bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒07‒12
forty-eight papers selected by
Sean Rudd
Karolinska Institutet


  1. Cell. 2020 Jul 09. pii: S0092-8674(20)30673-5. [Epub ahead of print]
    Olivieri M, Cho T, Álvarez-Quilón A, Li K, Schellenberg MJ, Zimmermann M, Hustedt N, Rossi SE, Adam S, Melo H, Heijink AM, Sastre-Moreno G, Moatti N, Szilard RK, McEwan A, Ling AK, Serrano-Benitez A, Ubhi T, Feng S, Pawling J, Delgado-Sainz I, Ferguson MW, Dennis JW, Brown GW, Cortés-Ledesma F, Williams RS, Martin A, Xu D, Durocher D.
      The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
    Keywords:  CRISPR; DNA damage; DNA repair; DNA-damaging agents; cancer therapeutics; functional genomics; genome stability; mechanism-of-action
    DOI:  https://doi.org/10.1016/j.cell.2020.05.040
  2. Cell Rep. 2020 Jul 07. pii: S2211-1247(20)30830-5. [Epub ahead of print]32(1): 107849
    Brannvoll A, Xue X, Kwon Y, Kompocholi S, Simonsen AKW, Viswalingam KS, Gonzalez L, Hickson ID, Oestergaard VH, Mankouri HW, Sung P, Lisby M.
      Replication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5'-to-3' helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.
    Keywords:  D-loop dissociation; DNA helicase; FANCD2; FANCJ; FANCM; Fanconi anemia; RAD51; homologous recombination; interstrand DNA crosslink repair; sister-chromatid exchange
    DOI:  https://doi.org/10.1016/j.celrep.2020.107849
  3. Proc Natl Acad Sci U S A. 2020 Jul 10. pii: 202003499. [Epub ahead of print]
    McLaughlin LJ, Stojanovic L, Kogan AA, Rutherford JL, Choi EY, Yen RC, Xia L, Zou Y, Lapidus RG, Baylin SB, Topper MJ, Rassool FV.
      Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.
    Keywords:  DNA methyltransferase inhibitors; Fanconi anemia; homologous recombination deficiency; poly(ADP-ribose) polymerase inhibitors; stimulator of interferon signaling
    DOI:  https://doi.org/10.1073/pnas.2003499117
  4. Nat Commun. 2020 Jul 07. 11(1): 3391
    Hanzlikova H, Prokhorova E, Krejcikova K, Cihlarova Z, Kalasova I, Kubovciak J, Sachova J, Hailstone R, Brazina J, Ghosh S, Cirak S, Gleeson JG, Ahel I, Caldecott KW.
      Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.
    DOI:  https://doi.org/10.1038/s41467-020-17069-9
  5. Mol Cell. 2020 Jul 02. pii: S1097-2765(20)30430-5. [Epub ahead of print]
    Sebastian R, Hosogane EK, Sun EG, Tran AD, Reinhold WC, Burkett S, Sturgill DM, Gudla PR, Pommier Y, Aladjem MI, Oberdoerffer P.
      The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.
    Keywords:  X chromosome; alternative end-joining; alternative splicing; female viability; genome instability; macro-histone; macroH2A1
    DOI:  https://doi.org/10.1016/j.molcel.2020.06.028
  6. Elife. 2020 Jul 06. pii: e57894. [Epub ahead of print]9
    Brunner A, Suryo Rahmanto A, Johansson H, Franco M, Viiliäinen J, Gazi M, Frings O, Fredlund E, Spruck C, Lehtiö J, Rantala JK, Larsson LG, Sangfelt O.
      Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.
