bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022‒05‒01
28 papers selected by
Sean Rudd
Karolinska Institutet
  1. Cancer cells use self-inflicted DNA breaks to evade growth limits imposed by genotoxic stress.
  2. Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.
  3. In crystallo observation of three metal ion promoted DNA polymerase misincorporation.
  4. Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair.
  5. Bloom helicase mediates formation of large single-stranded DNA loops during DNA end processing.
  6. Histone chaperone ASF1 acts with RIF1 to promote DNA end-joining in BRCA1-deficient cells.
  7. Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry.
  8. SUMO: A Swiss Army Knife for Eukaryotic Topoisomerases.
  9. Emerging roles of DNA topoisomerases in the regulation of R-loops.
  10. Structural and functional insight into mismatch extension by human DNA polymerase α.
  11. Discovery of novel 7,8-dihydropteridine-6(5H)-one-based DNA-PK inhibitors as potential anticancer agents via scaffold hopping strategy.
  12. Mapping of sister chromatid exchange events and genome alterations in single cells.
  13. DNA replication profiling by molecular combing on single DNA fibers.
  14. Targeting nucleotide metabolism: a promising approach to enhance cancer immunotherapy.
  15. PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy.
  16. Stretched skin cells divide without DNA replication.
  17. N-Carbamoyl Alanine-Mediated Selective Targeting for CHEK2-Null Colorectal Cancer.
  18. Role of PPAR-γ in diabetes-induced testicular dysfunction, oxidative DNA damage and repair in leptin receptor-deficient obese type 2 diabetic mice.
  19. BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.
  20. CRL4-DCAF8L2 E3 ligase promotes ubiquitination and degradation of BARD1.
  21. Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase.
  22. MiRNA-363-3p/DUSP10/JNK axis mediates chemoresistance by enhancing DNA damage repair in diffuse large B-cell lymphoma.
  23. Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.
  24. The role of nucleoside triphosphate hydrolase metallochaperones in making metalloenzymes.
  25. Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.
  26. Cytoplasmic PARP1 links the genome instability to the inhibition of antiviral immunity through PARylating cGAS.
  27. The paradoxical role of inositol in cancer: a consequence of the metabolic state of a tumor.
  28. Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.
  1. Science. 2022 Apr 29. 376(6592): 476-483
    Cancer cells use self-inflicted DNA breaks to evade growth limits imposed by genotoxic stress.
    Brian D Larsen, Jan Benada, Philip Yuk Kwong Yung, Ryan A V Bell, George Pappas, Vaclav Urban, Johanna K Ahlskog, Tia T Kuo, Pavel Janscak, Lynn A Megeney, Simon J Elsässer, Jiri Bartek, Claus S Sørensen.
      Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.
    DOI:  https://doi.org/10.1126/science.abi6378
  2. Nature. 2022 Apr 27.
    Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.
    Shangming Tang, Ema Stokasimov, Yuxiang Cui, David Pellman.
      Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridge5-8. These structures have fragile nuclear envelopes (NEs) that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here, we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by ADAR enzymes (adenine deaminases acting on RNA) to generate deoxyinosine (dI). dI is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, MPG (N-methyl-purine DNA glycosylase)11,12. These abasic sites are cleaved by the BER endonuclease, APE1 (apurinic/apyrimidinic endonuclease)12, creating single-strand DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the dI base from the DNA strand of RNA-DNA hybrids, which we demonstrate using pure proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with preexisting defects such as the DNA base abnormality described here.
    DOI:  https://doi.org/10.1038/s41586-022-04767-1
  3. Nat Commun. 2022 Apr 29. 13(1): 2346
    In crystallo observation of three metal ion promoted DNA polymerase misincorporation.
    Caleb Chang, Christie Lee Luo, Yang Gao.
      Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.
    DOI:  https://doi.org/10.1038/s41467-022-30005-3
  4. Front Oncol. 2022 ;12 850883
    Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair.
    Jeremy M Kelm, Amirreza Samarbakhsh, Athira Pillai, Pamela S VanderVere-Carozza, Hariprasad Aruri, Deepti S Pandey, Katherine S Pawelczak, John J Turchi, Navnath S Gavande.
      The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment.
