bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒11‒01
33 papers selected by
Sean Rudd
Karolinska Institutet

  1. Mol Cell. 2020 Oct 21. pii: S1097-2765(20)30691-2. [Epub ahead of print]
    Li F, Kozono D, Deraska P, Branigan T, Dunn C, Zheng XF, Parmar K, Nguyen H, DeCaprio J, Shapiro GI, Chowdhury D, D'Andrea AD.
      While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
    Keywords:  CHK1 inhibitor; CRISPR sgRNA screening; FAM122A; Fanconi Anemia; PABIR1; PP2A; WEE1
  2. Cancer Res. 2020 Oct 28. pii: canres.1666.2020. [Epub ahead of print]
    Ogrodzinski MP, Teoh ST, Lunt SY.
      Investigating metabolic rewiring in cancer can lead to the discovery of new treatment strategies for breast cancer subtypes that currently lack targeted therapies. In this study, we used MMTV-Myc-driven tumors to model breast cancer heterogeneity, investigating the metabolic differences between two histological subtypes, the epithelial-mesenchymal transition (EMT) and the papillary subtypes. A combination of genomic and metabolomic techniques identified differences in nucleotide metabolism between EMT and papillary subtypes: EMT tumors preferentially used the nucleotide salvage pathway, while papillary tumors preferred de novo nucleotide biosynthesis. CRISPR/Cas9 gene editing and mass spectrometry-based methods revealed that targeting the preferred pathway in each subtype resulted in greater metabolic impact than targeting the non-preferred pathway. Knocking out the preferred nucleotide pathway in each subtype has a deleterious effect on in vivo tumor growth, whereas knocking out the non-preferred pathway has a lesser effect or may even result in increased tumor growth. Collectively, these data suggest that significant differences in metabolic pathway utilization distinguish EMT and papillary subtypes of breast cancer and identify said pathways as a means to enhance subtype-specific diagnoses and treatment strategies.
  3. Nat Commun. 2020 Oct 30. 11(1): 5495
    Rageul J, Park JJ, Zeng PP, Lee EA, Yang J, Hwang S, Lo N, Weinheimer AS, Schärer OD, Yeo JE, Kim H.
      Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.
  4. Nucleic Acids Res. 2020 Oct 29. pii: gkaa934. [Epub ahead of print]
    Gavande NS, VanderVere-Carozza PS, Pawelczak KS, Mendoza-Munoz P, Vernon TL, Hanakahi LA, Summerlin M, Dynlacht JR, Farmer AH, Sears CR, Nasrallah NA, Garrett J, Turchi JJ.
      DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.
  5. J Biol Chem. 2020 Oct 30. pii: jbc.RA120.016457. [Epub ahead of print]
    Lee S, Heo J, Park CJ.
      Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32 - RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, while the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.
    Keywords:  DNA binding protein; DNA damage response; Fluorescence polarization; Replication Protein A; nuclear magnetic resonance (NMR); phosphorylation; protein-protein interaction
  6. Biomolecules. 2020 Oct 26. pii: E1483. [Epub ahead of print]10(11):
    Hanna BMF, Helleday T, Mortusewicz O.
      8-oxoguanine DNA glycosylase (OGG1) is the main DNA glycosylase responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG) from duplex DNA to initiate base excision repair. This glycosylase activity is relevant in many pathological conditions including cancer, inflammation, and neurodegenerative diseases. To have a better understanding of the role of OGG1, we previously reported TH5487, a potent active site inhibitor of OGG1. Here, we further investigate the consequences of inhibiting OGG1 with TH5487. TH5487 treatment induces accumulation of genomic 8-oxoG lesions. Furthermore, it impairs the chromatin binding of OGG1 and results in lower recruitment of OGG1 to regions of DNA damage. Inhibiting OGG1 with TH5487 interferes with OGG1's incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress. This study validates TH5487 as a potent OGG1 inhibitor that prevents the repair of 8-oxoG and alters OGG1-chromatin dynamics and OGG1's recruitment kinetics.
