bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒05‒17
twenty-six papers selected by
Sean Rudd
Karolinska Institutet


  1. Stem Cell Reports. 2020 Apr 28. pii: S2213-6711(20)30144-2. [Epub ahead of print]
      Human pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.
    Keywords:  DNA; damage; errors; human; mitotic; nucleosides; pluripotent; replication; stress
    DOI:  https://doi.org/10.1016/j.stemcr.2020.04.004
  2. Cell Rep. 2020 May 12. pii: S2211-1247(20)30593-3. [Epub ahead of print]31(6): 107640
      The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.
    Keywords:  BCX-1777; CyTOF; Immucillin H; SAMHD1; apoptosis; chronic lymphocytic leukemia; dGTP; dNTP; deoxyguanosine; forodesine
    DOI:  https://doi.org/10.1016/j.celrep.2020.107640
  3. EMBO Mol Med. 2020 May 13. e11217
      Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors.
    Keywords:   BCRA ; OXPHOS ; PARP inhibitors; cancer metabolism; metformin
    DOI:  https://doi.org/10.15252/emmm.201911217
  4. Mol Cell Oncol. 2020 ;7(3): 1733383
      Metabolic rearrangements and genome instability are two hallmarks of cancer. Recent evidence from our laboratory demonstrates that persistent DNA lesions hampering transcription may cause glucose rerouting through the pentose phosphate shunt and reductive stress. Here, we highlight the relevance of these findings for cancer and chemoresistance development.
    Keywords:  DNA damage; DNA repair; metabolism; pentose phosphate pathway; redox
    DOI:  https://doi.org/10.1080/23723556.2020.1733383
  5. Sci Rep. 2020 May 12. 10(1): 7838
      One-carbon metabolism fuels the high demand of cancer cells for nucleotides and other building blocks needed for increased proliferation. Although inhibitors of this pathway are widely used to treat many cancers, their global impact on anabolic and catabolic processes remains unclear. Using a combination of real-time bioenergetics assays and metabolomics approaches, we investigated the global effects of methotrexate on cellular metabolism. We show that methotrexate treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. The anti-proliferative and pro-respiratory effects of methotrexate are AMPK-dependent, as cells with reduced AMPK activity are less affected by methotrexate treatment. Conversely, the combination of methotrexate with the AMPK activator, phenformin, potentiates its anti-proliferative activity in cancer cells. These data highlight a reciprocal effect of methotrexate on anabolic and catabolic processes and implicate AMPK activation as a metabolic determinant of methotrexate response.
    DOI:  https://doi.org/10.1038/s41598-020-64460-z
  6. Nat Commun. 2020 May 15. 11(1): 2437
      DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.
    DOI:  https://doi.org/10.1038/s41467-020-16095-x
  7. J Cell Biol. 2020 Jun 01. pii: e201908192. [Epub ahead of print]219(6):
      BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.
    DOI:  https://doi.org/10.1083/jcb.201908192
  8. Cell Rep. 2020 May 12. pii: S2211-1247(20)30588-X. [Epub ahead of print]31(6): 107635
      Expanded CAG repeats form stem-loop secondary structures that lead to fork stalling and collapse. Previous work has shown that these collapsed forks relocalize to nuclear pore complexes (NPCs) in late S phase in a manner dependent on replication, the nucleoporin Nup84, and the Slx5 protein, which prevents repeat fragility and instability. Here, we show that binding of the Smc5/6 complex to the collapsed fork triggers Mms21-dependent sumoylation of fork-associated DNA repair proteins, and that RPA, Rad52, and Rad59 are the key sumoylation targets that mediate relocation. The SUMO interacting motifs of Slx5 target collapsed forks to the NPC. Notably, Rad51 foci only co-localize with the repeat after it is anchored to the nuclear periphery and Rad51 exclusion from the early collapsed fork is dependent on RPA sumoylation. This pathway may provide a mechanism to constrain recombination at stalled or collapsed forks until it is required for fork restart.
    Keywords:  CAG repeat; RPA sumoylation; Rad52 Rad59 sumoylation; Smc5-Smc6; fork collapse; fork relocation; nuclear pore complex; replication fork; trinucleotide repeat
    DOI:  https://doi.org/10.1016/j.celrep.2020.107635
  9. Nat Struct Mol Biol. 2020 May;27(5): 438-449
      The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.
    DOI:  https://doi.org/10.1038/s41594-020-0419-3
  10. Nat Struct Mol Biol. 2020 May;27(5): 424-437
      Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.