    Keywords:  DNA double-strand break (DSB) repair; DNA repair and DNA damage response (DDR); homology directed repair (HDR); non-PIKKs inhibitors; non-homologous end joining (NHEJ); polymerase theta-mediated end joining (TMEJ); single-strand annealing (SSA); synthetic lethality
    DOI:  https://doi.org/10.3389/fonc.2022.850883
  5. Nat Commun. 2022 Apr 26. 13(1): 2248
    Bloom helicase mediates formation of large single-stranded DNA loops during DNA end processing.
    Chaoyou Xue, Sameer J Salunkhe, Nozomi Tomimatsu, Ajinkya S Kawale, Youngho Kwon, Sandeep Burma, Patrick Sung, Eric C Greene.
      Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double-strand breaks (DSBs), to yield long 3' ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single-molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N-terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis-dependent strand annealing (SSA) instead of HR-mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.
    DOI:  https://doi.org/10.1038/s41467-022-29937-7
  6. J Biol Chem. 2022 Apr 23. pii: S0021-9258(22)00419-7. [Epub ahead of print] 101979
    Histone chaperone ASF1 acts with RIF1 to promote DNA end-joining in BRCA1-deficient cells.
    Mengfan Tang, Zhen Chen, Chao Wang, Xu Feng, Namsoo Lee, Min Huang, Huimin Zhang, Siting Li, Yun Xiong, Junjie Chen.
      Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends which plays critical roles in determining the DNA double-strand break (DSB) repair pathway choice between non-homologous end joining (NHEJ) and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to PARP inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed these functions of ASF1 are dependent on its interaction with RIF1, but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating DSB repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor (PARPi)-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARPi resistance.
    DOI:  https://doi.org/10.1016/j.jbc.2022.101979
  7. Sci Rep. 2022 Apr 28. 12(1): 6903
    Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry.
    Alexander Munden, Madison T Wright, Dongsheng Han, Reyhaneh Tirgar, Lars Plate, Jared T Nordman.
      Replication of the eukaryotic genome requires the formation of thousands of replication forks that must work in concert to accurately replicate the genetic and epigenetic information. Defining replication fork-associated proteins is a key step in understanding how genomes are replicated and repaired in the context of chromatin to maintain genome stability. To identify replication fork-associated proteins, we performed iPOND (Isolation of Proteins on Nascent DNA) coupled to quantitative mass spectrometry in Drosophila embryos and cultured cells. We identified 76 and 278 fork-associated proteins in post-MZT embryos and Drosophila cultured S2 cells, respectively. By performing a targeted screen of a subset of these proteins, we demonstrate that BRWD3, a targeting specificity factor for the DDB1/Cul4 ubiquitin ligase complex (CRL4), functions at or in close proximity to replication forks to promote fork progression and maintain genome stability. Altogether, our work provides a valuable resource for those interested in DNA replication, repair and chromatin assembly during development.
    DOI:  https://doi.org/10.1038/s41598-022-10821-9
  8. Front Mol Biosci. 2022 ;9 871161
    SUMO: A Swiss Army Knife for Eukaryotic Topoisomerases.
    Yilun Sun, John L Nitiss, Yves Pommier.
      Topoisomerases play crucial roles in DNA metabolism that include replication, transcription, recombination, and chromatin structure by manipulating DNA structures arising in double-stranded DNA. These proteins play key enzymatic roles in a variety of cellular processes and are also likely to play structural roles. Topoisomerases allow topological transformations by introducing transient breaks in DNA by a transesterification reaction between a tyrosine residue of the enzyme and DNA. The cleavage reaction leads to a unique enzyme intermediate that allows cutting DNA while minimizing the potential for damage-induced genetic changes. Nonetheless, topoisomerase-mediated cleavage has the potential for inducing genome instability if the enzyme-mediated DNA resealing is impaired. Regulation of topoisomerase functions is accomplished by post-translational modifications including phosphorylation, polyADP-ribosylation, ubiquitylation, and SUMOylation. These modifications modulate enzyme activity and likely play key roles in determining sites of enzyme action and enzyme stability. Topoisomerase-mediated DNA cleavage and rejoining are affected by a variety of conditions including the action of small molecules, topoisomerase mutations, and DNA structural forms which permit the conversion of the short-lived cleavage intermediate to persistent topoisomerase DNA-protein crosslink (TOP-DPC). Recognition and processing of TOP-DPCs utilizes many of the same post-translational modifications that regulate enzyme activity. This review focuses on SUMOylation of topoisomerases, which has been demonstrated to be a key modification of both type I and type II topoisomerases. Special emphasis is placed on recent studies that indicate how SUMOylation regulates topoisomerase function in unperturbed cells and the unique roles that SUMOylation plays in repairing damage arising from topoisomerase malfunction.