    Keywords:  8-oxoguanine incision; DNA oxidative damage; OGG1 glycosylase inhibitor; TH5487; base excision repair; chromatin dynamics; recruitment kinetics; γH2AX
  7. Prog Biophys Mol Biol. 2020 Oct 25. pii: S0079-6107(20)30111-5. [Epub ahead of print]
    Wilson DM, Deacon AM, Duncton MAJ, Pellicena P, Georgiadis MM, Yeh AP, Arvai AS, Moiani D, Tainer JA, Das D.
      Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with ∼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of ∼2-8% and crystal structures from ∼1.8 to 3.2 Å. We thus consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).
    Keywords:  APE1; Cancer therapeutics; DNA damage Response; DNA repair; FEN1; Fragment-based drug discovery; MRE11; Structure-based drug discovery; X-ray crystallography
  8. Trends Cancer. 2020 Oct 24. pii: S2405-8033(20)30263-6. [Epub ahead of print]
    Schrempf A, Slyskova J, Loizou JI.
      Targeted cancer therapies represent a milestone towards personalized treatment as they function via inhibition of cancer-specific alterations. Polymerase θ (POLQ), an error-prone translesion polymerase, also involved in DNA double-strand break (DSB) repair, is often upregulated in cancer. POLQ is synthetic lethal with various DNA repair genes, including known cancer drivers such as BRCA1/2, making it essential in homologous recombination-deficient cancers. Thus, POLQ represents a promising target in cancer therapy and efforts for the development of POLQ inhibitors are actively underway with first clinical trials due to start in 2021. This review summarizes the journey of POLQ from a backup DNA repair enzyme to a promising therapeutic target for cancer treatment.
    Keywords:  double-strand break repair; polymerase theta-mediated end joining; synthetic lethality; targeted cancer therapy
  9. Nat Rev Cancer. 2020 Oct 30.
    Rottenberg S, Disler C, Perego P.
      Platinum (Pt) compounds entered the clinic as anticancer agents when cisplatin was approved in 1978. More than 40 years later, even in the era of precision medicine and immunotherapy, Pt drugs remain among the most widely used anticancer drugs. As Pt drugs mainly target DNA, it is not surprising that recent insights into alterations of DNA repair mechanisms provide a useful explanation for their success. Many cancers have defective DNA repair, a feature that also sheds new light on the mechanisms of secondary drug resistance, such as the restoration of DNA repair pathways. In addition, genome-wide functional screening approaches have revealed interesting insights into Pt drug uptake. About half of cisplatin and carboplatin but not oxaliplatin may enter cells through the widely expressed volume-regulated anion channel (VRAC). The analysis of this heteromeric channel in tumour biopsies may therefore be a useful biomarker to stratify patients for initial Pt treatments. Moreover, Pt-based approaches may be improved in the future by the optimization of combinations with immunotherapy, management of side effects and use of nanodelivery devices. Hence, Pt drugs may still be part of the standard of care for several cancers in the coming years.
  10. Exp Mol Med. 2020 Oct 30.
    Zhao F, Kim W, Kloeber JA, Lou Z.
      DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3'-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.
  11. Int J Mol Sci. 2020 Oct 28. pii: E8039. [Epub ahead of print]21(21):
    Rzeszutek I, Betlej G.
      DNA damage is a common phenomenon promoted through a variety of exogenous and endogenous factors. The DNA damage response (DDR) pathway involves a wide range of proteins, and as was indicated, small noncoding RNAs (sncRNAs). These are double-strand break-induced RNAs (diRNAs) and DNA damage response small RNA (DDRNA). Moreover, RNA binding proteins (RBPs) and RNA modifications have also been identified to modulate diRNA and DDRNA function in the DDR process. Several theories have been formulated regarding the synthesis and function of these sncRNAs during DNA repair; nevertheless, these pathways' molecular details remain unclear. Here, we review the current knowledge regarding the mechanisms of diRNA and DDRNA biosynthesis and discuss the role of sncRNAs in maintaining genome stability.
