bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒08‒09
forty-four papers selected by
Sean Rudd
Karolinska Institutet

  1. Cell Metab. 2020 Jul 30. pii: S1550-4131(20)30367-3. [Epub ahead of print]
    van Gastel N, Spinelli JB, Sharda A, Schajnovitz A, Baryawno N, Rhee C, Oki T, Grace E, Soled HJ, Milosevic J, Sykes DB, Hsu PP, Vander Heiden MG, Vidoudez C, Trauger SA, Haigis MC, Scadden DT.
      Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.
    Keywords:  acute myeloid leukemia; aspartate; bone marrow niche; cell metabolism; chemotherapy; glutamine; mouse models; patient-derived xenografts; pyrimidine synthesis; tumor microenvironment
  2. Mol Cell. 2020 Jul 31. pii: S1097-2765(20)30476-7. [Epub ahead of print]
    Benslimane Y, Bertomeu T, Coulombe-Huntington J, McQuaid M, Sánchez-Osuna M, Papadopoli D, Avizonis D, Russo MST, Huard C, Topisirovic I, Wurtele H, Tyers M, Harrington L.
      Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.
    Keywords:  CRISPR-Cas9; DNA replication; SIRT1; cell proliferation; genome-wide screen; human cells; hydroxyurea; nucleotide pools; replicative stress; resveratrol
  3. Cell Rep. 2020 Aug 04. pii: S2211-1247(20)30970-0. [Epub ahead of print]32(5): 107985
    Gatti M, Imhof R, Huang Q, Baudis M, Altmeyer M.
      PARP inhibitors (PARPi) cause synthetic lethality in BRCA-deficient tumors. Whether specific vulnerabilities to PARPi exist beyond BRCA mutations and related defects in homology-directed repair (HDR) is not well understood. Here, we identify the ubiquitin E3 ligase TRIP12 as negative regulator of PARPi sensitivity. We show that TRIP12 controls steady-state PARP1 levels and limits PARPi-induced cytotoxic PARP1 trapping. Upon loss of TRIP12, elevated PARPi-induced PARP1 trapping causes increased DNA replication stress, DNA damage, cell cycle arrest, and cell death. Mechanistically, we demonstrate that TRIP12 binds PARP1 via a central PAR-binding WWE domain and, using its carboxy-terminal HECT domain, catalyzes polyubiquitylation of PARP1, triggering proteasomal degradation and preventing supra-physiological PARP1 accumulation. Further, in cohorts of breast and ovarian cancer patients, PARP1 abundance is negatively correlated with TRIP12 expression. We thus propose TRIP12 as regulator of PARP1 stability and PARPi-induced PARP trapping, with potential implications for PARPi sensitivity and resistance.
    Keywords:  BRCA mutations; HECT-type ubiquitin ligases; PAR-targeted protein ubiquitylation; PARP inhibitors; cancer; endogenous DNA lesions; genome instability; personalized cancer therapy; replication stress; synthetic lethality
  4. NAR Cancer. 2020 Jun;2(2): zcaa008
    Sharma N, Speed MC, Allen CP, Maranon DG, Williamson E, Singh S, Hromas R, Nickoloff JA.
      Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.
  5. Nat Genet. 2020 Aug 03.
    Mas-Ponte D, Supek F.
      Certain mutagens, including the APOBEC3 (A3) cytosine deaminase enzymes, can create multiple genetic changes in a single event. Activity of A3s results in striking 'mutation showers' occurring near DNA breakpoints; however, less is known about the mechanisms underlying the majority of A3 mutations. We classified the diverse patterns of clustered mutagenesis in tumor genomes, which identified a new A3 pattern: nonrecurrent, diffuse hypermutation (omikli). This mechanism occurs independently of the known focal hypermutation (kataegis), and is associated with activity of the DNA mismatch-repair pathway, which can provide the single-stranded DNA substrate needed by A3, and contributes to a substantial proportion of A3 mutations genome wide. Because mismatch repair is directed towards early-replicating, gene-rich chromosomal domains, A3 mutagenesis has a high propensity to generate impactful mutations, which exceeds that of other common carcinogens such as tobacco smoke and ultraviolet exposure. Cells direct their DNA repair capacity towards more important genomic regions; thus, carcinogens that subvert DNA repair can be remarkably potent.
  6. Nucleic Acids Res. 2020 Aug 04. pii: gkaa640. [Epub ahead of print]
    Olmedo-Pelayo J, Rubio-Contreras D, Gómez-Herreros F.
