurological development. PNP activity in various cells from two patients were 8-11% of the normal level. All siblings had normal blood uric acid and increased PNP substrates in the urine. PNP protein expression in cells from the two patients examined was similar to that observed in cells from healthy controls. The survival of lymphoblastoid B cells from 2 partial PNP-deficient patients after irradiation was similar to that of PNP-proficient cells and markedly higher than the survival of cells from a patient with absent PNP activity or a patient with ataxia telangiectasia. Conclusions: Patients with partial PNP deficiency can present in the third decade of life with mild-moderate immune abnormalities and typical development. Near-normal immunity might be achieved with relatively low PNP activity.
    Keywords:  deficiency; gene therapy; mutations; partial; purine nucleoside phosphorylase
    DOI:  https://doi.org/10.3389/fimmu.2020.01257
  42. Anticancer Agents Med Chem. 2020 Jul 20.
    Fused and Substituted Pyrimidine Derivatives as a Profound Anticancer Agent.
    Nahid Abbas, Gurubasavaraja S P Matada, Prasad S Dhiwar, Shilpa Patel, Giles Devasahayam.
      The rationale behind drug design is strategic utilization of heterocyclic fragments with specific physicochemical properties to form molecular targeted agents. Among the heterocyclic molecules, pyrimidine has proved to be a privileged pharmacophore for various biological cancer targets. The anticancer potential of small molecules with fused and substituted pyrimidine can be enhanced through bioisosteric replacements and altering their ADME parameters. Despite of several small molecules used in cancer chemotherapy, oncology therapeutics has various limitations. Especially in their routes of administration and their concurrent side effects. Such pernicious effects may be overcome, via selective biological targeting. In this review we have discussed the biological targets to inhibit cancer. Structural activity relationship of fused and substituted pyrimidine was studied. Eco friendly synthetic approaches for pyrimidine derivatives have been discussed. This review will give an insight to scientists and researchers of medicinal chemistry discipline to design small molecules having a pyrimidine scaffold with high anticancer potential.
    Keywords:  Apoptosis; SAR; antiproliferative; green chemistry; pyrimidine; structural activity relationship; synthesis
    DOI:  https://doi.org/10.2174/1871520620666200721104431
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