bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021–03–07
47 papers selected by
Sean Rudd, Karolinska Institutet
  1. Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism.
  2. A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.
  3. Aldehyde dehydrogenase inhibitors promote DNA damage in ovarian cancer and synergize with ATM/ATR inhibitors.
  4. Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein.
  5. Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line.
  6. The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.
  7. MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity.
  8. Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses.
  9. The role of DNA damage response in chemo- and radio-resistance of cancer cells: Can DDR inhibitors sole the problem?
  10. Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity.
  11. The Multiple Cellular Roles of SMUG1 in Genome Maintenance and Cancer.
  12. SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage.
  13. RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks.
  14. The N-terminal domain of uracil-DNA glycosylase: Roles for disordered regions.
  15. CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.
  16. Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.
  17. CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication.
  18. A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.
  19. A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.
  20. Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes.
  21. Identification and Validation of ERK5 as a DNA Damage Modulating Drug Target in Glioblastoma.
  22. Single-Strand Annealing in Cancer.
  23. OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.
  24. COMMD1, from the Repair of DNA Double Strand Breaks, to a Novel Anti-Cancer Therapeutic Target.
  25. SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks.
  26. On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response.
  27. EPHA2 Interacts with DNA-PKcs in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells.
  28. Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.
  29. Avoid the trap: targeting PARP1 beyond human malignancy.
  30. WNT inhibition creates a BRCA-like state in Wnt-addicted cancer.
  31. Rapid Detection and Signaling of DNA Damage by PARP-1.
  32. Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.
  33. NF-κB-induced R-loop accumulation and DNA damage select for nucleotide excision repair deficiencies in adult T cell leukemia.
  34. E2F1: Cause and Consequence of DNA Replication Stress.
  35. Altered DNA repair creates novel Alu/Alu repeat-mediated deletions.
  36. Reciprocal Links between Pre-messenger RNA 3'-End Processing and Genome Stability.
  37. The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.
  38. Molecular disruption of DNA polymerase β for platinum sensitisation and synthetic lethality in epithelial ovarian cancers.
  39. Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.
  40. Silencing DNA Polymerase β Induces Aneuploidy as a Biomarker of Poor Prognosis in Oral Squamous Cell Cancer.
  41. Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.
  42. 1-C Metabolism-Serine, Glycine, Folates-In Acute Myeloid Leukemia.
  43. Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics.
  44. Response and Toxicity to Cytarabine Therapy in Leukemia and Lymphoma: From Dose Puzzle to Pharmacogenomic Biomarkers.
  45. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.
  46. Role of the DDX11 DNA Helicase in Warsaw Breakage Syndrome Etiology.
  47. Key Enzymes in Pyrimidine Synthesis, CAD and CPS1, Predict Prognosis in Hepatocellular Carcinoma.
  1. EMBO Mol Med. 2021 Mar 05. e12461
    Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism.
    Goetz Hartleben, Kenji Schorpp, Yun Kwon, Barbara Betz, Foivos-Filippos Tsokanos, Zahra Dantes, Arlett Schäfer, Ina Rothenaigner, José Manuel Monroy Kuhn, Pauline Morigny, Lisa Mehr, Sean Lin, Susanne Seitz, Janina Tokarz, Anna Artati, Jerzy Adamsky, Oliver Plettenburg, Dominik Lutter, Martin Irmler, Johannes Beckers, Maximilian Reichert, Kamyar Hadian, Anja Zeigerer, Stephan Herzig, Mauricio Berriel Diaz.
      By accentuating drug efficacy and impeding resistance mechanisms, combinatorial, multi-agent therapies have emerged as key approaches in the treatment of complex diseases, most notably cancer. Using high-throughput drug screens, we uncovered distinct metabolic vulnerabilities and thereby identified drug combinations synergistically causing a starvation-like lethal catabolic response in tumor cells from different cancer entities. Domperidone, a dopamine receptor antagonist, as well as several tricyclic antidepressants (TCAs), including imipramine, induced cancer cell death in combination with the mitochondrial uncoupler niclosamide ethanolamine (NEN) through activation of the integrated stress response pathway and the catabolic CLEAR network. Using transcriptome and metabolome analyses, we characterized a combinatorial response, mainly driven by the transcription factors CHOP and TFE3, which resulted in cell death through enhanced pyrimidine catabolism as well as reduced pyrimidine synthesis. Remarkably, the drug combinations sensitized human organoid cultures to the standard-of-care chemotherapy paclitaxel. Thus, our combinatorial approach could be clinically implemented into established treatment regimen, which would be further facilitated by the advantages of drug repurposing.
    Keywords:  cancer metabolism; integrated stress response; metabolic vulnerabilities; pyrimidine metabolism; tricyclic antidepressants
    DOI:  https://doi.org/10.15252/emmm.202012461
  2. Cell Chem Biol. 2021 Mar 01. pii: S2451-9456(21)00058-1. [Epub ahead of print]
    A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.
    Duncan E Scott, Nicola J Francis-Newton, May E Marsh, Anthony G Coyne, Gerhard Fischer, Tommaso Moschetti, Andrew R Bayly, Timothy D Sharpe, Kalina T Haas, Lorraine Barber, Chiara R Valenzano, Rajavel Srinivasan, David J Huggins, Miyoung Lee, Amy Emery, Bryn Hardwick, Matthias Ehebauer, Claudio Dagostin, Alessandro Esposito, Luca Pellegrini, Trevor Perrior, Grahame McKenzie, Tom L Blundell, Marko Hyvönen, John Skidmore, Ashok R Venkitaraman, Chris Abell.
      BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
    Keywords:  BRCA2; DNA repair; RAD51; RAD51 inhibitor; cancer therapy; homologous recombination; protein-protein interaction inhibition; radiosensitizer; structure-guided drug discovery
    DOI:  https://doi.org/10.1016/j.chembiol.2021.02.006
  3. Theranostics. 2021 ;11(8): 3540-3551
    Aldehyde dehydrogenase inhibitors promote DNA damage in ovarian cancer and synergize with ATM/ATR inhibitors.
    Edward Grimley, Alexander J Cole, Thong T Luong, Stacy C McGonigal, Sarah Sinno, Dongli Yang, Kara A Bernstein, Ronald J Buckanovich.