      DNA topoisomerase II (TOP2) is a major DNA metabolic enzyme, with important roles in replication, transcription, chromosome segregation and spatial organisation of the genome. TOP2 is the target of a class of anticancer drugs that poison the DNA-TOP2 transient complex to generate TOP2-linked DNA double-strand breaks (DSBs). The accumulation of DSBs kills tumour cells but can also result in genome instability. The way in which topoisomerase activity contributes to transcription remains unclear. In this work we have investigated how transcription contributes to TOP2-dependent DSB formation, genome instability and cell death. Our results demonstrate that gene transcription is an important source of abortive TOP2 activity. However, transcription does not contribute significantly to apoptosis or cell death promoted by TOP2-induced DSBs. On the contrary: transcription-dependent breaks greatly contribute to deleterious mutations and translocations, and can promote oncogenic rearrangements. Importantly, we show that TOP2-induced genome instability is mediated by mutagenic canonical non-homologous end joining whereas homologous recombination protects cells against these insults. Collectively, these results uncover mechanisms behind deleterious effects of TOP2 abortive activity during transcription, with relevant implications for chemotherapy.
  7. NAR Cancer. 2020 Jun;2(2): zcaa006
    Dibitetto D, Sims JR, Ascenção CFR, Feng K, Kim D, Oberly S, Freire R, Smolka MB.
      Most cancer cells experience oncogene-induced replication stress and, as a result, exhibit high intrinsic activation of the ATR kinase. Although cancer cells often become more dependent on ATR for survival, the precise mechanism by which ATR signaling ensures cancer cell fitness and viability remains incompletely understood. Here, we find that intrinsic ATR signaling is crucial for the ability of cancer cells to promote DNA end resection, the first step in homology-directed DNA repair. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents the engagement of RAD51 as well as RAD52 at nuclear foci, leading to toxic DNA-PKcs signaling and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and engagement of RAD52. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.
  8. Essays Biochem. 2020 Aug 05. pii: EBC20190093. [Epub ahead of print]
    Kawale AS, Sung P.
      Homologous recombination (HR) is a major, conserved pathway of chromosome damage repair. It not only fulfills key functions in the removal of deleterious lesions such as DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs), but also in replication fork repair and protection. Several familial and acquired cancer predisposition syndromes stem from defects in HR. In particular, individuals with mutations in HR genes exhibit predisposition to breast, ovarian, pancreatic, and prostate cancers, and they also show signs of accelerated aging. However, aberrant and untimely HR events can lead to the loss of heterozygosity, genomic rearrangements, and cytotoxic nucleoprotein intermediates. Thus, it is critically important that HR be tightly regulated. In addition to DNA repair, HR is also involved in meiotic chromosome segregation and telomere maintenance in cells that lack telomerase. In this review, we focus on the role of HR in DSB repair (DSBR) and summarize the current state of the field.
    Keywords:  DNA end resection; DNA repair pathway choice; Homologous Recombination
  9. IUBMB Life. 2020 Aug 05.
    Bansal R, Hussain S, Chanana UB, Bisht D, Goel I, Muthuswami R.
      The ATP-dependent chromatin remodeling proteins play an important role in DNA repair. The energy released by ATP hydrolysis is used for myriad functions ranging from nucleosome repositioning and nucleosome eviction to histone variant exchange. In addition, the distant member of the family, SMARCAL1, uses the energy to reanneal stalled replication forks in response to DNA damage. Biophysical studies have shown that this protein has the unique ability to recognize and bind specifically to DNA structures possessing double-strand to single-strand transition regions. Mutations in SMARCAL1 have been linked to Schimke immuno-osseous dysplasia, an autosomal recessive disorder that exhibits variable penetrance and expressivity. It has long been hypothesized that the variable expressivity and pleiotropic phenotypes observed in the patients might be due to the ability of SMARCAL1 to co-regulate the expression of a subset of genes within the genome. Recently, the role of SMARCAL1 in regulating transcription has been delineated. In this review, we discuss the biophysical and functional properties of the protein that help it to transcriptionally co-regulate DNA damage response as well as to bind to the stalled replication fork and stabilize it, thus ensuring genomic stability. We also discuss the role of SMARCAL1 in cancer and the possibility of using this protein as a chemotherapeutic target.
    Keywords:  DNA damage response; SMARCAL1; SWI/SNF; helicases; transcription
  10. NAR Cancer. 2020 Jun;2(2): zcaa010
    Luo A, Gong Y, Kim H, Chen Y.
      Mechanistic understanding of hypoxia-responsive signaling pathways provides important insights into oxygen- and metabolism-dependent cellular phenotypes in diseases. Using SILAC-based quantitative proteomics, we provided a quantitative map identifying over 6300 protein groups in response to hypoxia in prostate cancer cells and identified both canonical and novel cellular networks dynamically regulated under hypoxia. Particularly, we identified SDE2, a DNA stress response modulator, that was significantly downregulated by hypoxia, independent of HIF (hypoxia-inducible factor) transcriptional activity. Mechanistically, hypoxia treatment promoted SDE2 polyubiquitination and degradation. Such regulation is independent of previously identified Arg/N-end rule proteolysis or the ubiquitin E3 ligase, CDT2. Depletion of SDE2 increased cellular sensitivity to DNA damage and inhibited cell proliferation. Interestingly, either SDE2 depletion or hypoxia treatment potentiated DNA damage-induced PCNA (proliferating cell nuclear antigen) monoubiquitination, a key step for translesion DNA synthesis. Furthermore, knockdown of SDE2 desensitized, while overexpression of SDE2 protected the hypoxia-mediated regulation of PCNA monoubiquitination upon DNA damage. Taken together, our quantitative proteomics and biochemical study revealed diverse hypoxia-responsive pathways that strongly associated with prostate cancer tumorigenesis and identified the functional roles of SDE2 and hypoxia in regulating DNA damage-induced PCNA monoubiquitination, suggesting a possible link between hypoxic microenvironment and the activation of error-prone DNA repair pathway in tumor cells.
