bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒10‒24
forty-seven papers selected by
Sean Rudd
Karolinska Institutet
  1. Mapping the genetic landscape of DNA double-strand break repair.
  2. Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.
  3. Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis.
  4. Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA.
  5. Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance.
  6. Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin.
  7. Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis.
  8. Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.
  9. Phospho-Ser784-VCP Drives Resistance of Pancreatic Ductal Adenocarcinoma to Genotoxic Chemotherapies and Predicts the Chemo-Sensitizing Effect of VCP Inhibitor.
  10. Class 1 Histone Deacetylases and Ataxia-Telangiectasia Mutated Kinase Control the Survival of Murine Pancreatic Cancer Cells upon dNTP Depletion.
  11. Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1.
  12. Dbf4-Dependent Kinase: DDK-ated to post-initiation events in DNA replication.
  13. DNA End Joining: G0-ing to the Core.
  14. Home and Away: The Role of Non-Coding RNA in Intracellular and Intercellular DNA Damage Response.
  15. The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability.
  16. cGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence.
  17. DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation.
  18. PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?
  19. Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast.
  20. Unravelling roles of error-prone DNA polymerases in shaping cancer genomes.
  21. The Intersection of Purine and Mitochondrial Metabolism in Cancer.
  22. Biogenesis of Iron-Sulfur Clusters and Their Role in DNA Metabolism.
  23. Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast.
  24. GAS6/AXL inhibition enhances ovarian cancer sensitivity to chemotherapy and PARP inhibition through increased DNA damage and enhanced replication stress.
  25. DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.
  26. Replication catastrophe is responsible for intrinsic PAR glycohydrolase inhibitor-sensitivity in patient-derived ovarian cancer models.
  27. Melatonin: A smart molecule in the DNA repair system.
  28. The pro-autophagic protein AMBRA1 coordinates cell cycle progression by regulating CCND (cyclin D) stability.
  29. ATR inhibition reverses the resistance of homologous recombination deficient MGMTlow/MMRproficient cancer cells to temozolomide.
  30. CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer.
  31. Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer.
  32. DEAD-Box RNA Helicases and Genome Stability.
  33. The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress.
  34. Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis.
  35. RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage.
  36. Genome instability independent of type I interferon signaling drives neuropathology caused by impaired ribonucleotide excision repair.
  37. Regulation of local GTP availability controls RAC1 activity and cell invasion.
  38. Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine.
  39. Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL).
  40. BET Proteins as Attractive Targets for Cancer Therapeutics.
  41. Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.
  42. Controllable genome editing with split-engineered base editors.
  43. Decomposing the mutational landscape of cancer genomes with RepairSig.
  44. RRM1 Expression as a Prognostic Biomarker for Unresectable or Recurrent Biliary Tract Cancer Treated with Gemcitabine plus Cisplatin.
  45. NeuroD1 promotes tumor cell proliferation and tumorigenesis by directly activating the pentose phosphate pathway in colorectal carcinoma.
  46. The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.
  47. Influence of stereochemistry on the activity of rapadocin, an isoform-specific inhibitor of the nucleoside transporter ENT1.
  1. Cell. 2021 Oct 15. pii: S0092-8674(21)01176-4. [Epub ahead of print]
    Mapping the genetic landscape of DNA double-strand break repair.
    Jeffrey A Hussmann, Jia Ling, Purnima Ravisankar, Jun Yan, Ann Cirincione, Albert Xu, Danny Simpson, Dian Yang, Anne Bothmer, Cecilia Cotta-Ramusino, Jonathan S Weissman, Britt Adamson.
      Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.
    Keywords:  CRISPR-Cas9; DNA repair; double-strand breaks; functional genomics; genome editing
    DOI:  https://doi.org/10.1016/j.cell.2021.10.002
  2. PLoS Genet. 2021 Oct 21. 17(10): e1009863
    Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.
    Michaela A Gold, Jenna M Whalen, Karine Freon, Zixin Hong, Ismail Iraqui, Sarah A E Lambert, Catherine H Freudenreich.
      Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2-7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.
    DOI:  https://doi.org/10.1371/journal.pgen.1009863
  3. Nat Commun. 2021 Oct 18. 12(1): 6060
    Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis.
    Jeannine Diesch, Marguerite-Marie Le Pannérer, René Winkler, Raquel Casquero, Matthias Muhar, Mark van der Garde, Michael Maher, Carolina Martínez Herráez, Joan J Bech-Serra, Michaela Fellner, Philipp Rathert, Nigel Brooks, Lurdes Zamora, Antonio Gentilella, Carolina de la Torre, Johannes Zuber, Katharina S Götze, Marcus Buschbeck.
      The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.
    DOI:  https://doi.org/10.1038/s41467-021-26258-z
  4. Nat Commun. 2021 Oct 19. 12(1): 6095
    Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA.
    Claudia Lancey, Muhammad Tehseen, Souvika Bakshi, Matthew Percival, Masateru Takahashi, Mohamed A Sobhy, Vlad S Raducanu, Kerry Blair, Frederick W Muskett, Timothy J Ragan, Ramon Crehuet, Samir M Hamdan, Alfredo De Biasio.
      Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.
    DOI:  https://doi.org/10.1038/s41467-021-26251-6
  5. Genes (Basel). 2021 Sep 29. pii: 1550. [Epub ahead of print]12(10):
    Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance.
    Félix Prado.
      The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.
    Keywords:  BRCA2; DNA damage tolerance; Rad51; Rad52; homologous recombination
    DOI:  https://doi.org/10.3390/genes12101550
  6. Front Genet. 2021 ;12 747734
    Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin.
    Anthony Sanchez, Doohyung Lee, Dae In Kim, Kyle M Miller.
      DNA double-strand breaks (DSBs) are hazardous to genome integrity and can promote mutations and disease if not handled correctly. Cells respond to these dangers by engaging DNA damage response (DDR) pathways that are able to identify DNA breaks within chromatin leading ultimately to their repair. The recognition and repair of DSBs by the DDR is largely dependent on the ability of DNA damage sensing factors to bind to and interact with nucleic acids, nucleosomes and their modified forms to target these activities to the break site. These contacts orientate and localize factors to lesions within chromatin, allowing signaling and faithful repair of the break to occur. Coordinating these events requires the integration of several signaling and binding events. Studies are revealing an enormously complex array of interactions that contribute to DNA lesion recognition and repair including binding events on DNA, as well as RNA, RNA:DNA hybrids, nucleosomes, histone and non-histone protein post-translational modifications and protein-protein interactions. Here we examine several DDR pathways that highlight and provide prime examples of these emerging concepts. A combination of approaches including genetic, cellular, and structural biology have begun to reveal new insights into the molecular interactions that govern the DDR within chromatin. While many questions remain, a clearer picture has started to emerge for how DNA-templated processes including transcription, replication and DSB repair are coordinated. Multivalent interactions with several biomolecules serve as key signals to recruit and orientate proteins at DNA lesions, which is essential to integrate signaling events and coordinate the DDR within the milieu of the nucleus where competing genome functions take place. Genome architecture, chromatin structure and phase separation have emerged as additional vital regulatory mechanisms that also influence genome integrity pathways including DSB repair. Collectively, recent advancements in the field have not only provided a deeper understanding of these fundamental processes that maintain genome integrity and cellular homeostasis but have also started to identify new strategies to target deficiencies in these pathways that are prevalent in human diseases including cancer.