    Keywords:  DNA damage response (DDR); double-strand breaks (DSBs), DNA repair; noncoding RNA
  12. Adv Sci (Weinh). 2020 Oct;7(20): 2000157
    Hu K, Li Y, Wu W, Xie L, Yan H, Cai Y, Chen D, Jiang Q, Lin L, Chen Z, Liao JY, Zhang Y, Koeffler HP, Yin D, Song E.
      Repair of DNA double-strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. However, the mechanisms how DNA repair induces transcriptional inhibition remain elusive. Here, it is identified that BRD7 participates in DNA damage response (DDR) and is recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joins the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruits E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM-mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11-RAD50-NBS1 (MRN), and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitizes cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR-mediated transcription repression, which may serve as a therapeutic target. The findings identify the missing link between DNA repair and transcription regulation that maintains genome integrity.
    Keywords:  ATM; BRD7; NuRD; PRC2; transcriptional repression
  13. Sci Rep. 2020 Oct 28. 10(1): 18419
    Kunz V, Bommert KS, Kruk J, Schwinning D, Chatterjee M, Stühmer T, Bargou R, Bommert K.
      Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase β, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.
  14. Chem Res Toxicol. 2020 Oct 28.
    Rioux KL, Delaney S.
      Genomic DNA is chemically reactive and therefore susceptible to damage by many exogenous and endogenous sources. Lesions produced from these damaging events can have various mutagenic and genotoxic consequences. This Perspective follows the journey of one particular lesion, 1,N6-ethenoadenine (εA), from its formation to replication and repair, and its role in cancerous tissues and inflammatory diseases. εA is generated by the reaction of adenine (A) with vinyl chloride or lipid peroxidation products. We present the miscoding properties of εA with an emphasis on how bacterial and mammalian cells can process lesions differently, leading to varied mutational spectra. But with information from these assays, we can better understand how the miscoding properties of εA lead to biological consequences and how genomic stability can be maintained via DNA repair mechanisms. We discuss how base excision repair (BER) and direct reversal repair (DRR) can minimize the biological consequences of εA lesions. Kinetic parameters of glycosylases and AlkB family enzymes are described, along with a discussion of the relative contributions of the BER and DRR pathways in the repair of εA. Because eukaryotic DNA is packaged in chromatin, we also discuss the impact of this packaging on BER and DRR, specifically in regards to repair of εA. Studying DNA lesions like εA in this context, from origin to biological implications, can provide crucial information to better understand prevention of mutagenesis and cancer.
  15. Front Cell Dev Biol. 2020 ;8 560098
    Tong ZB, Ai HS, Li JB.
      DNA packs into highly condensed chromatin to organize the genome in eukaryotes but occludes many regulatory DNA elements. Access to DNA within nucleosomes is therefore required for a variety of biological processes in cells including transcription, replication, and DNA repair. To cope with this problem, cells employ a set of specialized ATP-dependent chromatin-remodeling protein complexes to enable dynamic access to packaged DNA. In the present review, we summarize the recent advances in the functional and mechanistic studies on a particular chromatin remodeler SMARCAD1Fun30 which has been demonstrated to play a key role in distinct cellular processes and gained much attention in recent years. Focus is given to how SMARCAD1Fun30 regulates various cellular processes through its chromatin remodeling activity, and especially the regulatory role of SMARCAD1Fun30 in gene expression control, maintenance and establishment of heterochromatin, and DNA damage repair. Moreover, we review the studies on the molecular mechanism of SMARCAD1Fun30 that promotes the DNA end-resection on double-strand break ends, including the mechanisms of recruitment, activity regulation and chromatin remodeling.
    Keywords:  DNA damage; DNA end-resection; SMARCAD1/Fun30; chromatin remodeler; post-translational modification; single-stranded DNA
  16. Pigment Cell Melanoma Res. 2020 Oct 30.
    Zhuo M, Gorgun FM, Tyler DS, Englander EW.