  11. DNA Repair (Amst). 2020 Apr 27. pii: S1568-7864(20)30108-7. [Epub ahead of print] 102860
    Beecher M, Kumar N, Jang S, Rapić-Otrin V, Van Houten B.
      UV-damaged DNA binding protein (UV-DDB) is a heterodimeric complex, composed of DDB1 and DDB2, and is involved in global genome nucleotide excision repair. Mutations in DDB2 are associated with xeroderma pigmentosum complementation group E. UV-DDB forms a ubiquitin E3 ligase complex with cullin-4A and RBX that helps to relax chromatin around UV-induced photoproducts through the ubiquitination of histone H2A. After providing a brief historical perspective on UV-DDB, we review our current knowledge of the structure and function of this intriguing repair protein. Finally, this article discusses emerging data suggesting that UV-DDB may have other non-canonical roles in base excision repair and the etiology of cancer.
    Keywords:  Nucleotide excision repair; Single molecule; Structure-function; UV-DDB; XP-E; Xeroderma pigmentosum
  12. Commun Biol. 2020 Aug 05. 3(1): 428
    Hwang JW, Kim SN, Myung N, Song D, Han G, Bae GU, Bedford MT, Kim YK.
      PRMT5 participates in various cellular processes, including transcription regulation, signal transduction, mRNA splicing, and DNA repair; however, its mechanism of regulation is poorly understood. Here, we demonstrate that PRMT5 is phosphorylated at residue Y324 by Src kinase, a negative regulator of its activity. Either phosphorylation or substitution of the Y324 residue suppresses PRMT5 activity by preventing its binding with the methyl donor S-adenosyl-L-methionine. Additionally, we show that PRMT5 activity is associated with non-homologous end joining (NHEJ) repair by methylating and stabilizing p53-binding protein 1 (53BP1), which promotes cellular survival after DNA damage. Src-mediated phosphorylation of PRMT5 and the subsequent inhibition of its activity during the DNA damage process blocks NHEJ repair, leading to apoptotic cell death. Altogether, our findings suggest that PRMT5 regulates DNA repair through Src-mediated Y324 phosphorylation in response to DNA damage.
  13. Mutat Res. 2020 Jul 23. pii: S0027-5107(20)30049-X. [Epub ahead of print]821 111716
    Ma HT, Poon RYC.
      It is well established that Aurora kinases perform critical functions during mitosis. It has become increasingly clear that the Aurora kinases also perform a myriad of non-mitotic functions including DNA damage response. The available evidence indicates that inhibition Aurora kinase A (AURKA) may contribute to the G2 DNA damage checkpoint through AURKA's functions in PLK1 and CDC25B activation. Both AURKA and Aurora kinase B (AURKB) are also essential in mitotic DNA damage response that guard against DNA damage-induced chromosome segregation errors, including the control of abscission checkpoint and prevention of micronuclei formation. Dysregulation of Aurora kinases can trigger DNA damage in mitosis that is sensed in the subsequent G1 by a p53-dependent postmitotic checkpoint. Aurora kinases are themselves linked to the G1 DNA damage checkpoint through p53 and p73 pathways. Finally, several lines of evidence provide a connection between Aurora kinases and DNA repair and apoptotic pathways. Although more studies are required to provide a comprehensive picture of how cells respond to DNA damage, these findings indicate that both AURKA and AURKB are inextricably linked to pathways guarding against DNA damage. They also provide a rationale to support more detailed studies on the synergism between small-molecule inhibitors against Aurora kinases and DNA-damaging agents in cancer therapies.
    Keywords:  Cancer; Cancer therapies; DNA damage checkpoints; DNA repair; Mitosis
  14. Acta Naturae. 2020 Apr-Jun;12(2):12(2): 74-85
    Kuznetsova AA, Novopashina DS, Fedorova OS, Kuznetsov NA.
      Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5'-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine-purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.
    Keywords:  DNA repair; endoribonuclease activity; human AP endonuclease; substrate specificity
  15. Molecules. 2020 Jul 29. pii: E3438. [Epub ahead of print]25(15):
    Gmeiner WH.