    Keywords:  DNA damage; DNA repair; R-loops; chromatin; genome integrity; nucleic acids; protein domains
    DOI:  https://doi.org/10.3389/fgene.2021.747734
  7. Int J Biol Sci. 2021 ;17(14): 4047-4059
    Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis.
    Siyi Zhang, Tingting Zhou, Zhuo Wang, Fei Yi, Chunlu Li, Wendong Guo, Hongde Xu, Hongyan Cui, Xiang Dong, Jingwei Liu, Xiaoyu Song, Liu Cao.
      The faithful DNA replication is a critical event for cell survival and inheritance. However, exogenous or endogenous sources of damage challenge the accurate synthesis of DNA, which causes DNA lesions. The DNA lesions are obstacles for replication fork progression. However, the prolonged replication fork stalling leads to replication fork collapse, which may cause DNA double-strand breaks (DSB). In order to maintain genomic stability, eukaryotic cells evolve translesion synthesis (TLS) and template switching (TS) to resolve the replication stalling. Proliferating cell nuclear antigen (PCNA) trimer acts as a slide clamp and encircles DNA to orchestrate DNA synthesis and DNA damage tolerance (DDT). The post-translational modifications (PTMs) of PCNA regulate these functions to ensure the appropriate initiation and termination of replication and DDT. The aberrant regulation of PCNA PTMs will result in DSB, which causes mutagenesis and poor response to chemotherapy. Here, we review the roles of the PCNA PTMs in DNA duplication and DDT. We propose that clarifying the regulation of PCNA PTMs may provide insights into understanding the development of cancers.
    Keywords:  DDT; ISGylation; NEDDylation; SUMOylation; acetylation; phosphorylation; replication; ubiquitination
    DOI:  https://doi.org/10.7150/ijbs.64628
  8. Int J Mol Sci. 2021 Oct 11. pii: 10957. [Epub ahead of print]22(20):
    Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.
    Lisa Marie Krieger, Emil Mladenov, Aashish Soni, Marilen Demond, Martin Stuschke, George Iliakis.
      The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.
    Keywords:  DNA double-strand breaks; DNA-damage-response; alternative end-joining (alt-EJ); chromatin; chromatin dynamics; homologous recombination (HR); hypotonic stress (HypoS); ionizing radiation (IR); nonhomologous end-joining (NHEJ); single-strand annealing (SSA)
    DOI:  https://doi.org/10.3390/ijms222010957
  9. Cancers (Basel). 2021 Oct 11. pii: 5076. [Epub ahead of print]13(20):
    Phospho-Ser784-VCP Drives Resistance of Pancreatic Ductal Adenocarcinoma to Genotoxic Chemotherapies and Predicts the Chemo-Sensitizing Effect of VCP Inhibitor.
    Faliang Wang, Kiran Vij, Lin Li, Paarth Dodhiawala, Kian-Huat Lim, Jieya Shao.
      Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal prognosis due in large part to chemotherapy resistance. However, a small subset containing defects in the DNA damage response (DDR) pathways are chemotherapy-sensitive. Identifying intrinsic and therapeutically inducible DDR defects can improve precision and efficacy of chemotherapies for PDAC. DNA repair requires dynamic reorganization of chromatin-associated proteins, which is orchestrated by the AAA+ ATPase VCP. We recently discovered that the DDR function of VCP is selectively activated by Ser784 phosphorylation. In this paper, we show that pSer784-VCP but not total VCP levels in primary PDAC tumors negatively correlate with patient survival. In PDAC cell lines, different pSer784-VCP levels are induced by genotoxic chemotherapy agents and positively correlate with genome stability and cell survival. Causal effects of pSer784-VCP on DNA repair and cell survival were confirmed using VCP knockdown and functional rescue. Importantly, DNA damage-induced pSer784-VCP rather than total VCP levels in PDAC cell lines predict their chemotherapy response and chemo-sensitizing ability of selective VCP inhibitor NMS-873. Therefore, pSer784-VCP drives genotoxic chemotherapy resistance of PDAC, and can potentially be used as a predictive biomarker as well as a sensitizing target to enhance the chemotherapy response of PDAC.
    Keywords:  DNA damage response; chemotherapy predictive biomarker; chemotherapy resistance; pancreatic ductal adenocarcinoma; synthetic lethality
    DOI:  https://doi.org/10.3390/cancers13205076
  10. Cells. 2021 Sep 23. pii: 2520. [Epub ahead of print]10(10):
    Class 1 Histone Deacetylases and Ataxia-Telangiectasia Mutated Kinase Control the Survival of Murine Pancreatic Cancer Cells upon dNTP Depletion.
    Alexandra Nguyen, Melanie Dzulko, Janine Murr, Yun Yen, Günter Schneider, Oliver H Krämer.
      Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a dismal prognosis. Here, we show how an inhibition of de novo dNTP synthesis by the ribonucleotide reductase (RNR) inhibitor hydroxyurea and an inhibition of epigenetic modifiers of the histone deacetylase (HDAC) family affect short-term cultured primary murine PDAC cells. We used clinically relevant doses of hydroxyurea and the class 1 HDAC inhibitor entinostat. We analyzed the cells by flow cytometry and immunoblot. Regarding the induction of apoptosis and DNA replication stress, hydroxyurea and the novel RNR inhibitor COH29 are superior to the topoisomerase-1 inhibitor irinotecan which is used to treat PDAC. Entinostat promotes the induction of DNA replication stress by hydroxyurea. This is associated with an increase in the PP2A subunit PR130/PPP2R3A and a reduction of the ribonucleotide reductase subunit RRM2 and the DNA repair protein RAD51. We further show that class 1 HDAC activity promotes the hydroxyurea-induced activation of the checkpoint kinase ataxia-telangiectasia mutated (ATM). Unlike in other cell systems, ATM is pro-apoptotic in hydroxyurea-treated murine PDAC cells. These data reveal novel insights into a cytotoxic, ATM-regulated, and HDAC-dependent replication stress program in PDAC cells.
    Keywords:  ATM; DNA damage; HDAC; PDAC cells; RNR; apoptosis; cancer; replication stress
    DOI:  https://doi.org/10.3390/cells10102520
  11. DNA Repair (Amst). 2021 Sep 29. pii: S1568-7864(21)00188-9. [Epub ahead of print]108 103232
    Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1.