      Rationale: Aldehyde dehydrogenase (ALDH) enzymes are often upregulated in cancer cells and associated with therapeutic resistance. ALDH enzymes protect cells by metabolizing toxic aldehydes which can induce DNA double stand breaks (DSB). We recently identified a novel ALDH1A family inhibitor (ALDHi), 673A. We hypothesized that 673A, via inhibition of ALDH1A family members, could induce intracellular accumulation of genotoxic aldehydes to cause DSB and that ALDHi could synergize with inhibitors of the ATM and ATR, proteins which direct DSB repair. Methods: We used immunofluorescence to directly assess levels of the aldehyde 4-hydroxynonenal and comet assays to evaluate DSB. Western blot was used to evaluate activation of the DNA damage response pathways. Cell counts were performed in the presence of 673A and additional aldehydes or aldehyde scavengers. ALDH inhibition results were confirmed using ALDH1A3 CRISPR knockout. Synergy between 673A and ATM or ATR inhibitors was evaluated using the Chou-Talalay method and confirmed in vivo using cell line xenograft tumor studies. Results: The ALDHi 673A cellular accumulation of toxic aldehydes which induce DNA double strand breaks. This is exacerbated by addition of exogenous aldehydes such as vitamin-A (retinaldehyde) and ameliorated by aldehyde scavengers such as metformin and hydralazine. Importantly, ALDH1A3 knockout cells demonstrated increased sensitivity to ATM/ATR inhibitors. And, ALDHi synergized with inhibitors of ATM and ATR, master regulators of the DSB DNA damage response, both in vitro and in vivo. This synergy was evident in homologous recombination (HR) proficient cell lines. Conclusions: ALDHi can be used to induce DNA DSB in cancer cells and synergize with inhibitors the ATM/ATR pathway. Our data suggest a novel therapeutic approach to target HR proficient ovarian cancer cells.
    Keywords:  ATM; ATR; DNA damage; Ovarian cancer; aldehyde dehydrogenase
    DOI:  https://doi.org/10.7150/thno.51885
  4. Elife. 2021 Mar 01. pii: e65339. [Epub ahead of print]10
    Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein.
    Xiangrong Chen, Yusuf I Ali, Charlotte El Fisher, Raquel Arribas-Bosacoma, Mohan B Rajasekaran, Gareth Williams, Sarah Walker, Jessica R Booth, Jessica Jr Hudson, S Mark Roe, Laurence H Pearl, Simon E Ward, Frances Mg Pearl, Antony W Oliver.
      BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.
    Keywords:  Bloom syndrome; DNA repair; E. coli; Helicase; RECQ; allosteric; biochemistry; chemical biology; human; inhibitor; molecular biophysics; structural biology
    DOI:  https://doi.org/10.7554/eLife.65339
  5. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2020187118. [Epub ahead of print]118(10):
    Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line.
    Chunfang Gu, Juan Liu, Xiaojing Liu, Haibo Zhang, Ji Luo, Huanchen Wang, Jason W Locasale, Stephen B Shears.
      Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, far-reaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line.
    Keywords:  PPIP5K; inositol pyrophosphates; nucleotide synthesis; pentose phosphate pathway; serine–glycine–one-carbon metabolism
    DOI:  https://doi.org/10.1073/pnas.2020187118
  6. Cancers (Basel). 2021 Feb 28. pii: 1003. [Epub ahead of print]13(5):
    The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.
    Valentina Gaidano, Mohammad Houshmand, Nicoletta Vitale, Giovanna Carrà, Alessandro Morotti, Valerio Tenace, Stefania Rapelli, Stefano Sainas, Agnese Chiara Pippione, Marta Giorgis, Donatella Boschi, Marco Lucio Lolli, Daniela Cilloni, Alessandro Cignetti, Giuseppe Saglio, Paola Circosta.
      Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.
    Keywords:  DHODH; acute myeloid leukemia; apoptosis; cancer metabolism; differentiation; dipyridamole; pyrimidine depletion
    DOI:  https://doi.org/10.3390/cancers13051003
  7. Cell Rep. 2021 Mar 02. pii: S2211-1247(21)00122-4. [Epub ahead of print]34(9): 108808
    MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity.
    Timothy B Branigan, David Kozono, Amy E Schade, Peter Deraska, Hembly G Rivas, Larissa Sambel, Hunter D Reavis, Geoffrey I Shapiro, Alan D D'Andrea, James A DeCaprio.
      To identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.
    Keywords:  CDK1; CHK1; FOXM1; LIN54; MYBL2; MuvB; cell cycle checkpoint; non-small-cell lung cancer; prexasertib
    DOI:  https://doi.org/10.1016/j.celrep.2021.108808
  8. DNA Repair (Amst). 2021 Feb 20. pii: S1568-7864(21)00032-X. [Epub ahead of print]101 103076
    Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses.
    Aashish Soni, Emil Mladenov, George Iliakis.
      Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G2-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G2-phase. We report that in the absence of functional HRR, activation of the G2-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G2-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G2-checkpoint in HRR-deficient cells, suggesting that the G2-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G2-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G2-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G2-phase activate a weak but readily detectable ATM/ATR-dependent G2-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G2-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G2-checkpoint are closely intertwined in cells exposed to low IR-doses in G2-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G2-phase, and cells irradiated in S-phase utilize a different mechanistic setup.
    Keywords:  ATM; ATR; BRCA2; DNA double strand breaks (DSB); G(2)-checkpoint; Homologous recombination repair; Ionizing radiation (IR)
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103076
  9. DNA Repair (Amst). 2021 Feb 18. pii: S1568-7864(21)00030-6. [Epub ahead of print]101 103074
    The role of DNA damage response in chemo- and radio-resistance of cancer cells: Can DDR inhibitors sole the problem?
    Fatemeh Sadoughi, Liaosadat Mirsafaei, Parisa Maleki Dana, Jamal Hallajzadeh, Zatollah Asemi, Mohammad Ali Mansournia, Majid Montazer, Mohammad Hosseinpour, Bahman Yousefi.
      Up to now, many improvements have been made in providing more therapeutic strategies for cancer patients. The lack of susceptibility to common therapies like chemo- and radio-therapy is one of the reasons why we need more methods in the field of cancer therapy. DNA damage response (DDR) is a set of mechanisms which identifies DNA lesions and triggers the repair process for restoring DNA after causing an arrest in the cell cycle. The ability of DDR in maintaining the genome stability and integrity can be favorable to cancerous cells which are exposed to radiation therapy or are treated with chemotherapeutic agents. When DDR mechanisms are error-free in cancer cells, they can escape the expected cellular death and display resistance to treatment. In this regard, targeting different components of DDR can help to increase the susceptibility of advanced tumors to chemo- and radio-therapy.