      We review developments in fluorine chemistry contributing to the more precise use of fluorinated pyrimidines (FPs) to treat cancer. 5-Fluorouracil (5-FU) is the most widely used FP and is used to treat > 2 million cancer patients each year. We review methods for 5-FU synthesis, including the incorporation of radioactive and stable isotopes to study 5-FU metabolism and biodistribution. We also review methods for preparing RNA and DNA substituted with FPs for biophysical and mechanistic studies. New insights into how FPs perturb nucleic acid structure and dynamics has resulted from both computational and experimental studies, and we summarize recent results. Beyond the well-established role for inhibiting thymidylate synthase (TS) by the 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP), recent studies have implicated new roles for RNA modifying enzymes that are inhibited by 5-FU substitution including tRNA methyltransferase 2 homolog A (TRMT2A) and pseudouridylate synthase in 5-FU cytotoxicity. Furthermore, enzymes not previously implicated in FP activity, including DNA topoisomerase 1 (Top1), were established as mediating FP anti-tumor activity. We review recent literature summarizing the mechanisms by which 5-FU inhibits RNA- and DNA-modifying enzymes and describe the use of polymeric FPs that may enable the more precise use of FPs for cancer treatment in the era of personalized medicine.
    Keywords:  DNA repair; DNA topoisomerase 1; fluoropyrimidine; pseudouridine; ribothymidine; thymidylate synthase
  16. Cancers (Basel). 2020 Jul 31. pii: E2134. [Epub ahead of print]12(8):
    Wyld L, Bellantuono I, Tchkonia T, Morgan J, Turner O, Foss F, George J, Danson S, Kirkland JL.
      Cellular senescence is a key component of human aging that can be induced by a range of stimuli, including DNA damage, cellular stress, telomere shortening, and the activation of oncogenes. Senescence is generally regarded as a tumour suppressive process, both by preventing cancer cell proliferation and suppressing malignant progression from pre-malignant to malignant disease. It may also be a key effector mechanism of many types of anticancer therapies, such as chemotherapy, radiotherapy, and endocrine therapies, both directly and via bioactive molecules released by senescent cells that may stimulate an immune response. However, senescence may contribute to reduced patient resilience to cancer therapies and may provide a pathway for disease recurrence after cancer therapy. A new group of drugs, senotherapies, (drugs which interact with senescent cells to interfere with their pro-aging impacts by either selectively destroying senescent cells (senolytic drugs) or inhibiting their function (senostatic drugs)) are under active investigation to determine whether they can enhance the efficacy of cancer therapies and improve resilience to cancer treatments. Senolytic drugs include quercetin, navitoclax, and fisetin and preclinical and early phase clinical data are emerging of their potential role in cancer treatments, although none are yet in routine use clinically. This article provides a review of these issues.
    Keywords:  aging; cancer; frailty; geriatric oncology; senescence; senolytics; senotherapies
  17. Nucleic Acids Res. 2020 Aug 07. pii: gkaa663. [Epub ahead of print]
    Jing Y, Ding D, Tian G, Kwan KCJ, Liu Z, Ishibashi T, Li XD.
      Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.
  18. Cells. 2020 Jul 29. pii: E1804. [Epub ahead of print]9(8):
    Pereira CD, Martins F, Santos M, Müeller T, da Cruz E Silva OAB, Rebelo S.
      Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1's physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.
    Keywords:  DNA damage; DNA repair; LAP1; TRF2; bleomycin; hydrogen peroxide; nuclear envelope; nucleoplasm; protein phosphorylation; γ-H2AX
  19. Sci Adv. 2020 Jul;6(30): eaba3688
    Dhanwani R, Takahashi M, Mathews IT, Lenzi C, Romanov A, Watrous JD, Pieters B, Hedrick CC, Benedict CA, Linden J, Nilsson R, Jain M, Sharma S.
      Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production.
  20. Cancer Res. 2020 Aug 03. pii: canres.1830.2020. [Epub ahead of print]
    Krais JJ, Johnson N.
      Cancers that arise from BRCA1 germline mutations are deficient for homologous recombination (HR) DNA repair and are sensitive to DNA damaging agents such as platinum and PARP inhibitors (PARPi). In vertebrate organisms, knockout of critical HR genes including BRCA1 and BRCA2 is lethal because HR is required for genome replication. Thus, cancers must develop strategies to cope with loss of HR activity. Furthermore, as established tumors respond to chemotherapy selection pressure, additional genetic adaptations transition cancers to an HR-proficient state. In this review, we discuss biological mechanisms that influence the ability of BRCA1-mutant cancers to perform HR. Furthermore, we consider how the HR status fluctuates throughout the cancer life course, from tumor initiation to the development of therapy refractory disease.
  21. Br J Cancer. 2020 Aug 03.
    Dunlop CR, Wallez Y, Johnson TI, Bernaldo de Quirós Fernández S, Durant ST, Cadogan EB, Lau A, Richards FM, Jodrell DI.
      BACKGROUND: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC.METHODS: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo.
    RESULTS: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts.
    CONCLUSIONS: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.
  22. J Biol Chem. 2020 Aug 06. pii: jbc.RA120.015288. [Epub ahead of print]
    Nagareddy B, Khan A, Kim H.
      Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitutes a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multi-subunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely unknown. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue which when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.
    Keywords:  DNA repair; Fanconi anemia; acetylation; acetyltransferase; genome instability; post-translational modification (PTM); protein stability; proteolysis; the FA core complex; ubiquitylation (ubiquitination)
  23. Nat Rev Genet. 2020 Aug 06.
    Zong D, Oberdoerffer P, Batista PJ, Nussenzweig A.