    Matthew Day, Antony W Oliver, Laurence H Pearl.
      The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 - a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage.
    Keywords:  BRCT domains; Checkpoints; DNA damage response; Phosphopeptide binding; Protein-protein interactions; Specificity
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103232
  12. Cell Cycle. 2021 Oct 18. 1-13
    Dbf4-Dependent Kinase: DDK-ated to post-initiation events in DNA replication.
    Andrew Dolson, Safia Mahabub Sauty, Kholoud Shaban, Krassimir Yankulov.
      Dbf4-Dependent Kinase (DDK) has a well-established essential role at origins of DNA replication, where it phosphorylates and activates the replicative MCM helicase. It also acts in the response to mutagens and in DNA repair as well as in key steps during meiosis. Recent studies have indicated that, in addition to the MCM helicase, DDK phosphorylates several substrates during the elongation stage of DNA replication or upon replication stress. However, these activities of DDK are not essential for viability. Dbf4-Dependent Kinase is also emerging as a key factor in the regulation of genome-wide origin firing and in replication-coupled chromatin assembly. In this review, we summarize recent progress in our understanding of the diverse roles of DDK.
    Keywords:  Dbf4-dependent Kinase; cyclin dependent Kinase; origins of DNA replication; replication forks; replication stress
    DOI:  https://doi.org/10.1080/15384101.2021.1986999
  13. Biomolecules. 2021 Oct 09. pii: 1487. [Epub ahead of print]11(10):
    DNA End Joining: G0-ing to the Core.
    Richard L Frock, Cheyenne Sadeghi, Jodie Meng, Jing L Wang.
      Humans have evolved a series of DNA double-strand break (DSB) repair pathways to efficiently and accurately rejoin nascently formed pairs of double-stranded DNA ends (DSEs). In G0/G1-phase cells, non-homologous end joining (NHEJ) and alternative end joining (A-EJ) operate to support covalent rejoining of DSEs. While NHEJ is predominantly utilized and collaborates extensively with the DNA damage response (DDR) to support pairing of DSEs, much less is known about A-EJ collaboration with DDR factors when NHEJ is absent. Non-cycling lymphocyte progenitor cells use NHEJ to complete V(D)J recombination of antigen receptor genes, initiated by the RAG1/2 endonuclease which holds its pair of targeted DSBs in a synapse until each specified pair of DSEs is handed off to the NHEJ DSB sensor complex, Ku. Similar to designer endonuclease DSBs, the absence of Ku allows for A-EJ to access RAG1/2 DSEs but with random pairing to complete their repair. Here, we describe recent insights into the major phases of DSB end joining, with an emphasis on synapsis and tethering mechanisms, and bring together new and old concepts of NHEJ vs. A-EJ and on RAG2-mediated repair pathway choice.
    Keywords:  A-EJ; DSB repair; NHEJ; V(D)J recombination; end joining; functional redundancy; non-cycling; synapse; tethering; translocation
    DOI:  https://doi.org/10.3390/biom11101487
  14. Genes (Basel). 2021 Sep 23. pii: 1475. [Epub ahead of print]12(10):
    Home and Away: The Role of Non-Coding RNA in Intracellular and Intercellular DNA Damage Response.
    Annabelle Shaw, Monika Gullerova.
      Non-coding RNA (ncRNA) has recently emerged as a vital component of the DNA damage response (DDR), which was previously believed to be solely regulated by proteins. Many species of ncRNA can directly or indirectly influence DDR and enhance DNA repair, particularly in response to double-strand DNA breaks, which may hold therapeutic potential in the context of cancer. These include long non-coding RNA (lncRNA), microRNA, damage-induced lncRNA, DNA damage response small RNA, and DNA:RNA hybrid structures, which can be categorised as cis or trans based on the location of their synthesis relative to DNA damage sites. Mechanisms of RNA-dependent DDR include the recruitment or scaffolding of repair factors at DNA break sites, the regulation of repair factor expression, and the stabilisation of repair intermediates. DDR can also be communicated intercellularly via exosomes, leading to bystander responses in healthy neighbour cells to generate a population-wide response to damage. Many microRNA species have been directly implicated in the propagation of bystander DNA damage, autophagy, and radioresistance, which may prove significant for enhancing cancer treatment via radiotherapy. Here, we review recent developments centred around ncRNA and their contributions to intracellular and intercellular DDR mechanisms.
    Keywords:  DNA damage; RNA; bystander effect; exosomes; lncRNA; miRNA; repair
    DOI:  https://doi.org/10.3390/genes12101475
  15. Biology (Basel). 2021 Sep 28. pii: 977. [Epub ahead of print]10(10):
    The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability.
    Weihang Chai, Megan Chastain, Olga Shiva, Yuan Wang.
      The mammalian CTC1-STN1-TEN1 (CST) complex is an ssDNA-binding protein complex that has emerged as an important player in protecting genome stability and preserving telomere integrity. Studies have shown that CST localizes at stalled replication forks and is critical for protecting the stability of nascent strand DNA. Recent cryo-EM analysis reveals that CST subunits possess multiple OB-fold domains that can form a decameric supercomplex. While considered to be RPA-like, CST acts distinctly from RPA to protect genome stability. Here, we report that while the OB domain of STN1 shares structural similarity with the OB domain of RPA32, the STN1-OB domain contains an intrinsically disordered region (IDR) that is important for maintaining genome stability under replication stress. Single mutations in multiple positions in this IDR, including cancer-associated mutations, cause genome instabilities that are elevated by replication stress and display reduced cellular viability and increased HU sensitivity. While IDR mutations do not impact CST complex formation or CST interaction with its binding partner RAD51, they diminish RAD51 foci formation when replication is perturbed. Interestingly, the IDR is critical for STN1-POLα interaction. Collectively, our results identify the STN1 IDR as an important element in regulating CST function in genome stability maintenance.
    Keywords:  CST; STN1; genome instability; replication stress
    DOI:  https://doi.org/10.3390/biology10100977
  16. Protein Cell. 2021 Oct 22.
    cGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence.
    Xiaocui Li, Xiaojuan Li, Chen Xie, Sihui Cai, Mengqiu Li, Heping Jin, Shu Wu, Jun Cui, Haiying Liu, Yong Zhao.
      As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.
    Keywords:  DNA damage response; cGAS; chromosome end-to-end fusion; genome stability; mitosis; non-homologous end joining; telomeres
    DOI:  https://doi.org/10.1007/s13238-021-00879-y
  17. Front Genet. 2021 ;12 738230
    DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation.
    Gerarda van de Kamp, Tim Heemskerk, Roland Kanaar, Jeroen Essers.