    Keywords:  ATM; ATR; Chemo-resistance; DDR; PARP1; Radio-resistance
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103074
  10. Nucleic Acids Res. 2021 Feb 28. pii: gkab110. [Epub ahead of print]
    Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity.
    Joshua T Burgess, Chee Man Cheong, Amila Suraweera, Thais Sobanski, Sam Beard, Keyur Dave, Maddison Rose, Didier Boucher, Laura V Croft, Mark N Adams, Kenneth O'Byrne, Derek J Richard, Emma Bolderson.
      DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor-Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.
    DOI:  https://doi.org/10.1093/nar/gkab110
  11. Int J Mol Sci. 2021 Feb 17. pii: 1981. [Epub ahead of print]22(4):
    The Multiple Cellular Roles of SMUG1 in Genome Maintenance and Cancer.
    Sripriya Raja, Bennett Van Houten.
      Single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1) works to remove uracil and certain oxidized bases from DNA during base excision repair (BER). This review provides a historical characterization of SMUG1 and 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) one important substrate of this enzyme. Biochemical and structural analyses provide remarkable insight into the mechanism of this glycosylase: SMUG1 has a unique helical wedge that influences damage recognition during repair. Rodent studies suggest that, while SMUG1 shares substrate specificity with another uracil glycosylase UNG2, loss of SMUG1 can have unique cellular phenotypes. This review highlights the multiple roles SMUG1 may play in preserving genome stability, and how the loss of SMUG1 activity may promote cancer. Finally, we discuss recent studies indicating SMUG1 has moonlighting functions beyond BER, playing a critical role in RNA processing including the RNA component of telomerase.
    Keywords:  5-hmdU; DNA damage; SMUG1; base excision repair; cancer
    DOI:  https://doi.org/10.3390/ijms22041981
  12. Bio Protoc. 2020 Aug 05. 10(15): e3701
    SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage.
    Angela Altieri, Milena Dell'Aquila, Francesca Pentimalli, Antonio Giordano, Luigi Alfano.
      DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3' single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3' single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3' single-strand DNA tails, generated from DSB processing.
    Keywords:  BrdU; DNA end-resection; DNA repair; Homologous Recombination; Immunofluorescence
    DOI:  https://doi.org/10.21769/BioProtoc.3701
  13. Oncogenesis. 2021 Mar 05. 10(3): 24
    RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks.
    Qunsong Tan, Kaifeng Niu, Yuqi Zhu, Zixiang Chen, Yueyang Li, Mengge Li, Di Wei, Adayabalam S Balajee, Hongbo Fang, Yongliang Zhao.
      Ubiquitination-dependent DNA damage response (DDR) signals play a critical role in the cellular choice of DNA damage repair pathways. Human DNA helicase RecQL4 participates in DNA replication and repair, and loss of RecQL4 is associated with autosomal recessive genetic disorders characterized by genomic instability features. In an earlier study, RecQL4 was isolated as a stable complex that contained two ubiquitin ligases of the N-end rule (UBR1 and UBR2). However, it is unknown whether or not RecQL4 ubiquitination status is critical for its DNA repair function. Here, we report that RecQL4 directly interacts with RNF8 (a RING finger ubiquitin E3 ligase), and both co-localize at DNA double-strand break (DSB) sites. Our findings indicate that RNF8 ubiquitinates RecQL4 protein mainly at the lysine sites of 876, 1048, and 1101, thereby facilitating the dissociation of RecQL4 from DSB sites. RecQL4 mutant at ubiquitination sites had a significantly prolonged retention at DSBs, which hinders the recruitment of its direct downstream DSB repair proteins (CtIP & Ku80). Interestingly, reduced DSB repair capacity observed in RecQL4 depleted cells was restored only by the reconstitution of wild-type RecQL4, but not the ubiquitination mutant. Additionally, RecQL4 directly interacts with WRAP53β that is known to recruit RNF8 to DSBs and WRAP53β enhances the association of RecQL4 with RNF8. WRAP53β silencing resulted in a nearly diminished recruitment of RNF8 to DSBs and in a greatly attenuated dissociation of RecQL4 from the DSB sites. Collectively, our study demonstrates that the ubiquitination event mediated by RNF8 constitutes an essential component for RecQL4's function in DSB repair.
    DOI:  https://doi.org/10.1038/s41389-021-00315-0
  14. DNA Repair (Amst). 2021 Feb 18. pii: S1568-7864(21)00033-1. [Epub ahead of print]101 103077
    The N-terminal domain of uracil-DNA glycosylase: Roles for disordered regions.
    Jacob L Perkins, Linlin Zhao.
      The presence of uracil in DNA calls for rapid removal facilitated by the uracil-DNA glycosylase superfamily of enzymes, which initiates the base excision repair (BER) pathway. In humans, uracil excision is accomplished primarily by the human uracil-DNA glycosylase (hUNG) enzymes. In addition to BER, hUNG enzymes play a key role in somatic hypermutation to generate antibody diversity. hUNG has several isoforms, with hUNG1 and hUNG2 being the two major isoforms. Both isoforms contain disordered N-terminal domains, which are responsible for a wide range of functions, with minimal direct impact on catalytic efficiency. Subcellular localization of hUNG enzymes is directed by differing N-terminal sequences, with hUNG1 dedicated to mitochondria and hUNG2 dedicated to the nucleus. An alternative isoform of hUNG1 has also been identified to localize to the nucleus in mouse and human cell models. Furthermore, hUNG2 has been observed at replication forks performing both pre- and post-replicative uracil excision to maintain genomic integrity. Replication protein A (RPA) and proliferating cell nuclear antigen (PCNA) are responsible for recruitment to replication forks via protein-protein interactions with the N-terminus of hUNG2. These interactions, along with protein degradation, are regulated by various post-translational modifications within the N-terminal tail, which are primarily cell-cycle dependent. Finally, translocation on DNA is also mediated by interactions between the N-terminus and DNA, which is enhanced under molecular crowding conditions by preventing diffusion events and compacting tail residues. This review summarizes recent research supporting the emerging roles of the N-terminal domain of hUNG.
    Keywords:  Base excision repair; DNA damage; DNA repair; Deoxyuridine; Post-translational modification; Protein interaction
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103077
  15. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2015634118. [Epub ahead of print]118(10):
    CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.