      All organisms must safeguard the integrity of their DNA to avoid deleterious consequences of genome instability, which have been linked to human diseases such as autoimmune disorders, neurodegenerative diseases and cancer. Traditionally, genome maintenance has been viewed largely in terms of DNA-protein interactions. However, emerging evidence points to RNA as a key modulator of genome stability, with seemingly opposing roles in promoting chromosomal instability and protecting genome integrity. Unravelling the mechanistic and contextual basis of this duality will not only improve our understanding of the interfaces between RNA and the genome but will also provide important insights into how disrupted RNA metabolism contributes to disease origin, laying the foundation for targeted intervention.
  24. Sci Rep. 2020 Aug 06. 10(1): 13275
    Montenegro MF, González-Guerrero R, Sánchez-Del-Campo L, Piñero-Madrona A, Cabezas-Herrera J, Rodríguez-López JN.
      The therapeutic effect of irradiation is thought to come from DNA damage that affects rapidly proliferating cancer cells; however, resistant cells rapidly initiate mechanisms to repair such damage. While DNA repair mechanisms responsible for cancer cell survival following DNA damage are understood, less is known about the epigenetic mechanisms resulting in resistance to radiotherapy. Although changes in DNA methylation are related to mechanisms of long-term resistance, it is more likely that the methylation state of a series of proteins could be responsible for the first-line of defense of cancer cells against irradiation. In this study, we observed that irradiation of breast cancer cells was accompanied by an overproduction in S-adenosylmethionine, which increases the activity of cellular methylases. We found that by activating PRMT1, irradiation triggers a BRCA1-dependent program that results in efficient DNA repair and inhibition of apoptosis. Depletion of PRMT1 in irradiated cells resulted in a switch of BRCA1 functions from repair and survival in the nucleus to activation of cell death signals in the cytoplasm. We conclude that by modulating the cellular localization of BRCA1, PRMT1 is an important regulator of the oncogenic functions of BRCA1, contributing to the epigenetic defense of breast cancer cells against ionizing radiation.
  25. Commun Biol. 2020 Aug 03. 3(1): 421
    Singh VK, Rastogi A, Hu X, Wang Y, De S.
      Although a majority of somatic mutations in cancer are passengers, their mutational signatures provide mechanistic insights into mutagenesis and DNA repair processes. Mutational signature SBS8 is common in most cancers, but its etiology is debated. Incorporating genomic, epigenomic, and cellular process features for multiple cell-types we develop genome-wide composite epigenomic context-maps relevant for mutagenesis and DNA repair. Analyzing somatic mutation data from multiple cancer types in their epigenomic contexts, we show that SBS8 preferentially occurs in gene-poor, lamina-proximal, late replicating heterochromatin domains. While SBS8 is uncommon among mutations in non-malignant tissues, in tumor genomes its proportions increase with replication timing and speed, and checkpoint defects further promote this signature - suggesting that SBS8 probably arises due to uncorrected late replication errors during cancer progression. Our observations offer a potential reconciliation among different perspectives in the debate about the etiology of SBS8 and its relationship with other mutational signatures.
  26. Biomed Rep. 2020 Sep;13(3): 10
    Matsuda S, Murakami M, Ikeda Y, Nakagawa Y, Tsuji A, Kitagishi Y.
      Genomic perturbations due to inaccurate DNA replication, including inappropriate chromosomal segregation often underlie the development of cancer and neurodegenerative diseases. The incidence of these two diseases increases with age and exhibits an inverse association. Therefore, elderly subjects with cancer exhibit a reduced risk of a neurodegenerative disease, and vice versa. Both of these diseases are associated with aging and share several risk factors. Cells have multiple mechanisms to repair DNA damage and inaccurate replication. Previous studies have demonstrated that tumor suppressor proteins serve a critical role in the DNA damage response, which may result in genomic instability and thus induction of cellular apoptosis. Tumor suppressor genes, such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN), breast cancer susceptibility gene 1 (BRCA1) and TP53 reduce genomic susceptibility to cancer by repairing the damaged DNA. In addition, these genes work cooperatively to ensure the inhibition of the development of several types of cancer. PTEN, BRCA1 and TP53 have been recognized as the most frequently deleted and/or mutated genes in various types of human cancer. Recently, tumor suppressor genes have also been shown to be involved in the development of neurodegenerative diseases. The present review summarizes the recent findings of the functions of these tumor suppressors that are associated with genomic stability, and are involved in carcinogenic and neurodegenerative cell signaling. A summary is presented regarding the interactions of these tumor suppressors with their partners which results in transduction of downstream signals. The implications of these functions for cancer and neurodegenerative disease-associated biology are also highlighted.
    Keywords:  BRCA1; DNA repair; PTEN; TP53; aging; carcinogenesis; cell signaling; genomic stability; neurodegeneration; reactive oxygen species
  27. Nat Commun. 2020 Aug 06. 11(1): 3907
    De Magis A, Götz S, Hajikazemi M, Fekete-Szücs E, Caterino M, Juranek S, Paeschke K.