    Keywords:  DNA repair; SUMO; the ubiquitin–proteasome system; topoisomerase inhibitors; topoisomerases
    DOI:  https://doi.org/10.3389/fmolb.2022.871161
  9. Mutat Res Genet Toxicol Environ Mutagen. 2022 Apr-May;876-877:pii: S1383-5718(22)00011-0. [Epub ahead of print]876-877 503450
    Emerging roles of DNA topoisomerases in the regulation of R-loops.
    Parasvi S Patel, Rehna Krishnan, Razqallah Hakem.
      R-loops are comprised of a DNA:RNA hybrid and a displaced single-strand DNA (ssDNA) that reinvades the DNA duplex behind the moving RNA polymerase. Because they have several physiological functions within the cell, including gene expression, chromosomal segregation, and mitochondrial DNA replication, among others, R-loop homeostasis is tightly regulated to ensure normal functioning of cellular processes. Thus, several classes of enzymes including RNases, helicases, topoisomerases, as well as proteins involved in splicing and the biogenesis of messenger ribonucleoproteins, have been implicated in R-loop prevention, suppression, and resolution. There exist six topoisomerase enzymes encoded by the human genome that function to introduce transient DNA breaks to relax supercoiled DNA. In this mini-review, we discuss functions of DNA topoisomerases and their emerging role in transcription, replication, and regulation of R-loops, and we highlight how their role in maintaining genome stability can be exploited for cancer therapy.
    Keywords:  Cell cycle; DNA damage; DNA repair; DNA:RNA hybrids; Topoisomerases
    DOI:  https://doi.org/10.1016/j.mrgentox.2022.503450
  10. Proc Natl Acad Sci U S A. 2022 Apr 26. 119(17): e2111744119
    Structural and functional insight into mismatch extension by human DNA polymerase α.
    Andrey G Baranovskiy, Nigar D Babayeva, Alisa E Lisova, Lucia M Morstadt, Tahir H Tahirov.
      SignificanceDespite the important role of human DNA polymerase α (Polα) in genome mutagenesis, there are no structural studies of Polα infidelity. The functional studies are sparse, lack high-resolution approaches, and are performed at a low salt concentration. Here we report the structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template:primer containing a T-C mismatch at the growing primer terminus. Pre-steady-state and binding kinetics conducted at a physiological salt concentration revealed that Polα has a remarkably lower affinity to DNA and deoxynucleotide triphosphate (dNTP) than reported previously. Strikingly, we found that the incoming dNTP plays a crucial role in Polα interaction with DNA and in discrimination against a mismatched template:primer. This work is important for understanding the mechanism of Polα infidelity and provides a foundation for future studies.
    Keywords:  DNA polymerase α; DNA replication; crystal structure; kinetic studies; mismatch
    DOI:  https://doi.org/10.1073/pnas.2111744119
  11. Eur J Med Chem. 2022 Apr 20. pii: S0223-5234(22)00303-8. [Epub ahead of print]237 114401
    Discovery of novel 7,8-dihydropteridine-6(5H)-one-based DNA-PK inhibitors as potential anticancer agents via scaffold hopping strategy.
    Zongbao Ding, Wei Pan, Yao Xiao, Binbin Cheng, Gang Huang, Jianjun Chen.
      DNA-dependent protein kinase (DNA-PK) is an essential element in the DNA damage response (DDR) pathway and has been regarded as a druggable target for antineoplastic agents. Starting from AZD-7648, a potent DNA-PK inhibitor being investigated in phase II clinical trials for advanced cancer treatment, two series of DNA-PK inhibitors were rationally designed via scaffold hopping strategy, synthesized, and assessed for their biological activity. Most compounds exhibited potent biochemical activity on DNA-PK enzymatic assay with IC50 values below 300 nM. Among these compounds, DK1 showed the best DNA-PK-inhibitory potency (IC50 = 0.8 nM), slightly better than that of AZD-7648 (IC50 = 1.58 nM). Mode of action studies revealed that compound DK1 decreased the expression levels of γH2A.X and demonstrated synergistic antiproliferative activity against a series of cancer cell lines when used in combination with doxorubicin. Moreover, DK1 showed reasonable in vitro drug-like properties and favorable in vivo pharmacokinetics as an oral drug candidate. Importantly, the combination therapy of DK1 with DNA double-strand break (DSB)-inducing agent doxorubicin showed synergistic anticancer efficacy in the HL-60 xenograft model with a tumor growth inhibition (TGI) of 52.4% and 62.4% for tumor weight and tumor volume, respectively. In conclusion, DK1 is a novel DNA-PK inhibitor with great promise for further study.