    Keywords:  AZD1775; DNA-PK; PTEN; WEE1; basal-like breast cancer; cancer biology; cyclin E; mouse
    DOI:  https://doi.org/10.7554/eLife.57894
  7. Cancer Discov. 2020 Jul 08. pii: CD-19-1008. [Epub ahead of print]
    Aroua N, Boet E, Ghisi M, Nicolau-Travers ML, Saland E, Gwilliam R, de Toni F, Hosseini M, Mouchel PL, Farge T, Bosc C, Stuani L, Sabatier M, Mazed F, Larrue C, Jarrou L, Gandarillas S, Bardotti M, Picard M, Syrykh C, Laurent C, Gotanegre M, Bonnefoy N, Bellvert F, Portais JC, Nicot N, Azuaje F, Kaoma T, Joffre C, Tamburini J, Recher C, Vergez F, Sarry JE.
      Relapses driven by chemoresistant leukemic cell populations are the main cause of mortality for patients with acute myeloid leukemia (AML). Here, we show that the ectonucleotidase CD39 (ENTPD1) is upregulated in cytarabine (AraC)-resistant leukemic cells from both AML cell lines and patient samples in vivo and in vitro. CD39 cell surface expression and activity is increased in AML patients upon chemotherapy compared to diagnosis and enrichment in CD39-expressing blasts is a marker of adverse prognosis in the clinics. High CD39 activity promotes AraC resistance by enhancing mitochondrial activity and biogenesis through activation of a cAMP-mediated adaptive mitochondrial stress response. Finally, genetic and pharmacological inhibition of CD39 eATPase activity blocks the mitochondrial reprogramming triggered by AraC treatment and markedly enhances its cytotoxicity in AML cells in vitro and in vivo. Together, these results reveal CD39 as a new residual disease marker and a promising therapeutic target to improve chemotherapy response in AML.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1008
  8. Cell Death Dis. 2020 Jul 06. 11(7): 507
    Huang N, Xu C, Deng L, Li X, Bian Z, Zhang Y, Long S, Chen Y, Zhen N, Li G, Sun F.
      Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.
    DOI:  https://doi.org/10.1038/s41419-020-2708-5
  9. Curr Genet. 2020 Jul 04.
    Wang Z, Xiao W.
      Certain replication-blocking lesions can escape DNA repair and must be bypassed to prevent fork collapse and cell death. Budding yeast DNA-damage tolerance consists of translesion DNA synthesis (TLS) and template switch. TLS utilizes specialized DNA polymerases to insert nucleotides opposite the damage site, followed by extension, allowing continual replication in the presence of lesions on the template DNA. Meanwhile, Rev1 is additionally required for the subsequent extension step of TLS regardless of the initial insertion polymerase utilized. Here we assess relative contributions of two Y-family TLS polymerases, Rev1 and Polη, in bypassing lesions induced by various types of DNA-damaging agents. Our experimental results collectively indicate that yeast cells preferentially utilize relatively error-free TLS polymerase(s) to bypass given lesions, and that the mutagenic TLS polymerase may serve as a backup. Interestingly, if Polη is unable to serve as a TLS polymerase under certain circumstances, it may be counter-active. The cooperation among TLS polymerases may strike a balance between survival and stress-induced mutagenesis. These observations indicate that specialized Y-family DNA polymerases have evolved to deal with different types of environmental genotoxic stresses.
    Keywords:  DNA-damage tolerance; Polη; Rev1; Translesion DNA synthesis; Yeast
    DOI:  https://doi.org/10.1007/s00294-020-01092-w
  10. Cancer Sci. 2020 Jul 08.
    Hu Y, Guo M.
      Cancer cells are often characterized by abnormalities in DNA damage response including defects in cell cycle checkpoints and/or DNA repair. Synthetic Lethality between DNA damage repair (DDR) pathways has provided a paradigm for cancer therapy by targeting DDR. The successful example is that cancer cells with BRCA1/2 mutations are sensitized to poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors. Beyond the narrow scope of defects in the BRCA pathway, 'BRCAness' provides more opportunities for synthetic lethality strategy. In human pancreatic cancer, frequent mutations were found in cell cycle and DDR genes, including P16, P73, APC, MLH1, ATM, PALB2 and MGMT. Combined DDR inhibitors and chemotherapeutic agents are under preclinical or clinical trials. Promoter region methylation was found frequently in cell cycle and DDR genes. Epigenetics joins the Knudson's 'two hit' theory and 'BRCAness'. Aberrant epigenetic changes in cell cycle or DDR regulators may serve as a new avenue for synthetic Lethality strategy in pancreatic cancer.