      Nucleic acids can fold into G-quadruplex (G4) structures that can fine-tune biological processes. Proteins are required to recognize G4 structures and coordinate their function. Here we identify Zuo1 as a novel G4-binding protein in vitro and in vivo. In vivo in the absence of Zuo1 fewer G4 structures form, cell growth slows and cells become UV sensitive. Subsequent experiments reveal that these cellular changes are due to reduced levels of G4 structures. Zuo1 function at G4 structures results in the recruitment of nucleotide excision repair (NER) factors, which has a positive effect on genome stability. Cells lacking functional NER, as well as Zuo1, accumulate G4 structures, which become accessible to translesion synthesis. Our results suggest a model in which Zuo1 supports NER function and regulates the choice of the DNA repair pathway nearby G4 structures.
  28. Elife. 2020 Aug 07. pii: e58223. [Epub ahead of print]9
    Li BZ, Putnam CD, Kolodner RD.
      Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.
    Keywords:  S. cerevisiae; chromosomes; gene expression
  29. Mol Cell. 2020 Jul 28. pii: S1097-2765(20)30477-9. [Epub ahead of print]
    Zeinert RD, Baniasadi H, Tu BP, Chien P.
      During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.
    Keywords:  AAA+ protease; chaperone titration; proteotoxic stress; quality control; transposon sequencing
  30. J Biol Chem. 2020 Aug 06. pii: jbc.RA120.014161. [Epub ahead of print]
    Laverde EE, Lai Y, Leng F, Balakrishnan L, Freudenreich CH, Liu Y.
      Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNR can undergo somatic instability that is mediated by DNA damage and repair, and gene transcription. Recent studies have pointed towards a role for R-loops in causing TNR expansion and deletion, and it's been shown that base excision repair (BER) can result in CAG repeat deletions from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR-R-loops. We found that AP endonuclease 1 incised an abasic site on the non-template strand of a TNR R-loop, creating a double-flap intermediate containing an RNA-DNA hybrid that subsequently inhibited pol β DNA synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatments and prevention of repeat expansion diseases and cancer.
    Keywords:  DNA base damage; DNA damage; DNA endonuclease; DNA polymerase; DNA repair; DNA structure; R-loops; base excision repair; trinucleotide repeat instability
  31. FASEB J. 2020 Aug 04.
    Zacheja T, Toth A, Harami GM, Yang Q, Schwindt E, Kovács M, Paeschke K, Burkovics P.
      The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability is stalling of the replication fork which can result in DNA breakage. Several factors, such as DNA lesions or the formation of stable secondary structures (eg, G-quadruplexes) can lead to replication fork stalling. G-quadruplexes (G4s) are well-characterized stable secondary DNA structures that can form within specific single-stranded DNA sequence motifs and have been shown to block/pause the replication machinery. In most genomes several helicases have been described to regulate G4 unfolding to preserve genome integrity, however, different experiments raise the hypothesis that processing of G4s during DNA replication is more complex and requires additional, so far unknown, proteins. Here, we show that the Saccharomyces cerevisiae Mgs1 protein robustly binds to G4 structures in vitro and preferentially acts at regions with a strong potential to form G4 structures in vivo. Our results suggest that Mgs1 binds to G4-forming sites and has a role in the maintenance of genome integrity.
    Keywords:  DNA replication; G-quadruplex; genome stability; helicase; protein-DNA interaction
  32. Cancer Treat Res Commun. 2020 Jul 22. pii: S2468-2942(20)30036-8. [Epub ahead of print]24 100199
    Tripathi A, Balakrishna P, Agarwal N.
      Somatic or germline mutations in genes regulating DNA damage repair have been noted in around 20% of patients with advanced prostate cancer. Poly-ADP-ribose polymerase (PARP) inhibitors have shown encouraging efficacy in prostate cancer patients with DNA repair mutations. Two PARP inhibitors, olaparib, and rucaparib have recently received FDA approval for treatment of patients with advanced castration-resistant prostate cancer (CRPC), while several trials with other PARP inhibitors are ongoing. Here, we briefly summarize the current data supporting the efficacy of PARP inhibitors in advanced CRPC.
    Keywords:  BRCA; Castration-resistant prostate cancer; PARP inhibitor; Prostate cancer; Targeted therapy
  33. J Biol Chem. 2020 Aug 04. pii: jbc.RA120.012801. [Epub ahead of print]
    Liu X, Xie R, Yu LL, Chen SH, Yang X, Singh AK, Li H, Wu C, Yu X.
      The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose (ADPR) from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 Inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 ~ 2.41 μM in vitro. Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. And tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.
    Keywords:  ADP-ribosylation; ARH3; DNA damage response; DNA repair; cancer therapy; dePARylation; inhibitor
  34. Genes (Basel). 2020 Jul 30. pii: E866. [Epub ahead of print]11(8):
    Yudkina AV, Endutkin AV, Diatlova EA, Moor NA, Vokhtantsev IP, Grin IR, Zharkov DO.