      The superior dose distribution of particle radiation compared to photon radiation makes it a promising therapy for the treatment of tumors. However, the cellular responses to particle therapy and especially the DNA damage response (DDR) is not well characterized. Compared to photons, particles are thought to induce more closely spaced DNA lesions instead of isolated lesions. How this different spatial configuration of the DNA damage directs DNA repair pathway usage, is subject of current investigations. In this review, we describe recent insights into induction of DNA damage by particle radiation and how this shapes DNA end processing and subsequent DNA repair mechanisms. Additionally, we give an overview of promising DDR targets to improve particle therapy.
    Keywords:  DNA repair pathway; combination therapy; double strand break repair; end resection; linear energy transfer
    DOI:  https://doi.org/10.3389/fgene.2021.738230
  18. Biomolecules. 2021 Oct 19. pii: 1543. [Epub ahead of print]11(10):
    PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?
    Jun Che, Xin Hong, Hai Rao.
      DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion "i-Damage" system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.
    Keywords:  DNA damage tolerance; PCNA ubiquitylation; TLS; TS; pathway choice
    DOI:  https://doi.org/10.3390/biom11101543
  19. Mol Biol Cell. 2021 Oct 20. mbcE21040222
    Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast.
    Cory Haluska, Fengzhi Jin, Yanchang Wang.
      DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.
    DOI:  https://doi.org/10.1091/mbc.E21-04-0222
  20. Oncogene. 2021 Oct 18.
    Unravelling roles of error-prone DNA polymerases in shaping cancer genomes.
    Cyrus Vaziri, Igor B Rogozin, Qisheng Gu, Di Wu, Tovah A Day.
      Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the emerging challenge of determining how DNA damage response pathways-both tolerance and repair-act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase-whether error-free or error-prone-for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.
    DOI:  https://doi.org/10.1038/s41388-021-02032-9
  21. Cells. 2021 Sep 30. pii: 2603. [Epub ahead of print]10(10):
    The Intersection of Purine and Mitochondrial Metabolism in Cancer.
    Humberto De Vitto, Danushka B Arachchige, Brian C Richardson, Jarrod B French.
      Nucleotides are essential to cell growth and survival, providing cells with building blocks for DNA and RNA, energy carriers, and cofactors. Mitochondria have a critical role in the production of intracellular ATP and participate in the generation of intermediates necessary for biosynthesis of macromolecules such as purines and pyrimidines. In this review, we highlight the role of purine and mitochondrial metabolism in cancer and how their intersection influences cancer progression, especially in ovarian cancer. Additionally, we address the importance of metabolic rewiring in cancer and how the evolving landscape of purine synthesis and mitochondria inhibitors can be potentially exploited for cancer treatment.
    Keywords:  amino acids; cancers; metabolic reprogramming; mitochondrial metabolism; purines
    DOI:  https://doi.org/10.3390/cells10102603
  22. Front Cell Dev Biol. 2021 ;9 735678
    Biogenesis of Iron-Sulfur Clusters and Their Role in DNA Metabolism.
    Ruifeng Shi, Wenya Hou, Zhao-Qi Wang, Xingzhi Xu.
      Iron-sulfur (Fe/S) clusters (ISCs) are redox-active protein cofactors that their synthesis, transfer, and insertion into target proteins require many components. Mitochondrial ISC assembly is the foundation of all cellular ISCs in eukaryotic cells. The mitochondrial ISC cooperates with the cytosolic Fe/S protein assembly (CIA) systems to accomplish the cytosolic and nuclear Fe/S clusters maturation. ISCs are needed for diverse cellular functions, including nitrogen fixation, oxidative phosphorylation, mitochondrial respiratory pathways, and ribosome assembly. Recent research advances have confirmed the existence of different ISCs in enzymes that regulate DNA metabolism, including helicases, nucleases, primases, DNA polymerases, and glycosylases. Here we outline the synthesis of mitochondrial, cytosolic and nuclear ISCs and highlight their functions in DNA metabolism.
    Keywords:  DNA metabolism; DNA repair; DNA replication; genome stability; iron-sulfur (Fe-S) clusters
    DOI:  https://doi.org/10.3389/fcell.2021.735678
  23. Sci Rep. 2021 Oct 18. 11(1): 20582
    Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast.
    Paola L García-Medel, Antolín Peralta-Castro, Noe Baruch-Torres, Alma Fuentes-Pascacio, José A Pedroza-García, Alfredo Cruz-Ramirez, Luis G Brieba.
      PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3'-GTCG-5' DNA sequence, where the 3'-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.
    DOI:  https://doi.org/10.1038/s41598-021-00151-7
  24. Mol Cancer Res. 2021 Oct 20. pii: molcanres.0302.2021. [Epub ahead of print]
    GAS6/AXL inhibition enhances ovarian cancer sensitivity to chemotherapy and PARP inhibition through increased DNA damage and enhanced replication stress.
    Mary M Mullen, Elena Lomonosova, Michael D Toboni, Alyssa Oplt, Emily Cybulla, Barbara Blachut, Peinan Zhao, Hollie Noia, Daniel Wilke, Erinn B Rankin, Lindsay M Kuroki, Andrea R Hagemann, Ian S Hagemann, Carolyn K McCourt, Premal H Thaker, David G Mutch, Matthew A Powell, Nima Mosammaparast, Alessandro Vindigni, Katherine C Fuh.
      Over 80% of women with high-grade serous ovarian cancer develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to first-line platinum- and taxane-based chemotherapy or to poly (ADP-ribose) polymerase (PARP) inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors. We found that GAS6 levels in patient tumor and serum samples collected before chemotherapy correlated with ovarian cancer chemoresponse and patient survival. Compared to chemotherapy alone, AVB-500 plus carboplatin and/or paclitaxel led to decreased ovarian cancer cell survival in vitro and tumor burden in vivo. Cells treated with AVB-500 plus carboplatin had more DNA damage, slower DNA replication fork progression, and fewer RAD51 foci than cells treated with carboplatin alone, indicating AVB-500 impaired homologous recombination. Finally, treatment with the PARP inhibitor olaparib plus AVB-500 led to decreased ovarian cancer cell survival in vitro and less tumor burden in vivo. Importantly, this effect was seen in homologous recombination-proficient and homologous recombination-deficient ovarian cancer cells. Collectively, our findings suggest that GAS6 levels could be used to predict response to carboplatin and AVB-500 could be used to treat platinum-resistant, homologous recombination-proficient high-grade serous ovarian cancer. Implications: GAS6/AXL is a novel target to sensitize ovarian cancers to carboplatin and olaparib. Additionally, GAS6 levels can be associated with response to carboplatin treatment.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0302
  25. Genes (Basel). 2021 Sep 29. pii: 1548. [Epub ahead of print]12(10):
    DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.
    Andy Chun Hang Chen, Qian Peng, Sze Wan Fong, Kai Chuen Lee, William Shu Biu Yeung, Yin Lau Lee.
      Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.