    Keywords:  Anticancer agents; DNA-PK Inhibitors; Scaffold hopping
    DOI:  https://doi.org/10.1016/j.ejmech.2022.114401
  12. Methods. 2022 Apr 25. pii: S1046-2023(22)00102-5. [Epub ahead of print]
    Mapping of sister chromatid exchange events and genome alterations in single cells.
    Zeid Hamadeh, Vincent Hanlon, Peter M Lansdorp.
      Mammalian genomes encode over a hundred different helicases, many of which are implicated in the repair of DNA lesions by acting on DNA structures arising during DNA replication, recombination or transcription. Defining the in vivo substrates of such DNA helicases is a major challenge given the large number of helicases in the genome, the breadth of potential substrates in the genome and the degree of genetic pleiotropy among DNA helicases in resolving diverse substrates. Helicases such as WRN, BLM and RECQL5 are implicated in the resolution of error-free recombination events known as sister chromatid exchange events (SCEs). Single cell Strand-seq can be used to map the genomic location of individual SCEs at a resolution that exceeds that of classical cytogenetic techniques by several orders of magnitude. By mapping the genomic locations of SCEs in the absence of different helicases, it should in principle be possible to infer the substrate specificity of specific helicases. Here we describe how the genome can be interrogated for such DNA repair events using single-cell template strand sequencing (Strand-seq) and bioinformatic tools. SCEs and copy-number alterations were mapped to genomic locations at kilobase resolution in haploid KBM7 cells. Strategies, possibilities, and limitations of Strand-seq to study helicase function are illustrated using these cells before and after CRISPR/Cas9 knock out of WRN, BLM and/or RECQL5.
    Keywords:  DNA repair; RecQ helicases; Strand-seq; recombination; structural variants
    DOI:  https://doi.org/10.1016/j.ymeth.2022.04.013
  13. STAR Protoc. 2022 Jun 17. 3(2): 101290
    DNA replication profiling by molecular combing on single DNA fibers.
    Haiqing Fu, Mirit I Aladjem.
      In this protocol, the progression of DNA synthesis is profiled at a single-molecule resolution. DNA fibers are uniformly stretched on silanized coverslips, and replicating DNA can be traced with thymidine analogs using specific antibodies against distinct analogs. Single DNA fibers are visualized by an anti-single stranded DNA antibody. The protocol can be used to study DNA replication dynamics, the cellular response to replication stress, and replication fork progression at specific chromosomal regions when combined with fluorescent in situ hybridization. For complete details on the use and execution of this protocol, please refer to Conti et al. (2007), Fu et al. (2021), Kaykov et al. (2016), Redmond et al. (2018), and Schwob et al. (2009).
    Keywords:  Biotechnology and bioengineering; Cell Biology; Molecular Biology; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2022.101290
  14. J Hematol Oncol. 2022 Apr 27. 15(1): 45
    Targeting nucleotide metabolism: a promising approach to enhance cancer immunotherapy.
    Huai-Liang Wu, Yue Gong, Peng Ji, Yi-Fan Xie, Yi-Zhou Jiang, Guang-Yu Liu.
      Targeting nucleotide metabolism can not only inhibit tumor initiation and progression but also exert serious side effects. With in-depth studies of nucleotide metabolism, our understanding of nucleotide metabolism in tumors has revealed their non-proliferative effects on immune escape, indicating the potential effectiveness of nucleotide antimetabolites for enhancing immunotherapy. A growing body of evidence now supports the concept that targeting nucleotide metabolism can increase the antitumor immune response by (1) activating host immune systems via maintaining the concentrations of several important metabolites, such as adenosine and ATP, (2) promoting immunogenicity caused by increased mutability and genomic instability by disrupting the purine and pyrimidine pool, and (3) releasing nucleoside analogs via microbes to regulate immunity. Therapeutic approaches targeting nucleotide metabolism combined with immunotherapy have achieved exciting success in preclinical animal models. Here, we review how dysregulated nucleotide metabolism can promote tumor growth and interact with the host immune system, and we provide future insights into targeting nucleotide metabolism for immunotherapeutic treatment of various malignancies.