    Keywords:  DNA damage repair; cell cycle; epigenetics; pancreatic cancer; synthetic lethality
    DOI:  https://doi.org/10.1111/cas.14565
  11. DNA Repair (Amst). 2020 Jun 25. pii: S1568-7864(20)30122-1. [Epub ahead of print]94 102874
    Wang XS, Lee BJ, Zha S.
      Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.
    Keywords:  ATM; Class switch recombination; DNA-PK; Non-homologous end-joining; V(D)J recombination
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102874
  12. DNA Repair (Amst). 2020 Jun 23. pii: S1568-7864(20)30123-3. [Epub ahead of print]94 102875
    Stroik S, Hendrickson EA.
      Telomeres consist of repetitive tracts of DNA that shield a chromosome's contents from erosion and replicative attrition. However, telomeres are also late-replicating regions of the genome in which a myriad of replicative obstructions reside. The obstacles contained within telomeres, as well as their genomic location, drive replicative stalling and subsequent fork collapse in these regions. Consequently, large scale deletions, under-replicated DNA, translocations, and fusion events arise following telomere replication failure. Further, under-replicated DNA and telomere fusions that are permitted to enter mitosis will produce mitotic DNA bridges - known drivers of genetic loss and chromothripsis. Thus, aberrant telomere replication promotes genomic instability, which, in turn leads either to cellular death, senescence or oncogenic transformation. The importance of these issues for organismal well-being necessitates a need for resolute telomere maintenance. Here, we describe recent advances in identifying and understanding the molecular mechanisms that are in place in human cells to escort the replisome through the telomere's unwieldy structures and repetitive sequences. Finally, we review the pathways that combat the deleterious outcomes that occur when telomeric replication forks do collapse.
    Keywords:  Chromatin bridges; Homology-dependent repair; Replication; Restart; Stalled forks; Telomere
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102875
  13. Cancers (Basel). 2020 Jul 07. pii: E1821. [Epub ahead of print]12(7):
    Dang TT, Morales JC.
      It was recently shown that the 5' to 3' exoribonuclease XRN2 is involved in the DNA damage response. Importantly, loss of XRN2 abrogates DNA double stranded break repair via the non-homologous end-joining pathway. However, the mechanistic details of how XRN2 functions in the non-homologous end-joining repair process are unknown. In this study, we elucidated that XRN2-mediated RNA:DNA hybrid resolution is required to allow Ku70 binding to DNA ends. These data suggest that XRN2 is required for the initiation of non-homologous end-joining repair. Interestingly, we uncovered a role for XRN2 in the homologous recombination repair pathway. Loss of XRN2 lead to a decrease in the repair of double strand breaks by homologous recombination. Strikingly, when we removed RNA:DNA hybrids by RNaseH1 over-expression, homologous recombination was not restored. We found RNA:DNA hybrid formation at and downstream of the DSB site, suggesting that unregulated transcription inhibits homologous recombination repair. In summary, our results indicate a relation between RNA:DNA hybrid resolution and double strand break repair pathway choice.
    Keywords:  RNA:DNA hybrids; homologous recombination; non-homologous end-joining
    DOI:  https://doi.org/10.3390/cancers12071821
  14. Cancers (Basel). 2020 Jul 08. pii: E1838. [Epub ahead of print]12(7):
    Szatkowska M, Krupa R.