    Keywords:  DNA damage; FOXM1; SIRT1; cell cycle regulation; pluripotent stem cells (PSCs)
    DOI:  https://doi.org/10.3390/genes12101548
  26. J Exp Clin Cancer Res. 2021 Oct 16. 40(1): 323
    Replication catastrophe is responsible for intrinsic PAR glycohydrolase inhibitor-sensitivity in patient-derived ovarian cancer models.
    Camilla Coulson-Gilmer, Robert D Morgan, Louisa Nelson, Bethany M Barnes, Anthony Tighe, René Wardenaar, Diana C J Spierings, Helene Schlecht, George J Burghel, Floris Foijer, Sudha Desai, Joanne C McGrail, Stephen S Taylor.
      BACKGROUND: Patients with ovarian cancer often present at advanced stage and, following initial treatment success, develop recurrent drug-resistant disease. PARP inhibitors (PARPi) are yielding unprecedented survival benefits for women with BRCA-deficient disease. However, options remain limited for disease that is platinum-resistant and/or has inherent or acquired PARPi-resistance. PARG, the PAR glycohydrolase that counterbalances PARP activity, is an emerging target with potential to selectively kill tumour cells harbouring oncogene-induced DNA replication and metabolic vulnerabilities. Clinical development of PARG inhibitors (PARGi) will however require predictive biomarkers, in turn requiring an understanding of their mode of action. Furthermore, differential sensitivity to PARPi is key for expanding treatment options available for patients.METHODS: A panel of 10 ovarian cancer cell lines and a living biobank of patient-derived ovarian cancer models (OCMs) were screened for PARGi-sensitivity using short- and long-term growth assays. PARGi-sensitivity was characterized using established markers for DNA replication stress, namely replication fibre asymmetry, RPA foci, KAP1 and Chk1 phosphorylation, and pan-nuclear γH2AX, indicating DNA replication catastrophe. Finally, gene expression in sensitive and resistant cells was also examined using NanoString or RNAseq.
    RESULTS: PARGi sensitivity was identified in both ovarian cancer cell lines and patient-derived OCMs, with sensitivity accompanied by markers of persistent replication stress, and a pre-mitotic cell cycle block. Moreover, DNA replication genes are down-regulated in PARGi-sensitive cell lines consistent with an inherent DNA replication vulnerability. However, DNA replication gene expression did not predict PARGi-sensitivity in OCMs. The subset of patient-derived OCMs that are sensitive to single-agent PARG inhibition, includes models that are PARPi- and/or platinum-resistant, indicating that PARG inhibitors may represent an alternative treatment strategy for women with otherwise limited therapeutic options.
    CONCLUSIONS: We discover that a subset of ovarian cancers are intrinsically sensitive to pharmacological PARG blockade, including drug-resistant disease, underpinned by a common mechanism of replication catastrophe. We explore the use of a transcript-based biomarker, and provide insight into the design of future clinical trials of PARGi in patients with ovarian cancer. However, our results highlight the complexity of developing a predictive biomarker for PARGi sensitivity.
    Keywords:  DNA replication; Gene expression signature; High-grade serous ovarian cancer; PARG inhibitor; Predictive biomarkers; Replication stress
    DOI:  https://doi.org/10.1186/s13046-021-02124-0
  27. Cell Biochem Funct. 2021 Oct 21.
    Melatonin: A smart molecule in the DNA repair system.
    Seyed Mostafa Mir, Azadeh Aliarab, Golnaz Goodarzi, Moein Shirzad, Seyyed Mehdi Jafari, Durdi Qujeq, Sadra Samavarchi Tehrani, Jahanbakhsh Asadi.
      DNA repair is an important pathway for the protection of DNA molecules from destruction. DNA damage can be produced by oxidative reactive nitrogen or oxygen species, irritation, alkylating agents, depurination and depyrimidination; in this regard, DNA repair pathways can neutralize the negative effects of these factors. Melatonin is a hormone secreted from the pineal gland with an antioxidant effect by binding to oxidative factors. In addition, the effect of melatonin on DNA repair pathways has been proven by the literature. DNA repair is carried out by several mechanisms, of which homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) are of great importance. Because of the importance of DNA repair in DNA integrity and the anticancer effect of this pathway, we presented the effect of melatonin on DNA repair factors regarding previous studies conducted in this area.
    Keywords:  DNA damage; DNA repair; cancer; melatonin; signalling pathways
    DOI:  https://doi.org/10.1002/cbf.3672
  28. Autophagy. 2021 Oct 17. 1-3
    The pro-autophagic protein AMBRA1 coordinates cell cycle progression by regulating CCND (cyclin D) stability.
    Emiliano Maiani, Giacomo Milletti, Francesco Cecconi.
      The scaffold protein AMBRA1 regulates the early steps of autophagosome formation and cell growth, and its deficiency is associated with neurodevelopmental defects and cancer. In a recent study, we show that AMBRA1 is a key factor in the upstream branch of the MYCN-MYC and CDK4-CDK6-dependent regulation of G1/S phase transition. Indeed, in the developing neuroepithelium, in neural stem cells, and in cancer cells, we demonstrate that AMBRA1 regulates the expression of D-type cyclins by controlling both their proteasomal degradation and their MYCN-MYC-mediated transcription. Also, we show that this regulation axis maintains genome integrity during DNA replication, and we identify a possible line of treatment for tumors downregulating AMBRA1 and/or overexpressing CCND1 (cyclin D1), by demonstrating that AMBRA1-depleted cells carry an AMBRA1-loss-specific lethal sensitivity to CHEK1 inhibition. Interestingly, we show that this aspect is specific for AMBRA1 loss, because ATG7 knockdown does not display the same response to CHEK1 inhibitors. Hence, our findings underscore that the AMBRA1-CCND1 pathway represents a novel crucial mechanism of cell cycle regulation, deeply interconnected with genomic stability in development and cancer.
    Keywords:  AMBRA1; cancer; cell cycle regulation; cyclin D1; neurodevelopment; replication stress; synthetic lethality
    DOI:  https://doi.org/10.1080/15548627.2021.1985917
  29. Oncotarget. 2021 Oct 12. 12(21): 2114-2130
    ATR inhibition reverses the resistance of homologous recombination deficient MGMTlow/MMRproficient cancer cells to temozolomide.
    Lara H El Touny, Curtis Hose, John Connelly, Erik Harris, Anne Monks, Angie B Dull, Deborah F Wilsker, Melinda G Hollingshead, Michelle Gottholm-Ahalt, Sergio Y Alcoser, Michael E Mullendore, Ralph E Parchment, James H Doroshow, Beverly A Teicher, Annamaria Rapisarda.