      Major advances in cancer therapy rely on engagement of the patient's immune system and suppression of mechanisms that impede the antitumor immune response. Among the most notable is immune checkpoint blockade (ICB) therapy that releases immune cells from suppression. Although ICB has had significant success particularly in melanoma, it eradicates tumors in subsets of patients and sequencing data across different cancers suggest that tumors with high mutational loads are more likely to respond to ICB. This is consistent with the premise that greater tumoral mutational loads contribute to formation of neoantigens that spur the body's antitumor immune response. Prompted by strong evidence supporting the therapeutic benefits of neoantigens in the context of ICB, we have developed a mouse melanoma combination treatment, where intratumoral administration of DNA damaging drug transiently activates intrinsic mutagenic DNA damage tolerance pathway and improves success rates of ICB. Using the YUMM1.7 cells melanoma model, we demonstrate that intratumoral delivery of cisplatin activates translesion synthesis DNA polymerases-catalyzed DNA synthesis on damaged DNA, which when coupled with ICB regimen, elicits durable tumor regression. We expect that this new combination protocol affords insights with clinical relevance that will help expand the range of patients who benefit from ICB therapy.
    Keywords:  DNA damage; TLS DNA polymerases; cisplatin; immune checkpoint blockade; melanoma; tumor mutation load; tumor regression
  17. Front Mol Biosci. 2020 ;7 205
    Kakoti S, Sato H, Laskar S, Yasuhara T, Shibata A.
      Cancer therapy using immune checkpoint inhibitors (ICIs) is a promising clinical strategy for patients with multiple types of cancer. The expression of programmed cell death ligand-1 (PD-L1), an immune-suppressor ligand, in cancer cells is a factor that influences the efficacy of ICI therapy, particularly in the anti-programmed cell death protein-1 (PD-1)/PD-L1 antibody therapy. PD-L1 expression in cancer cells are associated with tumor mutation burden including microsatellite instability because the accumulation of mutations in the cancer genome can produce abnormal proteins via mutant mRNAs, resulting in neoantigen production and HLA-neoantigen complex presentation in cancer cells. HLA-neoantigen presentation promotes immune activity within tumor environment; therefore, known as hot tumor. Thus, as the fidelity of DNA repair affects the generation of genomic mutations, the status of DNA repair and signaling in cancer cells can be considered prior to ICI therapy. The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA) database analysis showed that tumor samples harboring mutations in any non-homologous end joining, homologous recombination, or DNA damage signaling genes exhibit high neoantigen levels. Alternatively, an urgent task is to understand how the DNA damage-associated cancer treatments change the status of immune activity in patients because multiple clinical trials on combination therapy are ongoing. Recent studies demonstrated that multiple pathways regulate PD-L1 expression in cancer cells. Here, we summarize the regulation of the immune response to ICI therapy, including PD-L1 expression, and also discuss the potential strategies to improve the efficacy of ICI therapy for poor responders from the viewpoint of DNA damage response before or after DNA damage-associated cancer treatment.
    Keywords:  DNA repair; ataxia telangiectasia and Rad3-related; ataxia-telangiectasia-mutated; homologous recombination; immune checkpoint inhibitor; non-homologous end joining
  18. DNA Repair (Amst). 2020 Oct 19. pii: S1568-7864(20)30256-1. [Epub ahead of print]96 102996
    Grandin N, Gallego ME, White CI, Charbonneau M.