      Ionizing radiation may be of both artificial and natural origin and causes cellular damage in living organisms. Radioactive isotopes have been used significantly in cancer therapy for many years. The formation of DNA double-strand breaks (DSBs) is the most dangerous effect of ionizing radiation on the cellular level. After irradiation, cells activate a DNA damage response, the molecular path that determines the fate of the cell. As an important element of this, homologous recombination repair is a crucial pathway for the error-free repair of DNA lesions. All components of DNA damage response are regulated by specific microRNAs. MicroRNAs are single-stranded short noncoding RNAs of 20-25 nt in length. They are directly involved in the regulation of gene expression by repressing translation or by cleaving target mRNA. In the present review, we analyze the biological mechanisms by which miRNAs regulate cell response to ionizing radiation-induced double-stranded breaks with an emphasis on DNA repair by homologous recombination, and its main component, the RAD51 recombinase. On the other hand, we discuss the ability of DNA damage response proteins to launch particular miRNA expression and modulate the course of this process. A full understanding of cell response processes to radiation-induced DNA damage will allow us to develop new and more effective methods of ionizing radiation therapy for cancers, and may help to develop methods for preventing the harmful effects of ionizing radiation on healthy organisms.
    Keywords:  DNA damage response; cancer therapy; double-strand DNA breaks; ionizing radiation; microRNA
    DOI:  https://doi.org/10.3390/cancers12071838
  15. Cell Rep. 2020 Jul 07. pii: S2211-1247(20)30831-7. [Epub ahead of print]32(1): 107850
    Socha A, Yang D, Bulsiewicz A, Yaprianto K, Kupculak M, Liang CC, Hadjicharalambous A, Wu R, Gygi SP, Cohn MA.
      The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs). Many FA proteins are recruited to ICLs in a timely fashion so that coordinated repair can occur. However, the mechanism of this process is poorly understood. Here, we report the purification of a FANCD2-containing protein complex with multiple subunits, including WRNIP1. Using live-cell imaging, we show that WRNIP1 is recruited to ICLs quickly after their appearance, promoting repair. The observed recruitment facilitates subsequent recruitment of the FANCD2/FANCI complex. Depletion of WRNIP1 sensitizes cells to ICL-forming drugs. We find that ubiquitination of WRNIP1 and the activity of its UBZ domain are required to facilitate recruitment of FANCD2/FANCI and promote repair. Altogether, we describe a mechanism by which WRNIP1 is recruited rapidly to ICLs, resulting in chromatin loading of the FANCD2/FANCI complex in an unusual process entailing ubiquitination of WRNIP1 and the activity of its integral UBZ domain.
    Keywords:  DNA repair; FANCD2/FANCI; Fanconi anemia; ICL repair; WRNIP1; cancer; genome stability; interstrand crosslink repair; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2020.107850
  16. DNA Repair (Amst). 2020 Jun 27. pii: S1568-7864(20)30150-6. [Epub ahead of print]94 102902
    Checa-Rodríguez C, Cepeda-García C, Ramón J, López-Saavedra A, Balestra FR, Domínguez-Sánchez MS, Gómez-Cabello D, Huertas P.
      Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.
    Keywords:  DNA end resection; DNA repair; KLF4; PRMT5; Recombination
    DOI:  https://doi.org/10.1016/j.dnarep.2020.102902
  17. Cancers (Basel). 2020 Jul 04. pii: E1793. [Epub ahead of print]12(7):
    Ambrosio S, Majello B.
      In recent years, a considerable correlation has emerged between autophagy and genome integrity. A range of mechanisms appear to be involved where autophagy participates in preventing genomic instability, as well as in DNA damage response and cell fate decision. These initial findings have attracted particular attention in the context of malignancy; however, the crosstalk between autophagy and DNA damage response is just beginning to be explored and key questions remain that need to be addressed, to move this area of research forward and illuminate the overall consequence of targeting this process in human therapies. Here we present current knowledge on the complex crosstalk between autophagy and genome integrity and discuss its implications for cancer cell survival and response to therapy.