      The therapeutic efficacy of temozolomide (TMZ) is hindered by inherent and acquired resistance. Biomarkers such as MGMT expression and MMR proficiency are used as predictors of response. However, not all MGMTlow/-ve/MMRproficient patients benefit from TMZ treatment, indicating a need for additional patient selection criteria. We explored the role of ATR in mediating TMZ resistance and whether ATR inhibitors (ATRi) could reverse this resistance in multiple cancer lines. We observed that only 31% of MGMTlow/-ve/MMRproficient patient-derived and established cancer lines are sensitive to TMZ at clinically relevant concentrations. TMZ treatment resulted in DNA damage signaling in both sensitive and resistant lines, but prolonged G2/M arrest and cell death were exclusive to sensitive models. Inhibition of ATR but not ATM, sensitized the majority of resistant models to TMZ and resulted in measurable DNA damage and persistent growth inhibition. Also, compromised homologous recombination (HR) via RAD51 or BRCA1 loss only conferred sensitivity to TMZ when combined with an ATRi. Furthermore, low REV3L mRNA expression correlated with sensitivity to the TMZ and ATRi combination in vitro and in vivo. This suggests that HR defects and low REV3L levels could be useful selection criteria for enhanced clinical efficacy of an ATRi plus TMZ combination.
    Keywords:  ATR; HR; MMR; REV3L; TMZ
    DOI:  https://doi.org/10.18632/oncotarget.28090
  30. Cancers (Basel). 2021 Oct 09. pii: 5056. [Epub ahead of print]13(20):
    CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer.
    Robert Cornelison, Kuntal Biswas, Danielle C Llaneza, Alexandra R Harris, Nisha G Sosale, Matthew J Lazzara, Charles N Landen.
      Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of 'STimulator of Interferon Genes' (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP-AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS-STING-TBK1-IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.
    Keywords:  CX-5461; RNA polymerase I; STING; chemoresistance; cytosolic DNA; ovarian cancer; patient-derived xenograft; ribosomal synthesis
    DOI:  https://doi.org/10.3390/cancers13205056
  31. Genome Med. 2021 Oct 18. 13(1): 166
    Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer.
    Yuchen Guo, Jun Wang, Bente Benedict, Chen Yang, Frank van Gemert, Xuhui Ma, Dongmei Gao, Hui Wang, Shu Zhang, Cor Lieftink, Roderick L Beijersbergen, Hein Te Riele, Xiaohang Qiao, Qiang Gao, Chong Sun, Wenxin Qin, René Bernards, Cun Wang.
      BACKGROUND: Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients.METHODS: Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects.
    RESULTS: Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe.
    CONCLUSIONS: Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.
    Keywords:  ATR-CHK1 signaling; Cell division cycle 7; Hepatocellular carcinoma; Replication stress
    DOI:  https://doi.org/10.1186/s13073-021-00981-0
  32. Genes (Basel). 2021 Sep 23. pii: 1471. [Epub ahead of print]12(10):
    DEAD-Box RNA Helicases and Genome Stability.
    Michael Cargill, Rasika Venkataraman, Stanley Lee.
      DEAD-box RNA helicases are important regulators of RNA metabolism and have been implicated in the development of cancer. Interestingly, these helicases constitute a major recurring family of RNA-binding proteins important for protecting the genome. Current studies have provided insight into the connection between genomic stability and several DEAD-box RNA helicase family proteins including DDX1, DDX3X, DDX5, DDX19, DDX21, DDX39B, and DDX41. For each helicase, we have reviewed evidence supporting their role in protecting the genome and their suggested mechanisms. Such helicases regulate the expression of factors promoting genomic stability, prevent DNA damage, and can participate directly in the response and repair of DNA damage. Finally, we summarized the pathological and therapeutic relationship between DEAD-box RNA helicases and cancer with respect to their novel role in genome stability.
    Keywords:  DEAD-box RNA helicases; cancer; genome stability
    DOI:  https://doi.org/10.3390/genes12101471
  33. Nat Metab. 2021 Oct;3(10): 1357-1371
    The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress.
    Chunmin Ma, Ke Zheng, Kun Jiang, Qin Zhao, Nannan Sha, Wang Wang, Man Yan, Tao Chen, Yuzheng Zhao, Yuhui Jiang.
      The multifunctional roles of metabolic enzymes allow for the integration of multiple signals to precisely transduce external stimuli into cell fate decisions. Elevation of 3-phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme for de novo serine biosynthesis, is broadly associated with human cancer development; although how PHGDH activity is regulated and its implication in tumorigenesis remains unclear. Here we show that glucose restriction induces the phosphorylation of PHGDH by p38 at Ser371, which promotes the translocation of PHGDH from the cytosol into the nucleus. Concurrently, AMPK phosphorylates PHGDH-Ser55, selectively increasing PHGDH oxidation of malate into oxaloacetate, thus generating NADH. In the nucleus, the altered PHGDH activity restricts NAD+ level and compartmentally repressed NAD+-dependent PARP1 activity for poly(ADP-ribosyl)ation of c-Jun, thereby leading to impaired c-Jun transcriptional activity linked to cell growth inhibition. Physiologically, nuclear PHGDH sustains tumour growth under nutrient stress, and the levels of PHGDH-Ser371 and PHGDH-Ser55 phosphorylation correlate with p38 and AMPK activity, respectively, in clinical human pancreatic cancer specimens. These findings illustrate a previously unidentified nutrient-sensing mechanism with the critical involvement of a non-canonical metabolic effect of PHGDH and underscore the functional importance of alternative PHGDH activity in tumorigenesis.
    DOI:  https://doi.org/10.1038/s42255-021-00456-x
  34. Genes (Basel). 2021 Sep 24. pii: 1487. [Epub ahead of print]12(10):
    Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis.
    Gustavo Carvalho, Alberto Díaz-Talavera, Patricia A Calvo, Luis Blanco, María I Martínez-Jiménez.
      PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.
    Keywords:  CTNA; DNA primase; NRTIs; PrimPol; anti-retroviral; ddC; dideoxynucleotides; polymerase; zalcitabine
    DOI:  https://doi.org/10.3390/genes12101487
  35. Cell Rep. 2021 Oct 19. pii: S2211-1247(21)01321-8. [Epub ahead of print]37(3): 109854
    RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage.
    Yongjie Xu, Yumeng Hu, Tao Xu, Kaowen Yan, Ting Zhang, Qin Li, Fen Chang, Xueyuan Guo, Jingyu Peng, Mo Li, Min Zhao, Hongying Zhen, Luzheng Xu, Duo Zheng, Li Li, Genze Shao.
      Despite the tremendous success of targeted and conventional therapies for lung cancer, therapeutic resistance is a common and major clinical challenge. RNF8 is a ubiquitin E3 ligase that plays essential roles in the DNA damage response; however, its role in the pathogenesis of lung cancer is unclear. Here, we report that RNF8 is overexpressed in lung cancer and positively correlates with the expression of p-Akt and poor survival of patients with non-small-cell lung cancer. In addition, we identify RNF8 as the E3 ligase for regulating the activation of Akt by K63-linked ubiquitination under physiological and genotoxic conditions, which leads to lung cancer cell proliferation and resistance to chemotherapy. Together, our study suggests that RNF8 could be a very promising target in precision medicine for lung cancer.