      In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.
    Keywords:  DNA damage; DNA polymerases; DNA repair; facilitated diffusion; molecular traffic; tight protein–DNA complexes
  35. Front Cell Dev Biol. 2020 ;8 531
    Al-Jomah N, Mukololo L, Anjum A, Al Madadha M, Patel R.
      Background: Pds5 is an abundant HEAT-repeat-containing protein that binds to cohesin and mediates sister chromatid cohesion. In vertebrates, Pds5A and Pds5B are known to protect DNA replication fork, as their loss leads to DNA damage. Pds5 interacts directly with Wapl, to remove cohesin during mitosis.Aim: To analyze the effects of the loss of Pds5 proteins-mediated DNA damage on the cell cycle checkpoints and to examine the possibility that Pds5 proteins have an overlapping function.
    Methods: We first analyzed the cell cycle regulation of Pds5 proteins and defects in S-phase; DNA damage was confirmed after Pds5A/B knockdown. The activation of cell cycle checkpoints and apoptosis were examined by the level of p-Chk1S317, MAD2 localization, and the level of pro-apoptotic markers, respectively.
    Results: Pds5 proteins dissociated from chromatin in a stepwise manner, and their loss led to activation of pro-apoptotic markers associated with the phosphorylation of Chk1S317 due to DNA damage. Depletion of either Pds5A or Pds5B alone increased Smc3 acetylation in perturbed cell cycle, while depletion of both proteins severely impaired Smc3 acetylation. Moreover, the loss of Pds5A/Pds5B activated the SAC in an ATR-Chk1-dependent manner and stabilized Wapl on chromatin. The depletion of Chk1 rescued the S-phase delay associated with Pds5 depletion and significantly increased mitotic catastrophe.
    Conclusion: Pds5A and Pds5B display overlapping functions in facilitating Smc3 acetylation. Somewhat paradoxically, they also have non-redundant functions in terms of cohesin removal due to the activated surveillance mechanism that leads to phosphorylation of Chk1S317.
    Keywords:  ATR; Chk1; DNA damage; Pds5A; Pds5B; cohesin; mitosis; spindle assembly checkpoints
  36. Nucleic Acids Res. 2020 Aug 05. pii: gkaa633. [Epub ahead of print]
    Bulock CR, Xing X, Shcherbakova PV.
      Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo-) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo- at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδ proofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδ proofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδ exonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.
  37. Nat Commun. 2020 Aug 06. 11(1): 3904
    Gao X, Wang Q, Wang Y, Liu J, Liu S, Liu J, Zhou X, Zhou L, Chen H, Pan L, Chen J, Wang D, Zhang Q, Shen S, Xiao Y, Wu Z, Cheng Y, Chen G, Krubra S, Qin J, Huang L, Zhang P, Wang C, Moses RE, Lonard DM, Malley BWO, Fares F, Zhang B, Li X, Li L, Xiao J.
      A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.
  38. Cell Metab. 2020 Jul 22. pii: S1550-4131(20)30359-4. [Epub ahead of print]
    Alissafi T, Kalafati L, Lazari M, Filia A, Kloukina I, Manifava M, Lim JH, Alexaki VI, Ktistakis NT, Doskas T, Garinis GA, Chavakis T, Boumpas DT, Verginis P.
      Regulatory T cells (Tregs) are vital for the maintenance of immune homeostasis, while their dysfunction constitutes a cardinal feature of autoimmunity. Under steady-state conditions, mitochondrial metabolism is critical for Treg function; however, the metabolic adaptations of Tregs during autoimmunity are ill-defined. Herein, we report that elevated mitochondrial oxidative stress and a robust DNA damage response (DDR) associated with cell death occur in Tregs in individuals with autoimmunity. In an experimental autoimmune encephalitis (EAE) mouse model of autoimmunity, we found a Treg dysfunction recapitulating the features of autoimmune Tregs with a prominent mtROS signature. Scavenging of mtROS in Tregs of EAE mice reversed the DDR and prevented Treg death, while attenuating the Th1 and Th17 autoimmune responses. These findings highlight an unrecognized role of mitochondrial oxidative stress in defining Treg fate during autoimmunity, which may facilitate the design of novel immunotherapies for diseases with disturbed immune tolerance.
    Keywords:  DNA damage response; autoimmunity; lysosome; metabolism; mitochondrial oxidative stress; regulatory T cell
  39. Nat Metab. 2020 Aug 03.
    Zhang T, Bauer C, Newman AC, Uribe AH, Athineos D, Blyth K, Maddocks ODK.