    Keywords:  DNA damage repair; DNA damage response; autophagy; cancer therapy
    DOI:  https://doi.org/10.3390/cancers12071793
  18. Essays Biochem. 2020 Jul 10. pii: EBC20200013. [Epub ahead of print]
    Grundy GJ, Parsons JL.
      Base excision repair (BER) has evolved to preserve the integrity of DNA following cellular oxidative stress and in response to exogenous insults. The pathway is a coordinated, sequential process involving 30 proteins or more in which single strand breaks are generated as intermediates during the repair process. While deficiencies in BER activity can lead to high mutation rates and tumorigenesis, cancer cells often rely on increased BER activity to tolerate oxidative stress. Targeting BER has been an attractive strategy to overwhelm cancer cells with DNA damage, improve the efficacy of radiotherapy and/or chemotherapy, or form part of a lethal combination with a cancer specific mutation/loss of function. We provide an update on the progress of inhibitors to enzymes involved in BER, and some of the challenges faced with targeting the BER pathway.
    Keywords:  Base excision repair; Cancer therapy; DNA damage; DNA repair
    DOI:  https://doi.org/10.1042/EBC20200013
  19. ACS Omega. 2020 Jun 30. 5(25): 15317-15324
    Perera L, Beard WA, Pedersen LG, Shock DD, Wilson SH.
      DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase β chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pβ and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.
    DOI:  https://doi.org/10.1021/acsomega.0c01345
  20. Cancers (Basel). 2020 Jul 06. pii: E1813. [Epub ahead of print]12(7):
    Engbrecht M, Mangerich A.
      The nucleolus has been known for a long time to fulfill crucial functions in ribosome biogenesis, of which cancer cells can become addicted to in order to produce sufficient amounts of proteins for cell proliferation. Recently, the nucleolus has emerged as a central regulatory hub in many other cancer-relevant processes, including stress sensing, DNA damage response, cell cycle control, and proteostasis. This fostered the idea that nucleolar processes can be exploited in cancer therapy. Interestingly, a significant proportion of poly(ADP-ribose) polymerase 1 (PARP1) molecules are localized in the nucleolus and PARP1 also plays crucial roles in many processes that are important in cancer biology, including genome maintenance, replication, transcription, and chromatin remodeling. Furthermore, during the last years, PARP1 came into focus in oncology since it represents a promising target of pharmacological PARP inhibitors in various types of cancers. Here, we provide an overview of our current understanding on the role of PARP1 in nucleolar functions and discuss potential implications in cancer biology and therapy.
    Keywords:  ARTDs; PARP; cancer; nucleolus; poly(ADP-ribosyl)ation
    DOI:  https://doi.org/10.3390/cancers12071813
  21. Cancer Cell Int. 2020 ;20 280
    Conroy LR, Lorkiewicz P, He L, Yin X, Zhang X, Rai SN, Clem BF.
      Background: Aberrant activity of cell cycle proteins is one of the key somatic events in non-small cell lung cancer (NSCLC) pathogenesis. In most NSCLC cases, the retinoblastoma protein tumor suppressor (RB) becomes inactivated via constitutive phosphorylation by cyclin dependent kinase (CDK) 4/6, leading to uncontrolled cell proliferation. Palbociclib, a small molecule inhibitor of CDK4/6, has shown anti-tumor activity in vitro and in vivo, with recent studies demonstrating a functional role for palbociclib in reprogramming cellular metabolism. While palbociclib has shown efficacy in preclinical models of NSCLC, the metabolic consequences of CDK4/6 inhibition in this context are largely unknown.Methods: In our study, we used a combination of stable isotope resolved metabolomics using [U-13C]-glucose and multiple in vitro metabolic assays, to interrogate the metabolic perturbations induced by palbociclib in A549 lung adenocarcinoma cells. Specifically, we assessed changes in glycolytic activity, the pentose phosphate pathway (PPP), and glutamine utilization. We performed these studies following palbociclib treatment with simultaneous silencing of RB1 to define the pRB-dependent changes in metabolism.