    Keywords:  Akt; DNA damage response; RNF8; chemoresistance; lung cancer; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2021.109854
  36. Neuron. 2021 Oct 08. pii: S0896-6273(21)00713-3. [Epub ahead of print]
    Genome instability independent of type I interferon signaling drives neuropathology caused by impaired ribonucleotide excision repair.
    Aditi, Susanna M Downing, Patrick A Schreiner, Young Don Kwak, Yang Li, Timothy I Shaw, Helen R Russell, Peter J McKinnon.
      Aicardi-Goutières syndrome (AGS) is a monogenic type I interferonopathy characterized by neurodevelopmental defects and upregulation of type I interferon signaling and neuroinflammation. Mutations in genes that function in nucleic acid metabolism, including RNASEH2, are linked to AGS. Ribonuclease H2 (RNASEH2) is a genome surveillance factor critical for DNA integrity by removing ribonucleotides incorporated into replicating DNA. Here we show that RNASEH2 is necessary for neurogenesis and to avoid activation of interferon-responsive genes and neuroinflammation. Cerebellar defects after RNASEH2B inactivation are rescued by p53 but not cGAS deletion, suggesting that DNA damage signaling, not neuroinflammation, accounts for neuropathology. Coincident inactivation of Atm and Rnaseh2 further affected cerebellar development causing ataxia, which was dependent upon aberrant activation of non-homologous end-joining (NHEJ). The loss of ATM also markedly exacerbates cGAS-dependent type I interferon signaling. Thus, DNA damage-dependent signaling rather than type I interferon signaling underlies neurodegeneration in this class of neurodevelopmental/neuroinflammatory disease.
    Keywords:  ATM; Aicardi-Goutières syndrome; Cerebellum; DNA damage; Microglia; Neurodegeneration; Neurodevelopment; Neuroinflammation; RNaseH2; cGAS/STING
    DOI:  https://doi.org/10.1016/j.neuron.2021.09.040
  37. Nat Commun. 2021 Oct 19. 12(1): 6091
    Regulation of local GTP availability controls RAC1 activity and cell invasion.
    Anna Bianchi-Smiraglia, David W Wolff, Daniel J Marston, Zhiyong Deng, Zhannan Han, Sudha Moparthy, Rebecca M Wombacher, Ashley L Mussell, Shichen Shen, Jialin Chen, Dong-Hyun Yun, Anderson O'Brien Cox, Cristina M Furdui, Edward Hurley, Maria Laura Feltri, Jun Qu, Thomas Hollis, Jules Berlin Nde Kengne, Bernard Fongang, Rui J Sousa, Mikhail E Kandel, Eugene S Kandel, Klaus M Hahn, Mikhail A Nikiforov.
      Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.
    DOI:  https://doi.org/10.1038/s41467-021-26324-6
  38. Int J Mol Sci. 2021 Oct 13. pii: 11025. [Epub ahead of print]22(20):
    Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine.
    Nadine Müller, Eveliina Ponkkonen, Thomas Carell, Andriy Khobta.
      Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2'-deoxy-(5-fC/5-caC) or their BER-resistant 2'-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis-element (5'-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5'-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches.
    Keywords:  5-carboxycytosine; 5-formylcytosine; DNA demethylation; base excision repair (BER); epigenetic marks; gene regulation; thymine DNA glycosylase (TDG)
    DOI:  https://doi.org/10.3390/ijms222011025
  39. Cells. 2021 Sep 29. pii: 2589. [Epub ahead of print]10(10):
    Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL).
    Zuhal Eraslan, Grigorios Papatzikas, Jean-Baptiste Cazier, Farhat L Khanim, Ulrich L Günther.
      BL and DLBCL are subtypes of B-cell lymphomas that arise from germinal centre B lymphocytes. Differentiation between BL and DLBCL is critical and can be challenging, as these two types of cancer share the same morphological, immunophenotypic, and genetic characteristics. In this study, we have examined metabolism in BL and DLBCL lymphomas and found distinctive differences in serine metabolism. We show that BL cells consume significantly more extracellular asparagine than DLBCL cells. Using a tracer-based approach, we find that asparagine regulates the serine uptake and serine synthesis in BL and DLBCL cells. Calculation of Differentially Expressed Genes (DEGs) from RNAseq datasets of BL and DLBCL patients show that BL cancers express the genes involved in serine synthesis at a higher level than DLBCL. Remarkably, combined use of an inhibitor of serine biosynthesis pathway and an anticancer drug asparaginase increases the sensitivity of BL cells to extracellular asparagine deprivation without inducing a change in the sensitivity of DLBCL cells to asparaginase. In summary, our study unravels metabolic differences between BL and DLBCL with diagnostic potential which may also open new avenues for treatment.
    Keywords:  metabolism; metabolomics; non-Hodgkin lymphomas
    DOI:  https://doi.org/10.3390/cells10102589
  40. Int J Mol Sci. 2021 Oct 14. pii: 11102. [Epub ahead of print]22(20):
    BET Proteins as Attractive Targets for Cancer Therapeutics.
    Joanna Sarnik, Tomasz Popławski, Paulina Tokarz.
      Transcriptional dysregulation is a hallmark of cancer and can be an essential driver of cancer initiation and progression. Loss of transcriptional control can cause cancer cells to become dependent on certain regulators of gene expression. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that regulate the expression of multiple genes involved in carcinogenesis. BET inhibitors (BETis) disrupt BET protein binding to acetylated lysine residues of chromatin and suppress the transcription of various genes, including oncogenic transcription factors. Phase I and II clinical trials demonstrated BETis' potential as anticancer drugs against solid tumours and haematological malignancies; however, their clinical success was limited as monotherapies. Emerging treatment-associated toxicities, drug resistance and a lack of predictive biomarkers limited BETis' clinical progress. The preclinical evaluation demonstrated that BETis synergised with different classes of compounds, including DNA repair inhibitors, thus supporting further clinical development of BETis. The combination of BET and PARP inhibitors triggered synthetic lethality in cells with proficient homologous recombination. Mechanistic studies revealed that BETis targeted multiple essential homologous recombination pathway proteins, including RAD51, BRCA1 and CtIP. The exact mechanism of BETis' anticancer action remains poorly understood; nevertheless, these agents provide a novel approach to epigenome and transcriptome anticancer therapy.
    Keywords:  BET; BETi; DNA repair; cancer; homologous recombination; transcription
    DOI:  https://doi.org/10.3390/ijms222011102
  41. Int J Mol Sci. 2021 Oct 09. pii: 10905. [Epub ahead of print]22(20):
    Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.
    Merle M Nicolai, Ann-Kathrin Weishaupt, Jessica Baesler, Vanessa Brinkmann, Anna Wellenberg, Nicola Winkelbeiner, Anna Gremme, Michael Aschner, Gerhard Fritz, Tanja Schwerdtle, Julia Bornhorst.
      Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism Caenorhabditis elegans to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) parg-1 or parg-2 (two orthologue of PARG), indicates that parg-1 accounts for most of the glycohydrolase activity in worms.
    Keywords:  Caenorhabditis elegans; DNA damage response; DNA repair; manganese; oxidative stress
    DOI:  https://doi.org/10.3390/ijms222010905
  42. Nat Chem Biol. 2021 Oct 18.
    Controllable genome editing with split-engineered base editors.
    Kiara N Berríos, Niklaus H Evitt, Rachel A DeWeerd, Diqiu Ren, Meiqi Luo, Aleksia Barka, Tong Wang, Caroline R Bartman, Yemin Lan, Abby M Green, Junwei Shi, Rahul M Kohli.
      DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.
    DOI:  https://doi.org/10.1038/s41589-021-00880-w
  43. Cell Syst. 2021 Oct 20. pii: S2405-4712(21)00377-X. [Epub ahead of print]12(10): 953-955
    Decomposing the mutational landscape of cancer genomes with RepairSig.
    Sara Bernardo, Mathilde Meyenberg, Joanna I Loizou.
      Mutational signatures are the outcomes of mutagenic processes that occur prior to, and during, tumorigenesis as a result of DNA damage, DNA repair, and DNA replication. In this issue of Cell Systems, Wojtowicz et al. introduce a new computational model aimed at deconstructing the mutational processes that shape cancer genomes.
    DOI:  https://doi.org/10.1016/j.cels.2021.09.009
  44. J Clin Med. 2021 Oct 11. pii: 4652. [Epub ahead of print]10(20):
    RRM1 Expression as a Prognostic Biomarker for Unresectable or Recurrent Biliary Tract Cancer Treated with Gemcitabine plus Cisplatin.
    Jung Won Chun, Boyoung Lee, Weon Seo Park, Nayoung Han, Eun Kyung Hong, Eun Young Park, Sung Sik Han, Sang-Jae Park, Tae Hyun Kim, Woo Jin Lee, Sang Myung Woo.
      The combination of gemcitabine plus cisplatin (GP) is regarded as a first-line treatment for patients with unresectable or recurrent biliary tract cancer (BTC). Several proteins including human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (DCK), cytidine deaminase (CDA), and ribonucleotide reductase subunit 1 (RRM1) are known to be involved in gemcitabine uptake and metabolism. This study was aimed to identify the predictive and prognostic values of these biomarkers in patients who treated with GP for advanced BTC. Tumor samples were obtained from 34 patients with unresectable or recurrent BTC who were treated with GP between August 2015 and February 2018. Intratumoral expression of hENT1, DCK, CDA and RRM1 was determined by immunohistochemistry and analyzed for association with chemotherapy response, progression-free survival (PFS) and overall survival (OS). Median OS was significantly longer in the RRM1-negative group than in the RRM1-positive (9.9 months vs. 5.9 months, p = 0.037). Multivariate adjustment analyses also demonstrated RRM1 expression as an independent prognostic factor for OS in patients treated with GP chemotherapy. Increased intratumoral expression of RRM1 on immunohistochemical staining may be a biomarker predicting poor survival in patients with GP chemotherapy for advanced BTC. Large-scale well-predefined prospective research is needed to validate the utility of biomarkers in clinical practice.
    Keywords:  biliary tract neoplasms; cisplatin; gemcitabine; ribonucleotide reductases
    DOI:  https://doi.org/10.3390/jcm10204652
  45. Oncogene. 2021 Oct 16.
    NeuroD1 promotes tumor cell proliferation and tumorigenesis by directly activating the pentose phosphate pathway in colorectal carcinoma.
    Zhuolin Li, Yuxin He, Yanjun Li, Juan Li, Hezhao Zhao, Guanbing Song, Makoto Miyagishi, Shourong Wu, Vivi Kasim.
      Tumor metabolic reprogramming ensures that cancerous cells obtain sufficient building blocks, energy, and antioxidants to sustain rapid growth and for coping with oxidative stress. Neurogenic differentiation factor 1 (NeuroD1) is upregulated in various types of tumors; however, its involvement in tumor cell metabolic reprogramming remains unclear. In this study, we report that NeuroD1 is positively correlated with glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP), in colorectal cancer cells. In addition, the regulation of G6PD by NeuroD1 alters tumor cell metabolism by stimulating the PPP, leading to enhanced production of nucleotides and NADPH. These, in turn, promote DNA and lipid biosynthesis in tumor cells, while decreasing intracellular levels of reactive oxygen species. Mechanistically, we showed that NeuroD1 binds directly to the G6PD promoter to activate G6PD transcription. Consequently, tumor cell proliferation and colony formation are enhanced, leading to increased tumorigenic potential in vitro and in vivo. These findings reveal a novel function of NeuroD1 as a regulator of G6PD, whereby its oncogenic activity is linked to tumor cell metabolic reprogramming and regulation of the PPP. Furthermore, NeuroD1 represents a potential target for metabolism-based anti-tumor therapeutic strategies.
    DOI:  https://doi.org/10.1038/s41388-021-02063-2
  46. BMC Biol. 2021 Oct 21. 19(1): 228
    The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.
    Marie-Lise Lacombe, Frederic Lamarche, Olivier De Wever, Teresita Padilla-Benavides, Alyssa Carlson, Imran Khan, Anda Huna, Sophie Vacher, Claire Calmel, Céline Desbourdes, Cécile Cottet-Rousselle, Isabelle Hininger-Favier, Stéphane Attia, Béatrice Nawrocki-Raby, Joël Raingeaud, Christelle Machon, Jérôme Guitton, Morgane Le Gall, Guilhem Clary, Cedric Broussard, Philippe Chafey, Patrice Thérond, David Bernard, Eric Fontaine, Malgorzata Tokarska-Schlattner, Patricia Steeg, Ivan Bièche, Uwe Schlattner, Mathieu Boissan.
      BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane.RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome.
    CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.
    Keywords:  Invasion; Metabolic reprogramming; Metastasis; Mitochondrial dynamics; NME4; Nucleoside diphosphate kinase; Prognosis biomarker; Retrograde signaling
    DOI:  https://doi.org/10.1186/s12915-021-01155-5
  47. Chem Sci. 2021 Sep 01. 12(34): 11484-11489
    Influence of stereochemistry on the activity of rapadocin, an isoform-specific inhibitor of the nucleoside transporter ENT1.
    Yuefan Wang, Hanjing Peng, Zufeng Guo, Brett R Ullman, Kana Yamamoto, Sam Y Hong, Jun O Liu.
      Rapadocin is a novel rapamycin-inspired polyketide-tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling.
    DOI:  https://doi.org/10.1039/d1sc02295d
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