      Cancer cells have high demands for non-essential amino acids (NEAAs), which are precursors for anabolic and antioxidant pathways that support cell survival and proliferation. It is well-established that cancer cells consume the NEAA cysteine, and that cysteine deprivation can induce cell death; however, the specific factors governing acute sensitivity to cysteine starvation are poorly characterized. Here, we show that that neither expression of enzymes for cysteine synthesis nor availability of the primary precursor methionine correlated with acute sensitivity to cysteine starvation. We observed a strong correlation between efflux of the methionine-derived metabolite methylthioadenosine (MTA) and sensitivity to cysteine starvation. MTA efflux results from genetic deletion of methylthioadenosine phosphorylase (MTAP), which is frequently deleted in cancers. We show that MTAP loss upregulates polyamine metabolism which, concurrently with cysteine withdrawal, promotes elevated reactive oxygen species and prevents cell survival. Our results reveal an unexplored metabolic weakness at the intersection of polyamine and cysteine metabolism.
  40. Science. 2020 Aug 07. 369(6504): 725-730
    Meanwell M, Silverman SM, Lehmann J, Adluri B, Wang Y, Cohen R, Campeau LC, Britton R.
      Nucleoside analogs are commonly used in the treatment of cancer and viral infections. Their syntheses benefit from decades of research but are often protracted, unamenable to diversification, and reliant on a limited pool of chiral carbohydrate starting materials. We present a process for rapidly constructing nucleoside analogs from simple achiral materials. Using only proline catalysis, heteroaryl-substituted acetaldehydes are fluorinated and then directly engaged in enantioselective aldol reactions in a one-pot reaction. A subsequent intramolecular fluoride displacement reaction provides a functionalized nucleoside analog. The versatility of this process is highlighted in multigram syntheses of d- or l-nucleoside analogs, locked nucleic acids, iminonucleosides, and C2'- and C4'-modified nucleoside analogs. This de novo synthesis creates opportunities for the preparation of diversity libraries and will support efforts in both drug discovery and development.
  41. Rev Physiol Biochem Pharmacol. 2020 Aug 07.
    Anderson JT, Huang KM, Lustberg MB, Sparreboom A, Hu S.
      Members of the solute carrier (SLC) family of transporters are responsible for the cellular influx of a broad range of endogenous compounds and xenobiotics. These proteins are highly expressed in the gastrointestinal tract and eliminating organs such as the liver and kidney, and are considered to be of particular importance in governing drug absorption and elimination. Many of the same transporters are also expressed in a wide variety of organs targeted by clinically important anticancer drugs, directly affect cellular sensitivity to these agents, and indirectly influence treatment-related side effects. Furthermore, targeted intervention strategies involving the use of transport inhibitors have been recently developed, and have provided promising lead candidates for combinatorial therapies associated with decreased toxicity. Gaining a better understanding of the complex interplay between transporter-mediated on-target and off-target drug disposition will help guide the further development of these novel treatment strategies to prevent drug accumulation in toxicity-associated organs, and improve the safety of currently available treatment modalities. In this report, we provide an update on this rapidly emerging field with particular emphasis on anticancer drugs belonging to the classes of taxanes, platinum derivatives, nucleoside analogs, and anthracyclines.
    Keywords:  Adverse drug reactions; Anticancer; Solute carrier; Toxicity
  42. Protein Sci. 2020 Aug 06.
    Gorelik A, Labriola JM, Illes K, Nagar B.
      The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown. To further our understanding of NTPDase4 function, we determined its crystal structure. The enzyme adopts a wide open, inactive conformation. Differences in the nucleotide-binding site relative to its homologs could account for its substrate selectivity. The putative membrane-interacting loop of cell-surface NTPDases is drastically altered in NTPDase4, potentially affecting its inter-domain dynamics at the Golgi membrane. This article is protected by copyright. All rights reserved.
    Keywords:  ENTPD4; NTPDase4; UDPase; X-ray crystallography; nucleotide metabolism
  43. Immunol Cell Biol. 2020 Aug 04.
    Chua SMH, Fraser JA.
      Purines play an integral role in cellular processes such as energy metabolism, cell signalling, and encoding the genetic makeup of all living organisms, ensuring that the purine metabolic pathway is maintained across all domains of life. To gain a deeper understanding of purine biosynthesis via the de novo biosynthetic pathway, the genes encoding purine metabolic enzymes from 35 archaea, 69 bacteria and 99 eukaryotic species were investigated. While the classic elements of the canonical purine metabolic pathway were utilised in all domains, a subset of familiar biochemical roles were found to be performed by unrelated proteins in some members of the Archaea and Bacteria. In the Bacteria, a major differentiating feature of de novo purine biosynthesis is the increasing prevalence of gene fusions, where two or more purine biosynthesis enzymes that perform consecutive biochemical functions in the pathway are encoded by a single gene. All species in the Eukaryota exhibited the most common fusions seen in the Bacteria, in addition to new gene fusions to potentially increase metabolic flux. This complexity is taken further in humans, where a reversible biomolecular assembly of enzymes known as the purinosome has been identified, allowing short-term regulation in response to metabolic cues whilst expanding on the benefits that can come from gene fusion. By surveying purine metabolism across all domains of life we have identified important features of the purine biosynthetic pathway that can potentially be exploited as prospective drug targets.
    Keywords:  Drug target; gene fusions; immunotherapy; infectious diseases; purine metabolism; purinosome