    DOI:  https://doi.org/10.1038/s41594-020-0408-6
  11. Open Biol. 2020 May;10(5): 190259
      Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.
    Keywords:  DNA decatenation; DNA replication; Topoisomerase II; chromosome segregation; ultrafine anaphase bridge
    DOI:  https://doi.org/10.1098/rsob.190259
  12. Mol Cell Oncol. 2020 ;7(3): 1698933
      DNA double-strand breaks (DSBs) could be deleterious and lead to age-related diseases, such as cancer. Recent evidence, however, associates DSBs with vital cellular processes. As discussed here, genome-wide mapping of DSBs revealed an unforeseen coupling mechanism between transcription and DNA repair at super-enhancers, as means of hypertranscription of oncogenic drivers.
    Keywords:  BLISS; DSBs; RAD51; breakome; super-enhancer
    DOI:  https://doi.org/10.1080/23723556.2019.1698933
  13. PLoS Genet. 2020 May 11. 16(5): e1008787
      During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.
    DOI:  https://doi.org/10.1371/journal.pgen.1008787
  14. Sci Rep. 2020 May 14. 10(1): 7959
      DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.
    DOI:  https://doi.org/10.1038/s41598-020-64868-7
  15. Leukemia. 2020 May 15.
      T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in T-ALL and is crucial for human T-ALL cell growth in vivo. Knockout of TET1 in mice and knockdown in human T cell did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was dependent on its catalytic property to maintain global 5-hydroxymethylcytosine (5hmC) marks, thereby regulate cell cycle, DNA repair genes, and T-ALL associated oncogenes. Furthermore, overexpression of the Tet1-catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of T-ALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in T-ALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in T-ALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks, and antagonized leukemic growth of T-ALL cells, opening a therapeutic avenue for this disease.
    DOI:  https://doi.org/10.1038/s41375-020-0864-3
  16. Nat Commun. 2020 May 15. 11(1): 2447
      Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.
    DOI:  https://doi.org/10.1038/s41467-020-16152-5
  17. Curr Genet. 2020 May 11.
      DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.
    Keywords:  DNA repair; Elg1; Genome stability; Homologous recombination; PCNA; Saccharomyces cerevisae
    DOI:  https://doi.org/10.1007/s00294-020-01082-y
  18. Mol Cell Oncol. 2020 ;7(3): 1743807
      Repetitive sequences can form a variety of alternative DNA structures (non-B DNA) that can modulate transcription, replication, and repair. However, non-B DNA-forming sequences can also stimulate mutagenesis, and are enriched at mutation hotspots in human cancer genomes. Interestingly, different types of non-B DNA stimulate mutagenesis via distinct repair processing mechanisms.
    Keywords:  DNA repair; DNA structure; Non-B DNA; cancer; mutagenesis
    DOI:  https://doi.org/10.1080/23723556.2020.1743807
  19. DNA Repair (Amst). 2020 Apr 30. pii: S1568-7864(20)30109-9. [Epub ahead of print]90 102861
      Non-growing quiescent cells face special challenges when repairing lesions produced by exogenous DNA damaging agents. These challenges include the global repression of transcription and translation and a compacted chromatin structure. We investigated how quiescent yeast cells regulated the repair of DNA lesions produced by UV irradiation. We found that UV lesions were excised and repaired in quiescent cells before their re-entry into S phase, and that lesion repair was correlated with high levels of Rad7, a recognition factor in the global genome repair sub-pathway of nucleotide excision repair (GGR-NER). UV exposure led to an increased frequency of mutations that included C->T transitions and T > A transversions. Mutagenesis was dependent on the error-prone translesion synthesis (TLS) DNA polymerase, Pol zeta, which was the only DNA polymerase present in detectable levels in quiescent cells. Across the genome of quiescent cells, UV-induced mutations showed an association with exons that contained H3K36 or H3K79 trimethylation but not with those bound by RNA polymerase II. Together, the data suggest that the distinct physiological state and chromatin structure of quiescent cells contribute to its regulation of UV damage repair.
    Keywords:  GGR-NER; Polymerase zeta; Quiescent cells; UV mutagenesis
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102861
  20. Nat Metab. 2019 Dec;1(12): 1209-1218
      The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes Parp9, remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam +/- mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.
    DOI:  https://doi.org/10.1038/s42255-019-0150-8
  21. Nat Commun. 2020 May 12. 11(1): 2375
      Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework.