    DOI:  https://doi.org/10.1186/s13045-022-01263-x
  15. Cell Biosci. 2022 Apr 27. 12(1): 50
    PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy.
    Thomas Fischer, Oliver Hartmann, Michaela Reissland, Cristian Prieto-Garcia, Kevin Klann, Nikolett Pahor, Christina Schülein-Völk, Apoorva Baluapuri, Bülent Polat, Arya Abazari, Elena Gerhard-Hartmann, Hans-Georg Kopp, Frank Essmann, Mathias Rosenfeldt, Christian Münch, Michael Flentje, Markus E Diefenbacher.
      BACKGROUND: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy.RESULTS: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model.
    CONCLUSION: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
    Keywords:  ATM; Cancer; DNA-PK; IR; NSCLC; PI3K; PTEN; Radiotherapy
    DOI:  https://doi.org/10.1186/s13578-022-00778-7
  16. Nature. 2022 May;605(7908): 31-32
    Stretched skin cells divide without DNA replication.
    Aki Stubb, Sara A Wickström.
      
    Keywords:  Cell biology; Developmental biology
    DOI:  https://doi.org/10.1038/d41586-022-00790-4
  17. ACS Omega. 2022 Apr 19. 7(15): 13095-13101
    N-Carbamoyl Alanine-Mediated Selective Targeting for CHEK2-Null Colorectal Cancer.
    Anas Ahmad, Ravi Prakash, Mohd Shahnawaz Khan, Nojood Altwaijry, Muhammad Nadeem Asghar, Syed Shadab Raza, Rehan Khan.
      Colorectal cancer (CRC) is one of the major causes of cancer-linked mortality worldwide. Selective therapeutic approaches toward cancer are the need of the hour to combat cancer. Synthetic lethality is a pragmatic targeted cancer therapy in which cancer cell-specific vulnerabilities such as genetic defects/somatic mutations are exploited for selective cancer therapy by targeting genetic interactors (synthetic lethal interactors) of such mutation/defects present in cancer cells. In this study, we investigated the synthetic lethal interaction between checkpoint kinase 2 (CHEK2) and peroxiredoxin-2 (PRDX2) in CRC cells to precisely target CRC cells having CHEK2 defects. We have performed siRNA-mediated silencing and n-carbamoyl alanine (NCA)-mediated inhibition of PRDX2 in CHEK2-null HCT116 cells to confirm the synthetic lethal (SL) interaction between PRDX2 and CHEK2 as the cell population reduced significantly after silencing/inhibition of PRDX2. Additionally, treatment with NCA resulted in an increased level of total ROS in both cell types (HCT116 and CHEK2-null HCT116 cells), which further confirms that inhibition of PRDX2 results in an increased ROS level, which are mainly responsible for DNA double-strand breaks (DSBs). ROS-induced DNA DSBs get repaired in HCT116 cells, in which CHEK2 is in the normal functional state, but these DNA DSBs persist in CHEK2-null HCT116 cells as confirmed by the immunofluorescence analysis of 53BP1 and γ-H2AX. Finally, CHEK2-null HCT116 cells undergo apoptosis due to persistent DNA damage as confirmed by immunofluorescence analysis of cleaved caspase-3. The findings of this study suggest that PRDX2 has a SL interaction with CHEK2, and this interaction can be exploited for the targeted cancer therapy using NCA as a drug inhibitor of PRDX2 for the therapy of colorectal cancer having CHEK2 defects. Further studies are warranted to confirm the interaction in the preclinical model.
    DOI:  https://doi.org/10.1021/acsomega.2c00527
  18. Chem Biol Interact. 2022 Apr 23. pii: S0009-2797(22)00163-6. [Epub ahead of print] 109958
    Role of PPAR-γ in diabetes-induced testicular dysfunction, oxidative DNA damage and repair in leptin receptor-deficient obese type 2 diabetic mice.
    Muneera Waheed Alfarhan, Heba Al-Hussaini, Narayana Kilarkaje.