    Results: Our studies revealed palbociclib does not affect glycolytic activity in A549 cells but decreases glucose metabolism through the PPP. This is in part via reducing activity of glucose 6-phosphate dehydrogenase, the rate limiting enzyme in the PPP. Additionally, palbociclib enhances glutaminolysis to maintain mitochondrial respiration and sensitizes A549 cells to the glutaminase inhibitor, CB-839. Notably, the effects of palbociclib on both the PPP and glutamine utilization occur in an RB-dependent manner.
    Conclusions: Together, our data define the metabolic impact of palbociclib treatment in A549 cells and may support the targeting CDK4/6 inhibition in combination with glutaminase inhibitors in NSCLC patients with RB-proficient tumors.
    Keywords:  Glutaminolysis; Lung cancer; Metabolism; PPP; Palbociclib; RB
    DOI:  https://doi.org/10.1186/s12935-020-01357-x
  22. Oncogene. 2020 Jul 09.
    Nair J, Huang TT, Murai J, Haynes B, Steeg PS, Pommier Y, Lee JM.
      High grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy in BRCA wild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1's regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.
    DOI:  https://doi.org/10.1038/s41388-020-1383-4
  23. Essays Biochem. 2020 Jul 10. pii: EBC20200007. [Epub ahead of print]
    Xu Y, Xu D.
      Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3' single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3' ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3' tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11-RAD50-NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.
    Keywords:  53BP1; BIR; BRCA1; PTIP; RIF1; shieldin
    DOI:  https://doi.org/10.1042/EBC20200007
  24. Mol Cell. 2020 Jul 01. pii: S1097-2765(20)30429-9. [Epub ahead of print]
    Huang J, Zhou Q, Gao M, Nowsheen S, Zhao F, Kim W, Zhu Q, Kojima Y, Yin P, Zhang Y, Guo G, Tu X, Deng M, Luo K, Qin B, Machida Y, Lou Z.
      DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequences of SPRTN deubiquitination remain unclear. Here we report that, in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. Deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model where two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and a healthy lifespan.
    Keywords:  DNA repair; DNA-protein crosslink; SPRTN; Top1cc; VCPIP1/VCIP135; acetylation; aging; genomic instability; metalloprotease; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2020.06.027
  25. Elife. 2020 Jul 09. pii: e56008. [Epub ahead of print]9
    Zhang W, Chen Y, Yang J, Zhang J, Yu J, Wang M, Zhao X, Wei K, Wan X, Xu X, Jiang Y, Chen J, Gao S, Mao Z.
      Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.
    Keywords:  cell biology; genetics; genomics; human; mouse
    DOI:  https://doi.org/10.7554/eLife.56008
  26. Nucleic Acids Res. 2020 Jul 07. pii: gkaa580. [Epub ahead of print]
    Bezalel-Buch R, Cheun YK, Roy U, Schärer OD, Burgers PM.
      DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.
    DOI:  https://doi.org/10.1093/nar/gkaa580
  27. Cancer Res. 2020 Jul 10. pii: canres.1672.2020. [Epub ahead of print]
    Patterson-Fortin J, D'Andrea AD.
      Repair of DNA double strand breaks (DSB) is performed by two major pathways: homology-dependent repair and classical non-homologous end joining. Recent studies have identified a third pathway: microhomology-mediated end-joining (MMEJ). MMEJ has similarities to homology-dependent repair in that repair is initiated with end resection, leading to single-stranded 3' ends which require microhomology upstream and downstream of the DSB. Importantly, the MMEJ pathway is commonly upregulated in cancers, especially in homologous recombination-deficient cancers which display a distinctive mutational signature. Here we review the molecular process of MMEJ as well as new targets and approaches exploiting the MMEJ pathway in cancer therapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1672
  28. FEBS J. 2020 Jul 07.
    Park S, Karatayeva N, Demin AA, Munashingha PR, Seo YS.