    Simona Giunta, Solène Hervé, Ryan R White, Therese Wilhelm, Marie Dumont, Andrea Scelfo, Riccardo Gamba, Cheng Kit Wong, Giulia Rancati, Agata Smogorzewska, Hironori Funabiki, Daniele Fachinetti.
      Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.
    Keywords:  CENP-A chromatin; DNA replication stress; centromere; chromosome translocations; genome instability
    DOI:  https://doi.org/10.1073/pnas.2015634118
  16. Oncol Rep. 2021 Apr;45(4): 1-8
    Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines.
    Tomonari Suetsugu, Ryutaro Mori, Manabu Futamura, Masahiro Fukada, Hideharu Tanaka, Itaru Yasufuku, Yuta Sato, Yoshinori Iwata, Takeharu Imai, Hisashi Imai, Yoshihiro Tanaka, Naoki Okumura, Nobuhisa Matsuhashi, Takao Takahashi, Kazuhiro Yoshida.
      Fluorouracil (5FU) is converted to its active metabolite fluoro‑deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)‑ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)‑thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU‑resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT‑RR pathway in SW48 cells but was scarcely synthesized through either the OPRT‑RR or TP‑TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP‑led 5FU resistance. These results indicated that fluoro‑deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT‑RR and TP‑TK pathways in LS174T cells but mainly through the TP‑TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross‑resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro‑thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU‑resistant cells.
    DOI:  https://doi.org/10.3892/or.2021.7978
  17. Mol Cells. 2021 Feb 28. 44(2): 101-115
    CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication.
    Hye-Ran Seo, Daun Jeong, Sunmi Lee, Han-Sae Lee, Shin-Ai Lee, Sang Won Kang, Jongbum Kwon.
      The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its halflife. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.
    Keywords:  BRCA1-associated protein 1; C-terminus of Hsp70-interacting protein; DNA replication; INO80 chromatin remodeler; ubiquitin
    DOI:  https://doi.org/10.14348/molcells.2021.2258
  18. Biomolecules. 2021 Feb 15. pii: 288. [Epub ahead of print]11(2):
    A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.
    Ioannis Emmanouilidis, Natalia Fili, Alexander W Cook, Yukti Hari-Gupta, Ália Dos Santos, Lin Wang, Marisa L Martin-Fernandez, Peter J I Ellis, Christopher P Toseland.
      Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.
    Keywords:  Cas9; DNA damage; DNA repair; double-strand break
    DOI:  https://doi.org/10.3390/biom11020288
  19. Int J Mol Sci. 2021 Feb 27. pii: 2365. [Epub ahead of print]22(5):
    A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.
    Elisa Mentegari, Federica Bertoletti, Miroslava Kissova, Elisa Zucca, Silvia Galli, Giulia Tagliavini, Anna Garbelli, Antonio Maffia, Silvia Bione, Elena Ferrari, Fabrizio d'Adda di Fagagna, Sofia Francia, Simone Sabbioneda, Liuh-Yow Chen, Joachim Lingner, Valerie Bergoglio, Jean-Sébastien Hoffmann, Ulrich Hübscher, Emmanuele Crespan, Giovanni Maga.
      Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
    Keywords:  DNA double-strand breaks (DSBs), extra-chromosomal telomeric repeats (ECTRs), promyelocytic leukemia (PML) bodies; alternative lengthening of telomeres (ALT), DNA polymerase λ; microhomology-mediated strand transfer (MMST) activity; telomere dysfunction-induced foci (TIFs), telomere stress
    DOI:  https://doi.org/10.3390/ijms22052365
  20. Mol Cell. 2021 Feb 18. pii: S1097-2765(21)00091-5. [Epub ahead of print]
    Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes.
    Martina Peritore, Karl-Uwe Reusswig, Susanne C S Bantele, Tobias Straub, Boris Pfander.
      In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.
    Keywords:  DNA binding; DNA double-stranded breaks; DNA end resection; histone; homologous recombination; nucleosome; nucleosome remodeller; single-stranded DNA
    DOI:  https://doi.org/10.1016/j.molcel.2021.02.005
  21. Cancers (Basel). 2021 Feb 24. pii: 944. [Epub ahead of print]13(5):
    Identification and Validation of ERK5 as a DNA Damage Modulating Drug Target in Glioblastoma.
    Natasha Carmell, Ola Rominiyi, Katie N Myers, Connor McGarrity-Cottrell, Aurelie Vanderlinden, Nikita Lad, Eva Perroux-David, Sherif F El-Khamisy, Malee Fernando, Katherine G Finegan, Stephen Brown, Spencer J Collis.
      Brain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)-the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease.
    Keywords:  DNA damage; ERK5; MAPK7; glioblastoma; sensitisation; temozolomide
    DOI:  https://doi.org/10.3390/cancers13050944
  22. Int J Mol Sci. 2021 Feb 22. pii: 2167. [Epub ahead of print]22(4):
    Single-Strand Annealing in Cancer.
    Janusz Blasiak.
      DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.
    Keywords:  BRCAness; CRISPR/Cas9; DNA double-strand break repair; RAD52; SSA; cancer; homologous recombination; single-strand annealing; synthetic lethality; therapeutic genome editing
    DOI:  https://doi.org/10.3390/ijms22042167
  23. J Biol Chem. 2021 Feb 25. pii: S0021-9258(21)00239-8. [Epub ahead of print] 100466
    OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.
    Qiong Wu, Yaping Huang, Liya Gu, Zhijie Chang, Guo-Min Li.
      DNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSα (MSH2-MSH6) and MutSβ (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or under-expression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy.
    Keywords:  DNA damage; DNA mismatch repair; OTUB1; deubiquitination; mutability; protein stability
    DOI:  https://doi.org/10.1016/j.jbc.2021.100466
  24. Cancers (Basel). 2021 Feb 16. pii: 830. [Epub ahead of print]13(4):
    COMMD1, from the Repair of DNA Double Strand Breaks, to a Novel Anti-Cancer Therapeutic Target.
    Amila Suraweera, Pascal H G Duijf, Christian Jekimovs, Karsten Schrobback, Cheng Liu, Mark N Adams, Kenneth J O'Byrne, Derek J Richard.