      In the budding yeast Saccharomyces cerevisiae, telomerase is constitutively active and is essential for chromosome end protection and illimited proliferation of cell populations. However, upon inactivation of telomerase, alternative mechanims of telomere maintenance allow proliferation of only extremely rare survivors. S. cerevisiae type I and type II survivors differ by the nature of the donor sequences used for repair by homologous recombination of the uncapped terminal TG1-3 telomeric sequences. Type I amplifies the subtelomeric Y' sequences and is more efficient than type II, which amplifies the terminal TG1-3 repeats. However, type II survivors grow faster than type I survivors and can easily outgrow them in liquid cultures. The mechanistic interest of studying S. cerevisiae telomeric recombination is reinforced by the fact that type II recombination is the equivalent of the alternative lengthening of telomeres (ALT) pathway that is used by 5-15 % of cancer types as an alternative to telomerase reactivation. In budding yeast, only around half of the 32 telomeres harbor Y' subtelomeric elements. We report here that in strains harboring Y' elements on all telomeres, type II survivors are not observed, most likely due to an increase in the efficiency of type I recombination. However, in a temperature-sensitive cdc13-1 mutant grown at semi-permissive temperature, the increased amount of telomeric TG1-3 repeats could overcome type II inhibition by the subtelomeric Y' sequences. Strikingly, in the 100 % Y' strain the replicative senescence crisis normally provoked by inactivation of telomerase completely disappeared and the severity of the crisis was proportional to the percentage of chromosome-ends lacking Y' subtelomeric sequences. The present study highlights the fact that the nature of subtelomeric elements can influence the selection of the pathway of telomere maintenance by recombination, as well as the response of the cell to telomeric damage caused by telomerase inactivation.
    Keywords:  Budding yeast; Rad52; Replicative senescence; Subtelomeric Y’ elements; Telomerase-independent telomere maintenance; Telomere recombination
  19. Genes Dev. 2020 Oct 29.
    Pinzaru AM, Kareh M, Lamm N, Lazzerini-Denchi E, Cesare AJ, Sfeir A.
      Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
    Keywords:  CRISPRi; MiDAS; POT1; nuclear F-actin; nuclear periphery; nuclear pore; replication stress; telomeres
  20. Semin Cell Dev Biol. 2020 Oct 24. pii: S1084-9521(19)30132-6. [Epub ahead of print]
    Balzano E, Pelliccia F, Giunta S.
      Repeat sequences account for over half of the human genome and represent a significant source of variation that underlies physiological and pathological states. Yet, their study has been hindered due to limitations in short-reads sequencing technology and difficulties in assembly. A important category of repetitive DNA in the human genome is comprised of tandem repeats (TRs), where repetitive units are arranged in a head-to-tail pattern. Compared to other regions of the genome, TRs carry between 10 and 10,000 fold higher mutation rate. There are several mutagenic mechanisms that can give rise to this propensity toward instability, but their precise contribution remains speculative. Given the high degree of homology between these sequences and their arrangement in tandem, once damaged, TRs have an intrinsic propensity to undergo aberrant recombination with non-allelic exchange and generate harmful rearrangements that may undermine the stability of the entire genome. The dynamic mutagenesis at TRs has been found to underlie individual polymorphism associated with neurodegenerative and neuromuscular disorders, as well as complex genetic diseases like cancer and diabetes. Here, we review our current understanding of the surveillance and repair mechanisms operating within these regions, and we describe how alterations in these protective processes can readily trigger mutational signatures found at TRs, ultimately resulting in the pathological correlation between TRs instability and human diseases. Finally, we provide a viewpoint to counter the detrimental effects that TRs pose in light of their selection and conservation, as important drivers of human evolution.
    Keywords:  DNA damage response; DNA repair; Mutagenesis; Repeats damage; Tandem disorders; Tandem repeats
  21. Int J Mol Sci. 2020 Oct 22. pii: E7842. [Epub ahead of print]21(21):
    Wu Y, Li S.
      Cyclic GMP-AMP synthase (cGAS) is the synthase that generates the second messenger cyclic GMP-AMP (cGAMP) upon DNA binding. cGAS was first discovered as the cytosolic DNA sensor that detects DNA exposed in the cytoplasm either from pathogens or cellular damage. Activated cGAS instigates the signaling cascades to activate type I interferon (IFN) expression, critical for host defense and autoimmune diseases. In addition, cGAS plays a role in senescence, DNA repair, apoptosis, and tumorigenesis. Recently, various post-translational modifications (PTMs) of cGAS have been reported, such as phosphorylation, ubiquitination, acetylation, glutamylation, and sumoylation. These PTMs profoundly affect cGAS functions. Thus, here we review the recent reported PTMs of cGAS and how these PTMs regulate cGAS enzymatic activity, DNA binding, and protein stability, and discuss the potential future directions.