      Dna2 can efficiently process 5' flaps containing DNA secondary structure using coordinated action of the three biochemical activities; the N-terminally encoded DNA-binding activity and the C-terminally encoded endonuclease and helicase activities. In this study, we investigated the crosstalk among the three functional domains using a variety of dna2 mutant alleles and enzymes derived thereof. We found that disruption of the catalytic activities of Dna2 activated Dna2-dependent checkpoint, residing in the N-terminal domain. This checkpoint activity contributed to growth defects of dna2 catalytic mutants, revealing the presence of an intramolecular functional crosstalk in Dna2. The N-terminal domain of Dna2 bound specifically to substrates that mimic DNA replication fork intermediates, including Holliday junctions. Using site-directed mutagenesis of the N-terminal domain of Dna2, we discovered that five consecutive basic amino acid residues were essential for the ability of Dna2 to bind hairpin DNA in vitro. Mutant cells expressing the dna2 allele containing all five basic residues substituted with alanine displayed three distinct phenotypes: (i) temperature-sensitive growth defects, (ii) bypass of S phase arrest, and (iii) increased sensitivity to DNA-damaging agents. Taken together, our results indicate that the interplay between the N-terminal regulatory and C-terminal catalytic domains of Dna2 plays an important role in vivo, especially when cells are placed under replication stress.
    Keywords:  DNA binding protein; DNA replication; DNA secondary structure; Dna2; checkpoint control
    DOI:  https://doi.org/10.1111/febs.15475
  29. Cell Death Dis. 2020 Jul 09. 11(7): 519
    Liu K, Wang Y, Zhu Q, Li P, Chen J, Tang Z, Shen Y, Cheng X, Lu LY, Liu Y.
      HORMAD1 is a meiosis-specific protein that promotes synapsis and recombination of homologous chromosomes in meiotic prophase. Originally identified as a cancer/testis antigen, HORMAD1 is also aberrantly expressed in several cancers. However, the functions of HORMAD1 in cancer cells are still not clear. Here, we show that HORMAD1 is aberrantly expressed in a wide variety of cancers and compromises DNA mismatch repair in cancer cells. Mechanistically, HORMAD1 interacts with MCM8-MCM9 complex and prevents its efficient nuclear localization. As a consequence, HORMAD1-expressing cancer cells have reduced MLH1 chromatin binding and DNA mismatch repair defects. Consistently, HORMAD1 expression is associated with increased mutation load and genomic instability in many cancers. Taken together, our study provides mechanistic insights into HORMAD1's functions in cancer cells, which can potentially be exploited for targeted therapy of HORMAD1-expressing cancers.
    DOI:  https://doi.org/10.1038/s41419-020-2736-1
  30. Free Radic Biol Med. 2020 Jul 07. pii: S0891-5849(20)31107-2. [Epub ahead of print]
    Bao Y, Tong L, Song B, Liu G, Zhu Q, Lu X, Zhang J, Lu Y, Wen H, Tian Y, Sun Y, Zhu WG.
      Cancer therapeutics produce reactive oxygen species (ROS) that damage the cancer genome and lead to cell death. However, cancer cells can resist ROS-induced cytotoxicity and survive. We show that nuclear-localized uracil-DNA N-glycosylase isoform 2 (UNG2) has a critical role in preventing ROS-induced DNA damage and enabling cancer-cell resistance. Under physiological conditions, UNG2 is targeted for rapid degradation via an interaction with the E3 ligase UHRF1. In response to ROS, however, UNG2 protein in cancer cells exhibits a remarkably extended half-life. Upon ROS exposure, UNG2 is deacetylated at lysine 78 by histone deacetylases, which prevents the UNG2-UHRF1 interaction. Accumulated UNG2 protein can thus excise the base damaged by ROS and enable the cell to survive these otherwise toxic conditions. Consequently, combining HDAC inhibitors (to permit UNG2 degradation) with genotoxic agents (to produce cytotoxic cellular levels of ROS) leads to a robust synergistic killing effect in cancer cells in vitro. Altogether, these data support the application of a novel approach to cancer treatment based on promoting UNG2 degradation by altering its acetylation status using an HDAC inhibitor.