      Lung cancer has the highest incidence and mortality among all cancers, with non-small cell lung cancer (NSCLC) accounting for 85-90% of all lung cancers. Here we investigated the function of COMMD1 in the repair of DNA double strand breaks (DSBs) and as a prognostic and therapeutic target in NSCLC. COMMD1 function in DSB repair was investigated using reporter assays in COMMD1-siRNA-depleted cells. The role of COMMD1 in NSCLC was investigated using bioinformatic analysis, qRT-PCR and immunoblotting of control and NSCLC cells, tissue microarrays, cell viability and cell cycle experiments. DNA repair assays demonstrated that COMMD1 is required for the efficient repair of DSBs and reporter assays showed that COMMD1 functions in both non-homologous-end-joining and homologous recombination. Bioinformatic analysis showed that COMMD1 is upregulated in NSCLC, with high levels of COMMD1 associated with poor patient prognosis. COMMD1 mRNA and protein were upregulated across a panel of NSCLC cell lines and siRNA-mediated depletion of COMMD1 decreased cell proliferation and reduced cell viability of NSCLC, with enhanced death after exposure to DNA damaging-agents. Bioinformatic analyses demonstrated that COMMD1 levels positively correlate with the gene ontology DNA repair gene set enrichment signature in NSCLC. Taken together, COMMD1 functions in DSB repair, is a prognostic maker in NSCLC and is potentially a novel anti-cancer therapeutic target for NSCLC.
    Keywords:  COMMD1; DNA double strand break repair; genomic stability; non-small cell lung cancer; novel therapeutic target
    DOI:  https://doi.org/10.3390/cancers13040830
  25. Bio Protoc. 2019 Sep 20. 9(18): e3377
    SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks.
    Sunetra Roy, Katharina Schlacher.
      The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5'-ethylene-2'-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (e.g., IF) or assay an average change of millions of cells without single-cell resolution (e.g., iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed "in situ protein interaction with nascent DNA replication forks (SIRF)". This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.
    Keywords:  DNA replication; Fork protection; Genome instability; IPOND; Proximity ligation assay; SIRF; Stalled replication forks
    DOI:  https://doi.org/10.21769/BioProtoc.3377
  26. Cells. 2021 Feb 27. pii: 507. [Epub ahead of print]10(3):
    On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response.
    Isadora Carolina Betim Pavan, Andressa Peres de Oliveira, Pedro Rafael Firmino Dias, Fernanda Luisa Basei, Luidy Kazuo Issayama, Camila de Castro Ferezin, Fernando Riback Silva, Ana Luisa Rodrigues de Oliveira, Lívia Alves Dos Reis Moura, Mariana Bonjiorno Martins, Fernando Moreira Simabuco, Jörg Kobarg.
      NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIβ, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases.
    Keywords:  DNA damage response; cell cycle; kinase; protein kinase
    DOI:  https://doi.org/10.3390/cells10030507
  27. Cancers (Basel). 2021 Feb 28. pii: 1010. [Epub ahead of print]13(5):
    EPHA2 Interacts with DNA-PKcs in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells.
    Vitaliy O Kaminskyy, Petra Hååg, Metka Novak, Ákos Végvári, Vasiliki Arapi, Rolf Lewensohn, Kristina Viktorsson.
      Ephrin (EFN)/ Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.
    Keywords:  DNA damage response; DNA-PKcs; EphA2; ionizing radiation; non-small cell lung cancer
    DOI:  https://doi.org/10.3390/cancers13051010
  28. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2021120118. [Epub ahead of print]118(10):
    Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.
    Ke Shi, Nicholas H Moeller, Surajit Banerjee, Jennifer L McCann, Michael A Carpenter, Lulu Yin, Ramkumar Moorthy, Kayo Orellana, Daniel A Harki, Reuben S Harris, Hideki Aihara.
      Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.
    Keywords:  DNA damage repair; DNA deamination; deoxyinosine; deoxyuridine; endonuclease
    DOI:  https://doi.org/10.1073/pnas.2021120118
  29. Cell Chem Biol. 2021 Feb 23. pii: S2451-9456(21)00056-8. [Epub ahead of print]
    Avoid the trap: targeting PARP1 beyond human malignancy.
    Chiho Kim, Chuo Chen, Yonghao Yu.
      PARP1 is a poly(ADP-ribose) polymerase (PARP) enzyme that plays a critical role in regulating DNA damage response. The main enzymatic function of PARP1 is to catalyze a protein post-translational modification known as poly(ADP-ribosyl)ation (PARylation). Human cancers with homologous recombination deficiency are highly sensitive to PARP1 inhibitors. PARP1 is aberrantly activated in many non-oncological diseases, leading to the excessive NAD+ depletion and PAR formation, thus causing cell death and tissue damage. PARP1 deletion offers a profound protective effect in the relevant animal models. However, many of the current PARP1 inhibitors also induce PARP1 trapping, which drives subsequent DNA damage, innate immune response and cytotoxicity. This minireview provides an overview of the basic biology of PARP1 trapping, and its implications in disease. Furthermore, we also discuss the recent development of PARP1 PROTAC compounds, and their utility as "non-trapping" PARP1 degraders for the potential amelioration of non-oncological diseases driven by aberrant PARP1 activation.
    Keywords:  BRCA; NAD(+); PARP; breast cancer; cell death; ischemia reperfusion injury; neurodegeneration; ovarian cancer; phase transition; poly(ADP-ribose); stroke
    DOI:  https://doi.org/10.1016/j.chembiol.2021.02.004
  30. EMBO Mol Med. 2021 Mar 04. e13349
    WNT inhibition creates a BRCA-like state in Wnt-addicted cancer.
    Amanpreet Kaur, Jun Yi Stanley Lim, Sugunavathi Sepramaniam, Siddhi Patnaik, Nathan Harmston, May Ann Lee, Enrico Petretto, David M Virshup, Babita Madan.
      Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt-addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt-addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51, are dependent on Wnt/β-catenin signaling in Wnt-high cancers, and treatment with a PORCN inhibitor creates a BRCA-like state. This coherent regulation of DNA repair genes occurs in part via a Wnt/β-catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt-high APCmin mutant polyps. Our findings suggest a general paradigm that Wnt/β-catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents.
    Keywords:  BRCA1; DNA repair; ETC-1922159; FANCD2; homologous recombination
    DOI:  https://doi.org/10.15252/emmm.202013349
  31. Trends Biochem Sci. 2021 Mar 02. pii: S0968-0004(21)00028-1. [Epub ahead of print]
    Rapid Detection and Signaling of DNA Damage by PARP-1.
    Nootan Pandey, Ben E Black.