    Keywords:  acetylation; cGAS; glutamylation; innate immunity; phosphorylation; post-translational modification; sumoylation; ubiquitination
  22. Nucleic Acids Res. 2020 Oct 26. pii: gkaa912. [Epub ahead of print]
    Formosa T, Winston F.
      FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.
  23. Pathol Int. 2020 Oct 28.
    Sanoyama I, Sakurai Y, Ichinoe M, Hoshino A, Kesen Y, Kato T, Numata Y, Umezawa A, Jiang SX, Murakumo Y.
      REV7 is involved in multiple biological processes including DNA damage tolerance, cell cycle regulation and gene expression, and is an accessory subunit of the mutation-prone DNA polymerase ζ. It has been reported that REV7 expression is associated with poor prognosis in several human cancers. The aim of this study is to investigate the significance of REV7 in lung carcinogenesis. Immunohistochemical analyses of surgically resected lung cancer specimens revealed that REV7 shows an increased expression in small cell lung carcinomas (SCLCs) when compared with other histological types of lung carcinoma. Association between REV7 expression levels and clinicopathological factors was investigated using SCLC cases with or without surgical resection. Our analyses revealed that high REV7 expression significantly correlated with tumor cell proliferation, assessed by Ki-67 labeling indices, and was negatively associated with distant metastasis and extensive-stage disease. No significant association was detected between REV7 expression and other factors, including prognosis or response to chemoradiotherapy in SCLC. Increase in REV7 expression in SCLC was confirmed using SCLC cell lines. In addition, siRNA-mediated depletion of REV7 activated the apoptotic pathway and suppressed cell growth in SCLC cells. These results suggest that REV7 plays an important role in tumor cell survival and proliferation in SCLC.
    Keywords:  REV7; apoptosis; immunohistochemistry; proliferation; small cell lung carcinoma
  24. Clin Pharmacol Ther. 2020 Oct 29.
    JiangP C, Yang W, MoriyamaP T, LiuP C, SmithP C, YangP W, QianP M, LiP Z, TulstrupP M, SchmiegelowP K, CrewsP KR, ZhangP H, PuiP CH, EvansP W, RellingP M, Bhatia S, YangP JJ.
      6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide metabolites (TGN). Recent genome-wide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms (SNPs) nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.
    Keywords:   NT5C2 ; acute lymphoblastic leukemia; single nucleotide polymorphisms; thiopurine metabolism
  25. Br J Cancer. 2020 Oct 27.
    Lozano R, Castro E, Aragón IM, Cendón Y, Cattrini C, López-Casas PP, Olmos D.
      Over the past years, several studies have demonstrated that defects in DNA damage response and repair (DDR) genes are present in a significant proportion of patients with prostate cancer. These alterations, particularly mutations in BRCA2, are known to be associated with an increased risk of developing prostate cancer and more aggressive forms of the disease. There is growing evidence that certain DDR gene aberrations confer sensitivity to poly-(ADP ribose) polymerase inhibitors and/or platinum chemotherapy, while other defects might identify cases that are more likely to benefit from immune checkpoint inhibition. The potential prognostic impact and relevance for treatment selection together with the decreasing costs and broader accessibility to next-generation sequencing have already resulted in the increased frequency of genetic profiling of prostate tumours. Remarkably, almost half of all DDR genetic defects can occur in the germline, and prostate cancer patients identified as mutation carriers, as well as their families, will require appropriate genetic counselling. In this review, we summarise the current knowledge regarding the biology and clinical implications of DDR defects in prostate cancer, and outline how this evidence is prompting a change in the treatment landscape of the disease.
  26. Trends Mol Med. 2020 Oct 26. pii: S1471-4914(20)30262-8. [Epub ahead of print]
    Culp-Hill R, D'Alessandro A, Pietras EM.