      The testis expresses peroxisome proliferator-activated receptor-γ (PPAR-γ), but its involvement in regulating diabetes-induced testicular dysfunction and DNA damage repair is not known. Pioglitazone-induced activation of PPAR-γ for 12 weeks in db/db obese diabetic mice increases bodyweights and reduces blood glucose levels, but PPAR-γ inhibition by 2-chloro-5-nitro-N-phenylbenzamide does not alter these parameters; instead, improves testis and epididymis weights and sperm count. Neither activation nor inhibition of PPAR-γ normalizes the diabetes-induced seminiferous epithelial degeneration. The PPAR-γ activation normalizes testicular lipid peroxidation, but its inhibition reduces lipid peroxidation and oxidative DNA damage (8-oxo-dG) in diabetic mice. As a response to diabetes-induced oxidative DNA damage, the base-excision repair (BER) mechanism proteins- 8-oxoguanine DNA glycosylases (OGG1/2) and X-ray repair cross-complementing protein-1 (XRCC1) increase, whereas the redox-factor-1 (REF1), DNA polymerase (pol) δ and poly (ADP-ribose) polymerase-1 (PARP1) show a tendency to increase suggesting an attempt to repair the oxidative DNA damage. The PPAR-γ stimulation inhibits OGG2, DNA pol δ, and XRCC1 in diabetic mice testes, but PPAR-γ inhibition reduces oxidative DNA damage and normalizes BER protein levels. In conclusion, type 2 diabetes negatively affects testicular structure and function and increases oxidative DNA damage and BER protein levels due to increased DNA damage. The PPAR-γ modulation does not significantly affect the structural changes in the testis. The PPAR-γ stimulation aggravates diabetes-induced effects on testis, including oxidative DNA damage and BER proteins, but PPAR-γ inhibition marginally recovers these diabetic effects indicating the involvement of the receptor in the reproductive effects of diabetes.
    Keywords:  Base-excision repair; DNA damage repair; Diabetes mellitus; PPAR-γ; Pioglitazone
    DOI:  https://doi.org/10.1016/j.cbi.2022.109958
  19. DNA Repair (Amst). 2022 Apr 16. pii: S1568-7864(22)00064-7. [Epub ahead of print]115 103331
    BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.
    Christopher Parker, Adam C Chambers, Dustin J Flanagan, Jasmine Wing Yu Ho, Tracey J Collard, Greg Ngo, Duncan M Baird, Penny Timms, Rhys G Morgan, Owen J Sansom, Ann C Williams.
      The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.
    Keywords:  Colorectal cancer/DNA Damage response/Therapy response/Chemoradiotherapy/Homologous recombination
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103331
  20. Biochem Biophys Res Commun. 2022 Apr 23. pii: S0006-291X(22)00642-8. [Epub ahead of print]611 107-113
    CRL4-DCAF8L2 E3 ligase promotes ubiquitination and degradation of BARD1.
    Jingcheng Deng, Ting Zhang, Fei Liu, Qianying Han, Qin Li, Xueyuan Guo, Yanfang Ma, Li Li, Genze Shao.
      BARD1 is a tumor suppressor that is necessary for the functioning and stability of BRCA1, with which it forms a heterodimer and participates in the repair of DNA double-strand breaks. The cellular level of BARD1 and its interaction with BRCA1 are crucial for BRCA1/BARD1 function in homologous recombination and tumor suppression. However, the regulatory mechanism underpinning the stability of BARD1 is largely unclear. In this study, we identified DCAF8L2, a DDB1-Cullin associated factor (DCAF) associated with CRL4 E3 ligase, as a negative regulator of BARD1. Mechanistically, DCAF8L2 interacts with and targets BARD1 for ubiquitination and degradation. In addition, the interaction of DCAF8L2 with BARD1 through the RING domain could compete with the dimerization of BRCA1 and BARD1, leading to increased cellular uncoupling of BARD1 and BRCA1, subjecting the latter to degradation. The overexpression of DCAF8L2 compromises the homologous recombination process and confers cells with increased sensitivity to DNA damage. Furthermore, DCAF8L2 was aberrantly expressed in breast cancer cell lines. Our findings suggest that DCAF8L2 may play an oncogenic role in the pathogenesis of breast cancer, possibly by negative regulation of BARD1.