      Poly(ADP-ribosyl) polymerase-1 (PARP-1) is an abundant ADP-ribosyl transferase that regulates various biological processes. PARP-1 is widely recognized as a first-line responder molecule in DNA damage response (DDR). Here, we review the full cycle of detecting DNA damage by PARP-1, PARP-1 activation upon DNA binding, and PARP-1 release from a DNA break. We also discuss the allosteric consequence upon binding of PARP inhibitors (PARPi) and the opportunity to tune its release from a DNA break. It is now possible to harness this new understanding to design novel PARPi for treating diseases where cell toxicity caused by PARP-1 'trapping' on DNA is either the desired consequence or entirely counterproductive.
    Keywords:  DNA damage response; PARPi; PARylation; poly(ADP-ribose) glycohydrolase
    DOI:  https://doi.org/10.1016/j.tibs.2021.01.014
  32. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2003014118. [Epub ahead of print]118(10):
    Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.
    Pei-Yun Tsai, Min-Sik Lee, Unmesh Jadhav, Insia Naqvi, Shariq Madha, Ashley Adler, Meeta Mistry, Sergey Naumenko, Caroline A Lewis, Daniel S Hitchcock, Frederick R Roberts, Peter DelNero, Thomas Hank, Kim C Honselmann, Vicente Morales Oyarvide, Mari Mino-Kenudson, Clary B Clish, Ramesh A Shivdasani, Nada Y Kalaany.
      Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.
    Keywords:  epigenetics; glutamine synthetase; mTORC1; nutrient deprivation; pancreatic cancer
    DOI:  https://doi.org/10.1073/pnas.2003014118
  33. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2005568118. [Epub ahead of print]118(10):
    NF-κB-induced R-loop accumulation and DNA damage select for nucleotide excision repair deficiencies in adult T cell leukemia.
    Yunlong He, Nagesh Pasupala, Huijun Zhi, Batsuhk Dorjbal, Imran Hussain, Hsiu-Ming Shih, Sharmistha Bhattacharyya, Roopa Biswas, Milos Miljkovic, Oliver John Semmes, Thomas A Waldmann, Andrew L Snow, Chou-Zen Giam.
      Constitutive NF-κB activation (NF-κBCA) confers survival and proliferation advantages to cancer cells and frequently occurs in T/B cell malignancies including adult T cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1). Counterintuitively, NF-κBCA by the HTLV-1 transactivator/oncoprotein Tax induces a senescence response, and HTLV-1 infections in culture mostly result in senescence or cell-cycle arrest due to NF-κBCA How NF-κBCA induces senescence, and how ATL cells maintain NF-κBCA and avert senescence, remain unclear. Here we report that NF-κBCA by Tax increases R-loop accumulation and DNA double-strand breaks, leading to senescence. R-loop reduction via RNase H1 overexpression, and short hairpin RNA silencing of two transcription-coupled nucleotide excision repair (TC-NER) endonucleases that are critical for R-loop excision-Xeroderma pigmentosum F (XPF) and XPG-attenuate Tax senescence, enabling HTLV-1-infected cells to proliferate. Our data indicate that ATL cells are often deficient in XPF, XPG, or both and are hypersensitive to ultraviolet irradiation. This TC-NER deficiency is found in all ATL types. Finally, ATL cells accumulate R-loops in abundance. Thus, TC-NER deficits are positively selected during HTLV-1 infection because they facilitate the outgrowth of infected cells initially and aid the proliferation of ATL cells with NF-κBCA later. We suggest that TC-NER deficits and excess R-loop accumulation represent specific vulnerabilities that may be targeted for ATL treatment.
    Keywords:  DNA damage; NF-κB activation; R-loop; adult T cell leukemia; transcription-coupled nucleotide excision repair
    DOI:  https://doi.org/10.1073/pnas.2005568118
  34. Front Mol Biosci. 2020 ;7 599332
    E2F1: Cause and Consequence of DNA Replication Stress.
    Shahd Fouad, David Hauton, Vincenzo D'Angiolella.
      In mammalian cells, cell cycle entry occurs in response to the correct stimuli and is promoted by the transcriptional activity of E2F family members. E2F proteins regulate the transcription of S phase cyclins and genes required for DNA replication, DNA repair, and apoptosis. The activity of E2F1, the archetypal and most heavily studied E2F family member, is tightly controlled by the DNA damage checkpoints to modulate cell cycle progression and initiate programmed cell death, when required. Altered tumor suppressor and oncogenic signaling pathways often result in direct or indirect interference with E2F1 regulation to ensure higher rates of cell proliferation independently of external cues. Despite a clear link between dysregulated E2F1 activity and cancer progression, literature on the contribution of E2F1 to DNA replication stress phenotypes is somewhat scarce. This review discusses how dysfunctional tumor suppressor and oncogenic signaling pathways promote the disruption of E2F1 transcription and hence of its transcriptional targets, and how such events have the potential to drive DNA replication stress. In addition to the involvement of E2F1 upstream of DNA replication stress, this manuscript also considers the role of E2F1 as a downstream effector of the response to this type of cellular stress. Lastly, the review introduces some reflections on how E2F1 activity is integrated with checkpoint control through post-translational regulation, and proposes an exploitable tumor weakness based on this axis.
    Keywords:  DNA replication stress; E2F1; cyclin E; cyclin F; retinoblastoma; ribonucleotide reductase; ubiquitin proteasome system
    DOI:  https://doi.org/10.3389/fmolb.2020.599332
  35. Hum Mutat. 2021 Mar 06.
    Altered DNA repair creates novel Alu/Alu repeat-mediated deletions.
    Maria E Morales, Tiffany Kaul, JaNiece Walker, Chelsea Everett, Travis White, Prescott Deininger.
      Alu elements are the most abundant source of non-allelic homology that influences genetic instability in the human genome. When there is a DNA double-stranded break, the Alu element's high copy number, moderate length and distance and mismatch between elements uniquely influence recombination processes. We utilize a reporter-gene assay to show the complex influence of Alu mismatches on Alu-related Repeat-Mediated Deletions (RMDs). The Alu/Alu heteroduplex intermediate can result in a non-allelic homologous recombination. Alternatively, the heteroduplex can result in various DNA breaks around the Alu elements caused by competing nucleases. These breaks can undergo Alt-NHEJ to cause deletions focused around the Alu elements. Formation of these heteroduplex intermediates is largely RAD52 dependent. Cells with low ERCC1 levels utilize more of these alternatives resolutions, while cells with MSH2 defects tend to have more RMDs with a specific increase in the homologous recombination events. Therefore, Alu elements are expected to create different forms of deletions in various cancers depending on a number of these DNA repair defects. This article is protected by copyright. All rights reserved.