      Acute myeloid leukemia (AML) is a cancer derived from the myeloid lineage of blood cells, characterized by overproduction of leukemic blasts. Although therapeutic improvements have made a significant impact on the outcomes of patients with AML, survival rates remain low due to a high incidence of relapse. Similar to how wildfires can reignite from hidden embers not extinguished from an initial round of firefighting, leukemic stem cells (LSCs) are the embers remaining after completion of traditional chemotherapeutic treatments. LSCs exhibit a unique metabolic profile and contain metabolically distinct subpopulations. In this review, we detail the metabolic features of LSCs and how these characteristics promote resistance to traditional chemotherapy. We also discuss new therapeutic approaches that target metabolic vulnerabilities of LSC to selectively eradicate them.
    Keywords:  AML; LSC; acute myeloid leukemia; leukemic stem cells; mitochondrial metabolism; oxidative phosphorylation
  27. ACS Chem Biol. 2020 Oct 27.
    Schreier VN, Loehr MO, Deng T, Lattmann E, Hajnal A, Neuhauss SCF, Luedtke NW.
      Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.
  28. BMC Cancer. 2020 Oct 28. 20(1): 1037
    Zeng Z, Cao Z, Tang Y.
      BACKGROUND: The E2F family of transcription factor 2 (E2F2) plays an important role in the development and progression of various tumors, but its association with hepatocellular carcinoma (HCC) remains unknown. Our study aimed to investigate the role and clinical significance of E2F2 in HCC.METHODS: HCC raw data were extracted from The Cancer Genome Atlas (TCGA). Wilcoxon signed-rank test, Kruskal-Wallis test and logistic regression were applied to analyze the relationship between the expression of E2F2 and clinicopathologic characteristics. Cox regression and Kaplan-Meier were employed to evaluate the correlation between clinicopathologic features and survival. The biological function of E2F2 was annotated by Gene Set Enrichment Analysis (GSEA).
    RESULTS: The expression of E2F2 was increased in HCC samples. The expression of elevated E2F2 in HCC samples was prominently correlated with histologic grade (OR = 2.62 for G3-4 vs. G1-2, p = 1.80E-05), clinical stage (OR = 1.74 for III-IV vs. I-II, p = 0.03), T (OR = 1.64 for T3-4 vs.T1-2, p = 0.04), tumor status (OR = 1.88 for with tumor vs. tumor free, p = 3.79E-03), plasma alpha fetoprotein (AFP) value (OR = 3.18 for AFP ≥ 400 vs AFP<20, p = 2.16E-04; OR = 2.50 for 20 ≤ AFP<400 vs AFP<20, p = 2.56E-03). Increased E2F2 had an unfavorable OS (p = 7.468e- 05), PFI (p = 3.183e- 05), DFI (p = 0.001), DSS (p = 4.172e- 05). Elevated E2F2 was independently bound up with OS (p = 0.004, hazard ratio [HR] = 2.4 (95% CI [1.3-4.2])), DFI (P = 0.029, hazard ratio [HR] = 2.0 (95% CI [1.1-3.7])) and PFI (P = 0.005, hazard ratio [HR] = 2.2 (95% CI [1.3-3.9])). GSEA disclosed that cell circle, RNA degradation, pyrimidine metabolism, base excision repair, aminoacyl tRNA biosynthesis, DNA replication, p53 signaling pathway, nucleotide excision repair, ubiquitin-mediated proteolysis, citrate cycle TCA cycle were notably enriched in E2F2 high expression phenotype.
    CONCLUSIONS: Elevated E2F2 can be a promising independent prognostic biomarker and therapeutic target for HCC. Additionally, cell cycle, pyrimidine metabolism, DNA replication, p53 signaling pathway, ubiquitin-mediated proteolysis, the citrate cycle TCA cycle may be the key pathway by which E2F2 participates in the initial and progression of HCC.
    Keywords:  E2F2; Hepatocellular carcinoma; Prognosis
  29. Expert Opin Drug Metab Toxicol. 2020 Oct 27.
    Ehren R, Schijvens AM, Hackl A, Schreuder MF, Weber LT.