    Keywords:  BARD1; BRCA1; Cullin 4 E3 ligase; DCAF8L2; Ubiquitination
    DOI:  https://doi.org/10.1016/j.bbrc.2022.04.100
  21. Protein Sci. 2022 May;31(5): e4314
    Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase.
    David Fernández-Justel, Íñigo Marcos-Alcalde, Federico Abascal, Nerea Vidaña, Paulino Gómez-Puertas, Alberto Jiménez, José L Revuelta, Rubén M Buey.
      IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.
    Keywords:  (p)ppGpp; IMP dehydrogenase; allosteric regulation; bacterial GTP homeostasis; protein structure and function; purine nucleotide biosynthesis
    DOI:  https://doi.org/10.1002/pro.4314
  22. Leukemia. 2022 Apr 29.
    MiRNA-363-3p/DUSP10/JNK axis mediates chemoresistance by enhancing DNA damage repair in diffuse large B-cell lymphoma.
    Wenping Zhou, Yuanlin Xu, Jiuyang Zhang, Peipei Zhang, Zhihua Yao, Zheng Yan, Haiying Wang, Junfeng Chu, Shuna Yao, Shuang Zhao, Shujun Yang, Yongjun Guo, Jinxin Miao, Kangdong Liu, Wing C Chan, Qingxin Xia, Yanyan Liu.
      Anthracycline-based chemotherapy resistance represents a major challenge in diffuse large B-cell lymphoma (DLBCL). MiRNA and gene expression profiles (n = 47) were determined to uncover potential chemoresistance mechanisms and therapeutic approaches. An independent correlation between high expression of miRNA-363-3p and chemoresistance was observed and validated in a larger cohort (n = 106). MiRNA-363-3p was shown to reduce doxorubicin-induced apoptosis and tumor shrinkage in in vitro and in vivo experiments by ectopic expression and CRISPR/Cas9-mediated knockout in DLBCL cell lines. DNA methylation was found to participate in transcriptional regulation of miRNA-363-3p. Further investigation revealed that dual specificity phosphatase 10 (DUSP10) is a target of miRNA-363-3p and its suppression promotes the phosphorylation of c-Jun N-terminal kinase (JNK). The miRNA-363-3p/DUSP10/JNK axis was predominantly associated with negative regulation of homologous recombination (HR) and DNA repair pathways. Ectopic expression of miRNA-363-3p more effectively repaired doxorubicin-induced double-strand break (DSB) while enhancing non-homologous end joining repair and reducing HR repair. Targeting JNK and poly (ADP-ribose) polymerase 1 significantly inhibited doxorubicin-induced DSB repair, increased doxorubicin-induced cell apoptosis and tumor shrinkage, and improved the survival of tumor-bearing mice. In conclusion, the miRNA-363-3p/DUSP10/JNK axis is a novel chemoresistance mechanism in DLBCL that may be reversed by targeted therapy.
    DOI:  https://doi.org/10.1038/s41375-022-01565-6
  23. J Biol Chem. 2022 Apr 22. pii: S0021-9258(22)00416-1. [Epub ahead of print] 101976
    Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.
    Alexander Kröning, Johannes van den Boom, Matthias Kracht, Anja F Kueck, Hemmo Meyer.
      The protease SPRTN degrades DNA protein-crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability, and for designing strategies that target p97 in combination cancer therapy.
    Keywords:  AAA+ ATPase; Cdc48; DNA-protein crosslink; SPRTN; VCP; Wss1; p97; protein unfolding
    DOI:  https://doi.org/10.1016/j.jbc.2022.101976
  24. Metallomics. 2022 Apr 29. pii: mfac030. [Epub ahead of print]
    The role of nucleoside triphosphate hydrolase metallochaperones in making metalloenzymes.
    Francesca A Vaccaro, Catherine L Drennan.
      Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate (ATP) or guanosine triphosphate (GTP), to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.
    Keywords:  ATPases; GTPases; metallochaperones; metallocofactors; metalloenzyme maturation; metals in biology
    DOI:  https://doi.org/10.1093/mtomcs/mfac030
  25. Sci Adv. 2022 Apr 29. 8(17): eabm2827
    Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.
    Nadine M Shaban, Rui Yan, Ke Shi, Sofia N Moraes, Adam Z Cheng, Michael A Carpenter, Jason S McLellan, Zhiheng Yu, Reuben S Harris.
      Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å2 binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.
    DOI:  https://doi.org/10.1126/sciadv.abm2827
  26. Mol Cell. 2022 Apr 14. pii: S1097-2765(22)00290-8. [Epub ahead of print]
    Cytoplasmic PARP1 links the genome instability to the inhibition of antiviral immunity through PARylating cGAS.
    Fei Wang, Mengmeng Zhao, Boran Chang, Yilong Zhou, Xiangyang Wu, Mingtong Ma, Siyu Liu, Yajuan Cao, Mengge Zheng, Yifang Dang, Junfang Xu, Li Chen, Tianhao Liu, Fen Tang, Yefei Ren, Zhu Xu, Zhiyong Mao, Kai Huang, Minhua Luo, Jingsong Li, Haipeng Liu, Baoxue Ge.
      Virus infection modulates both host immunity and host genomic stability. Poly(ADP-ribose) polymerase 1 (PARP1) is a key nuclear sensor of DNA damage, which maintains genomic integrity, and the successful application of PARP1 inhibitors for clinical anti-cancer therapy has lasted for decades. However, precisely how PARP1 gains access to cytoplasm and regulates antiviral immunity remains unknown. Here, we report that DNA virus induces a reactive nitrogen species (RNS)-dependent DNA damage and activates DNA-dependent protein kinase (DNA-PK). Activated DNA-PK phosphorylates PARP1 on Thr594, thus facilitating the cytoplasmic translocation of PARP1 to inhibit the antiviral immunity both in vitro and in vivo. Mechanistically, cytoplasmic PARP1 interacts with and directly PARylates cyclic GMP-AMP synthase (cGAS) on Asp191 to inhibit its DNA-binding ability. Together, our findings uncover an essential role of PARP1 in linking virus-induced genome instability with inhibition of host immunity, which is of relevance to cancer, autoinflammation, and other diseases.
    Keywords:  DNA damage response; DNA-dependent protein kinase; PARylation; antiviral immunity; cyclic GMP-AMP synthase; inducible nitric oxide synthase; poly(ADP-ribose) polymerase 1; type I interferon
    DOI:  https://doi.org/10.1016/j.molcel.2022.03.034
  27. Cancer Metastasis Rev. 2022 Apr 25.
    The paradoxical role of inositol in cancer: a consequence of the metabolic state of a tumor.
    Kendall C Case, Michael W Schmidtke, Miriam L Greenberg.
      Inositol is an essential nutrient, obtained either by uptake from the environment or by de novo synthesis from glucose. Inositol and its derivatives exhibit tumor-suppressive effects, potentially mediated by inhibition of the ERK-MAPK or PI3K-Akt pathways. Accordingly, many cancers have been documented to silence expression of the ISYNA1 gene, which encodes the rate-limiting enzyme of inositol synthesis. Paradoxically, recent studies have also reported upregulation of ISYNA1 in some cancers. Upregulation may reflect a compensatory response brought about by defective inositol uptake or oncogenic mutations that preclude its tumor-suppressive effects. In these scenarios, de novo synthesis of inositol may be upregulated to promote cell proliferation. The role of inositol in cancer is further complicated by its ability to inhibit the master metabolic regulator AMPK, which upon activation can either decrease cell proliferation and metastasis or promote cell survival. Due to its potential dual role in cancer, inositol homeostasis must be tightly regulated in tumor cells. Thus, whether inositol acts to suppress or promote tumor progression is determined by the metabolic profile and oncogenic background of the cancer.
    Keywords:  AMPK; Cancer; ISYNA1; Inositol; Metabolism; PI3K-Akt
    DOI:  https://doi.org/10.1007/s10555-022-10032-8
  28. Sci Immunol. 2022 Apr 29. 7(70): eabm8161
    Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.
    Xuyong Chen, Benjamin Sunkel, Meng Wang, Siwen Kang, Tingting Wang, J N Rashida Gnanaprakasam, Lingling Liu, Teresa A Cassel, David A Scott, Ana M Muñoz-Cabello, Jose Lopez-Barneo, Jun Yang, Andrew N Lane, Gang Xin, Benjamin Z Stanton, Teresa W-M Fan, Ruoning Wang.
      Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.
    DOI:  https://doi.org/10.1126/sciimmunol.abm8161
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