    Keywords:  Alu elements; DNA double-stranded break repair; heteroduplex rejection; homologous recombination; repeat-mediated deletions
    DOI:  https://doi.org/10.1002/humu.24193
  36. Trends Biochem Sci. 2021 Feb 27. pii: S0968-0004(21)00023-2. [Epub ahead of print]
    Reciprocal Links between Pre-messenger RNA 3'-End Processing and Genome Stability.
    Martin Dutertre, Rym Sfaxi, Stéphan Vagner.
      The 3'-end processing of most pre-messenger RNAs (pre-mRNAs) involves RNA cleavage and polyadenylation and is coupled to transcription termination. In both yeast and human cells, pre-mRNA 3'-end cleavage is globally inhibited by DNA damage. Recently, further links between pre-mRNA 3'-end processing and the control of genome stability have been uncovered, as reviewed here. Upon DNA damage, various genes related to the DNA damage response (DDR) escape 3'-end processing inhibition or are regulated through alternative polyadenylation (APA). Conversely, various pre-mRNA 3'-end processing factors prevent genome instability and are found at sites of DNA damage. Finally, the reciprocal link between pre-mRNA 3'-end processing and genome stability control seems important because it is conserved in evolution and involved in disease development.
    Keywords:  DNA damage; RNA cleavage; genomic instability; polyadenylation; transcription termination
    DOI:  https://doi.org/10.1016/j.tibs.2021.01.009
  37. Methods Protoc. 2021 Feb 16. pii: 14. [Epub ahead of print]4(1):
    The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.
    Maria Rita Fabbrizi, Jonathan R Hughes, Jason L Parsons.
      The comet assay is a versatile, simple, and sensitive gel electrophoresis-based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1-2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.
    Keywords:  DNA damage; DNA repair; comet assay; complex DNA damage; ionising radiation; protons
    DOI:  https://doi.org/10.3390/mps4010014
  38. Oncogene. 2021 Mar 05.
    Molecular disruption of DNA polymerase β for platinum sensitisation and synthetic lethality in epithelial ovarian cancers.
    Reem Ali, Adel Alblihy, Islam M Miligy, Muslim L Alabdullah, Mansour Alsaleem, Michael S Toss, Mashael Algethami, Tarek Abdel-Fatah, Paul Moseley, Stephen Chan, Nigel P Mongan, Satya Narayan, Emad A Rakha, Srinivasan Madhusudan.
      Targeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.
    DOI:  https://doi.org/10.1038/s41388-021-01710-y
  39. Cancers (Basel). 2021 Feb 13. pii: 787. [Epub ahead of print]13(4):
    Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.
    Cinzia Caggiano, Francesca Cavallo, Teresa Giannattasio, Gioia Cappelletti, Pellegrino Rossi, Paola Grimaldi, Darren R Feldman, Maria Jasin, Marco Barchi.
      Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line's resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.
    Keywords:  53BP1; DNA-PKcs; HR; NHEJ; TGCT; cisplatin resistance; olaparib (AZD2281)
    DOI:  https://doi.org/10.3390/cancers13040787
  40. Int J Mol Sci. 2021 Feb 27. pii: 2402. [Epub ahead of print]22(5):
    Silencing DNA Polymerase β Induces Aneuploidy as a Biomarker of Poor Prognosis in Oral Squamous Cell Cancer.
    Hui-Ching Wang, Leong-Perng Chan, Chun-Chieh Wu, Shu-Jyuan Chang, Sin-Hua Moi, Chi-Wen Luo, Mei-Ren Pan.
      Most patients with oral squamous cell cancer (OSCC) have a locally advanced stage at diagnosis. The treatment strategies are diverse, including surgery, radiotherapy and chemotherapy. Despite multimodality treatment, the response rate is unsatisfactory. DNA repair and genetic instability are highly associated with carcinogenesis and treatment outcomes in oral squamous cell cancer, affecting cell growth and proliferation. Therefore, focusing on DNA repair and genetic instability interactions could be a potential target for improving the outcomes of OSCC patients. DNA polymerase-β (POLB) is an important enzyme in base excision repair and contributes to gene instability, leading to tumorigenesis and cancer metastasis. The aim of our study was to confirm POLB regulates the growth of OSCC cells through modulation of cell cycle and chromosomal instability. We analyzed a tissue array from 133 OSCC patients and discovered that low POLB expression was associated with advanced tumor stage and poor overall survival. In multivariate Cox proportional hazards regression analysis, low POLB expression and advanced lymph node status were significantly associated with poor survival. By performing in vitro studies on model cell lines, we demonstrated that POLB silencing regulated cell cycles, exacerbated mitotic abnormalities and enhanced cell proliferation. After POLB depletion, OSCC cells showed chromosomal instability and aneuploidy. Thus, POLB is an important maintainer of karyotypic stability in OSCC cells.
    Keywords:  DNA polymerase β; aneuploidy; cell cycles; oral cancer
    DOI:  https://doi.org/10.3390/ijms22052402
  41. Biochem Biophys Rep. 2021 Jul;26 100954
    Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.
    Avani Mehta, Prateek Raj, Sandeep Sundriyal, Balasubramanian Gopal, Umesh Varshney.
      Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.
    DOI:  https://doi.org/10.1016/j.bbrep.2021.100954
  42. Pharmaceuticals (Basel). 2021 Feb 26. pii: 190. [Epub ahead of print]14(3):
    1-C Metabolism-Serine, Glycine, Folates-In Acute Myeloid Leukemia.
    Kanwal Mahmood, Ashkan Emadi.
      Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Studies in hematological malignancies have shown alterations in fatty acid, folate, and amino acid metabolism pathways in cancer cells. One-carbon (1-C) metabolism is essential for numerous cancer cell functions, including protein and nucleic acid synthesis and maintaining cellular redox balance, and inhibition of the 1-C pathway has yielded several highly active drugs, such as methotrexate and 5-FU. Glutamine depletion has also emerged as a therapeutic approach for cancers that have demonstrated dependence on glutamine for survival. Recent studies have shown that in response to glutamine deprivation leukemia cells upregulate key enzymes in the serine biosynthesis pathway, suggesting that serine upregulation may be a targetable compensatory mechanism. These new findings may provide opportunities for novel cancer treatments.
    Keywords:  amino acid metabolism; amino acid restriction in cancer; amino-acid-degrading enzymes; cancer therapy; leukemia
    DOI:  https://doi.org/10.3390/ph14030190
  43. Cells. 2021 Feb 18. pii: 433. [Epub ahead of print]10(2):
    Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics.