      INTRODUCTION: Mycophenolate mofetil (MMF) is an ester prodrug of the immunosuppressant mycophenolic acid (MPA) and is recommended and widely used for maintenance immunosuppressive therapy in solid organ and stem-cell transplantation as well as in immunological kidney diseases. MPA is a potent, reversible, non-competitive inhibitor of the inosine monophosphate dehydrogenase (IMPDH), a crucial enzyme in the de novo purine synthesis in T- and B-lymphocytes, thereby inhibiting cell-mediated immunity and antibody formation. The use of therapeutic drug monitoring (TDM) of MMF is still controversial as outcome data of clinical trials are equivocal.AREAS COVERED: This review covers in great depth the existing literature on TDM of MMF in the field of pediatric (kidney) transplantation. In addition, the relevance of TDM in immunological kidney diseases, in particular childhood nephrotic syndrome is highlighted.
    EXPERT OPINION: TDM of MMF has the potential to optimize therapy in pediatric transplantation as well as in nephrotic syndrome. Limited sampling strategies to estimate MPA exposure increase its feasibility. Future perspectives rather encompass approaches reflecting total immunosuppressive load than single drug TDM.
    Keywords:  IMPDH; limited sampling strategies; mycophenolic acid; pediatric transplantation; pharmacodynamics; pharmacogenetics; pharmacokinetic; trough level
  30. Int J Mol Sci. 2020 Oct 28. pii: E8040. [Epub ahead of print]21(21):
    Honsho M, Abe Y, Imoto Y, Chang ZF, Mandel H, Falik-Zaccai TC, Fujiki Y.
      Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology.
    Keywords:  GTP; NDP kinase; constriction; nme3 patient; peroxisome
  31. Bioorg Med Chem Lett. 2020 Oct 21. pii: S0960-894X(20)30723-X. [Epub ahead of print] 127612
    Penjarla S, Kumar Sabui S, Sudhakar Reddy D, Banerjee S, Yella Reddy P, Penta S, Sanghvi YS.
      Various tetrazole and oxadiazole C-nucleoside analogues were synthesized starting from pure α- or β-glycosyl-cyanide. The synthesis of glycosyl-cyanide as key precursor was optimized on gram-scale to furnish crystalline starting material for the assembly of C-nucleosides. 1,2,4-Oxadizole C-nucleosides were synthesized via two independent routes. First, both anomers of glycosyl-cyanide were transformed into tetrazole nucleosides followed by acylative rearrangement to furnish 1,2,4-oxadiazoles in high yields. Second, the glycosyl-cyanide was converted into an amidoxime which upon ring closure offered an alternative pathway for the assembly of 1,2,4-oxadizoles in an efficient manner. These protocols offer an easy access to otherwise difficult to synthesize C-nucleosides in good yield and protecting group compatibility. These C-nucleosides were evaluated for their antitumor activity. This work paves a path for facile assembly of library of new chemical entities useful for drug discovery.
    Keywords:  1; 2; 4-oxadiazole; Anomers; Antitumor; C-Nucleoside; Cyano-sugar; Hoffer’s Chloro-sugar
  32. Antimicrob Agents Chemother. 2020 Oct 29. pii: AAC.01652-20. [Epub ahead of print]
    Zandi K, Amblard F, Musall K, Downs-Bowen J, Kleinbard R, Oo A, Cao D, Liang B, Russell OO, McBrayer T, Bassit L, Kim B, Schinazi RF.
      Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2, and thus needed confirmation. Here, we evaluated a panel compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance sofosbuvir demonstrated no antiviral effect against CoV-2 and its triphosphate did not inhibit CoV-2 RNA polymerase.
  33. Genome Biol Evol. 2020 Oct 30. pii: evaa229. [Epub ahead of print]
    Ou Z, Ouzounis C, Wang D, Sun W, Li J, Chen W, Marlière P, Danchin A.
      In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve towards likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3'-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate (UTP) that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d'être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3'-deoxy-3',4'-didehydro-CTP (ddhCTP) as an extremely efficient antiviral nucleotide.
    Keywords:  ABCE1; Maxwell’s demon; Nsp1; cytoophidia; phosphoribosyltransferase; queuine