    DOI:  https://doi.org/10.1038/s41467-020-16078-y
  22. Cells. 2020 May 13. pii: E1210. [Epub ahead of print]9(5):
      Drug repurposing appears to offer an attractive alternative in finding new anticancer agents. Their applicability seems to have multiple benefits, among which are the potential of immediate efficacy assessment in clinical trials and the existence of patient safety and tolerability evidence. Nevertheless, their effective application in terms of tumor-type targeting requires accurate knowledge of their exact mechanism of action. In this review, we present such a successful drug, namely Disulfiram (commercially known as Antabuse), and discuss its recently uncovered mode of anticancer action through DNA damage.
    Keywords:  DNA damage; cancer; disulfiram; drug repurposing
    DOI:  https://doi.org/10.3390/cells9051210
  23. Chem Res Toxicol. 2020 May 11.
      The cellular outcomes of chemical exposure are as much about the cellular response to the chemical as it is an effect of the chemical. We are growing in our understanding of the genotoxic interaction between chemistry and biology. For example, recent data has revealed the biological basis for mutation induction curves for a methylating chemical, which has been shown to be dependent on the repair capacity of the cells. However, this is just one endpoint in the toxicity pathway from chemical exposure to cell death. Much remains to be known in order for us to predict how cells will respond to a certain dose. Methylating agents, a subset of alkylating agents, are of particular interest because of the variety of adverse genetic endpoints that can result, not only at increasing doses but also, over time. For instance, methylating agents are mutagenic, their potency for this endpoint, is determined by the cellular repair capacity of an enzyme called methylguanine DNA-methyltransferase (MGMT), for example. However, the adducts can become clastogenic. Erroneous biological processing will convert mutagenic adducts into clastogenic events in the form of double strand breaks (DSBs). How the cell responds to DSBs, via a cascade of protein kinases, called the DNA damage response (DDR), will determine if the damage is repaired effectively, via homologous recombination, or with errors, via non-homologous end joining. Alternatively, the cell dies via apoptosis, or enters senescence. The fate of cells may be determined by the extent of damage and the resulting strength of DDR signalling. Therefore, thresholds of damage may exist that determine cell fate. Such thresholds would be dependent on each of the repair and response mechanisms that these methyl adducts stimulate. The molecular mechanism of how methyl adducts kill cells is still to be fully resolved. If we are able to quantify each of these thresholds of damage, then we can ascertain; of the many adducts that are induced, what proportion of them are mutagenic, what proportion are clastogenic and how many of these clastogenic events are toxic. This review examines the possibility of dose and damage thresholds for an SN1-type methylating agent, from the perspective of the underlying evolutionary mechanisms that may be accountable.
    DOI:  https://doi.org/10.1021/acs.chemrestox.0c00052
  24. Cancer Sci. 2020 May 16.
      MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and non-amplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter and thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the non-amplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.
    Keywords:  MYCN; antifolate; chemotherapy; neuroblastoma; raltitrexed
    DOI:  https://doi.org/10.1111/cas.14485
  25. Cell Rep. 2020 May 12. pii: S2211-1247(20)30585-4. [Epub ahead of print]31(6): 107632
      Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. Here, we show that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. We discover a feedback mechanism involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Our findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer.
    Keywords:  DDR; DNA damage response; Drosophila intestinal stem cell; ISC; MEF; O-GlcNAc transferase; O-GlcNAcase; O-GlcNAcylation; OGA; OGT; mESC; mouse embryonic fibroblast; mouse embryonic stem cell
    DOI:  https://doi.org/10.1016/j.celrep.2020.107632
  26. Front Oncol. 2020 ;10 658
      The bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a mitochondrial one-carbon folate metabolic enzyme whose role in cancer was not known until recently. MTHFD2 is highly expressed in embryos and a wide range of tumors but has low or absent expression in most adult differentiated tissues. Elevated MTHFD2 expression is associated with poor prognosis in both hematological and solid malignancy. Its depletion leads to suppression of multiple malignant phenotypes including proliferation, invasion, migration, and induction of cancer cell death. The non-metabolic functions of this enzyme, especially in cancers, have thus generated considerable research interests. This review summarizes current knowledge on both the metabolic functions and non-enzymatic roles of MTHFD2. Its expression, potential functions, and regulatory mechanism in cancers are highlighted. The development of MTHFD2 inhibitors and their implications in pre-clinical models are also discussed.
    Keywords:  cancer metabolism; epigenetic modification; metabolic enzyme; oncogenicity; one carbon metabolism
    DOI:  https://doi.org/10.3389/fonc.2020.00658