    Bijesh George, P Mukundan Pillai, Aswathy Mary Paul, Revikumar Amjesh, Kim Leitzel, Suhail M Ali, Oleta Sandiford, Allan Lipton, Pranela Rameshwar, Gabriel N Hortobagyi, Madhavan Radhakrishna Pillai, Rakesh Kumar.
      To define the growing significance of cellular targets and/or effectors of cancer drugs, we examined the fitness dependency of cellular targets and effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular effectors and/or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules for their actions were approved. Additionally, our analysis recognized 43 cellular molecules as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues for repurposing certain approved oncology drugs in such cancer types. For example, we found a widespread upregulation and fitness dependency of several components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these molecules and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the intended cellular effectors of drug might not be good fitness genes and that this study offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types.
    Keywords:  Mevalonate and Purine biosynthesis; breast cancer hard-to-treat cancers; cancer fitness genes; oncology drugs; repurposing
    DOI:  https://doi.org/10.3390/cells10020433
  44. Cancers (Basel). 2021 Feb 25. pii: 966. [Epub ahead of print]13(5):
    Response and Toxicity to Cytarabine Therapy in Leukemia and Lymphoma: From Dose Puzzle to Pharmacogenomic Biomarkers.
    Raffaele Di Francia, Stefania Crisci, Angela De Monaco, Concetta Cafiero, Agnese Re, Giancarla Iaccarino, Rosaria De Filippi, Ferdinando Frigeri, Gaetano Corazzelli, Alessandra Micera, Antonio Pinto.
      Cytarabine is a pyrimidine nucleoside analog, commonly used in multiagent chemotherapy regimens for the treatment of leukemia and lymphoma, as well as for neoplastic meningitis. Ara-C-based chemotherapy regimens can induce a suboptimal clinical outcome in a fraction of patients. Several studies suggest that the individual variability in clinical response to Leukemia & Lymphoma treatments among patients, underlying either Ara-C mechanism resistance or toxicity, appears to be associated with the intracellular accumulation and retention of Ara-CTP due to genetic variants related to metabolic enzymes. Herein, we reported (a) the latest Pharmacogenomics biomarkers associated with the response to cytarabine and (b) the new drug formulations with optimized pharmacokinetics. The purpose of this review is to provide readers with detailed and comprehensive information on the effects of Ara-C-based therapies, from biological to clinical practice, maintaining high the interest of both researcher and clinical hematologist. This review could help clinicians in predicting the response to cytarabine-based treatments.
    Keywords:  Ara-C; mechanism of resistance; pharmacogenetics; target therapy
    DOI:  https://doi.org/10.3390/cancers13050966
  45. Nat Commun. 2021 03 02. 12(1): 1384
    Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.
    Liwei Chen, Jung Eun Park, Peter Paa, Priscilla D Rajakumar, Hong-Ting Prekop, Yi Ting Chew, Swathi N Manivannan, Wei Leong Chew.
      Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.
    DOI:  https://doi.org/10.1038/s41467-021-21559-9
  46. Int J Mol Sci. 2021 Feb 25. pii: 2308. [Epub ahead of print]22(5):
    Role of the DDX11 DNA Helicase in Warsaw Breakage Syndrome Etiology.
    Diana Santos, Mohammad Mahtab, Ana Boavida, Francesca M Pisani.
      Warsaw breakage syndrome (WABS) is a genetic disorder characterized by sister chromatid cohesion defects, growth retardation, microcephaly, hearing loss and other variable clinical manifestations. WABS is due to biallelic mutations of the gene coding for the super-family 2 DNA helicase DDX11/ChlR1, orthologous to the yeast chromosome loss protein 1 (Chl1). WABS is classified in the group of "cohesinopathies", rare hereditary diseases that are caused by mutations in genes coding for subunits of the cohesin complex or protein factors having regulatory roles in the sister chromatid cohesion process. In fact, among the cohesion regulators, an important player is DDX11, which is believed to be important for the functional coupling of DNA synthesis and cohesion establishment at the replication forks. Here, we will review what is known about the molecular and cellular functions of human DDX11 and its role in WABS etiopathogenesis, even in light of recent findings on the role of cohesin and its regulator network in promoting chromatin loop formation and regulating chromatin spatial organization.
    Keywords:  DDX11; DNA helicase; DNA replication; G-quadruplexes; cohesinopathies; sister chromatid cohesion
    DOI:  https://doi.org/10.3390/ijms22052308
  47. Cancers (Basel). 2021 Feb 11. pii: 744. [Epub ahead of print]13(4):
    Key Enzymes in Pyrimidine Synthesis, CAD and CPS1, Predict Prognosis in Hepatocellular Carcinoma.
    Dirk Andreas Ridder, Mario Schindeldecker, Arndt Weinmann, Kristina Berndt, Lana Urbansky, Hagen Roland Witzel, Stefan Heinrich, Wilfried Roth, Beate Katharina Straub.
      Patients with hepatocellular carcinoma (HCC) have a highly variable clinical course. Therefore, there is an urgent need to identify new prognostic markers to determine prognosis and select specific therapies. Recently, it has been demonstrated that dysregulation of the urea cycle (UC) is a common phenomenon in multiple types of cancer. Upon UC dysregulation, nitrogen is diverted toward the multifunctional enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD), and increases pyrimidine synthesis. In this study, we investigated the role of CAD and carbamoyl-phosphate synthetase 1 (CPS1), a rate-limiting enzyme of the UC highly expressed in hepatocytes, in HCC. We created a tissue microarray to analyze expression of both enzymes by immunohistochemistry in a large and well-characterized overall cohort of 871 HCCs of 561 patients that underwent surgery. CAD was induced in recurrent HCCs, and high expression predicted shorter overall survival. CPS1 was downregulated in HCC and further reduced in recurrent tumors and distant metastases. Additionally, low CPS1 was associated with short overall survival. A combined score of both enzymes was an independent prognostic marker in a multivariate Cox regression model (HR = 1.37, 95% confidence interval 1.06-1.75, p = 0.014). Inhibition of pyrimidine synthesis may represent a novel therapeutic strategy for HCC.
    Keywords:  HCC; biomarker; cad; cps1; hepatocellular carcinoma; prognosis; pyrimidine; urea cycle dysregulation
    DOI:  https://doi.org/10.3390/cancers13040744
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