    Keywords:  HDAC inhibitor; Oxidative DNA damage; ROS; UHRF1; UNG2
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.06.010
  31. Chem Res Toxicol. 2020 Jul 08.
    Yeom M, Hong JK, Kim JK, Guengerich FP, Choi JY.
      Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of ten human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1-445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants (kcat/Km) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H2O2 in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H2O2 sensitivity in POLI-deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H2O2 sensitivity in POLI-deficient cells, while the R209Q, K228I, and Q386R variants-all impaired in polymerase and dRP lyase activity-failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.
    DOI:  https://doi.org/10.1021/acs.chemrestox.0c00127
  32. Cancers (Basel). 2020 Jul 02. pii: E1769. [Epub ahead of print]12(7):
    Varanda AB, Martins-Logrado A, Ferreira MG, Fior R.
      Poly (ADP-ribose) polymerase (PARP) inhibition in BRCA-mutated cells results in an incapacity to repair DNA damage, leading to cell death caused by synthetic lethality. Within the treatment options for advanced triple negative breast cancer, the PARP inhibitor olaparib is only given to patients with BRCA1/2 mutations. However, these patients may show resistance to this drug and BRCA1/2 wild-type tumors can show a striking sensitivity, making BRCA status a poor biomarker for treatment choice. Aiming to investigate if the zebrafish model can discriminate sensitivities to olaparib, we developed zebrafish xenografts with different BRCA status and measured tumor response to treatment, as well as its impact on angiogenesis and metastasis. When challenged with olaparib, xenografts revealed sensitivity phenotypes independent of BRCA. Moreover, its combination with ionizing radiation increased the cytotoxic effects, showing potential as a combinatorial regimen. In conclusion, we show that the zebrafish xenograft model may be used as a sensitivity profiling platform for olaparib in monotherapy or in combinatorial regimens. Hence, this model presents as a promising option for the future establishment of patient-derived xenografts for personalized medicine approaches beyond BRCA status.
    Keywords:  BRCA1/2; PARP; breast cancer; ionizing radiation; olaparib; radiotherapy; xenografts; zebrafish
    DOI:  https://doi.org/10.3390/cancers12071769
  33. J Biol Chem. 2020 Jul 09. pii: jbc.RA120.013547. [Epub ahead of print]
    Howard MJ, Horton JK, Zhao ML, Wilson SH.
      DNA polymerase (pol) b catalyzes two reactions at DNA gaps generated during base excision repair; gap-filling DNA synthesis and lyase-dependent 5' end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol b for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of wild-type pol b, pol l and pol μ, and several variants of pol b, for 1-nt gap containing and undamaged DNA.  Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol b, 35, 68 and 72, to alanine (pol b K∆3A), increased the binding affinity for non-specific DNA ~11-fold as compared to wild-type.  Wild-type pol μ, lacking homologous lysines, displayed similar non-specific DNA binding behavior as pol b K∆3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol b K∆3A to localize to sites of laser-induced DNA damage was strongly decreased, as compared to wild-type pol b.  These data suggest that the three lysines in the lyase active site destabilize pol b when bound to DNA non-specifically, promoting DNA scanning and providing binding specificity for gapped DNA.
    Keywords:  DNA binding protein; DNA damage; DNA polymerase; DNA-protein interaction; base excision repair (BER)
    DOI:  https://doi.org/10.1074/jbc.RA120.013547
  34. Cells. 2020 Jul 03. pii: E1612. [Epub ahead of print]9(7):
    Campos-Contreras ADR, Díaz-Muñoz M, Vázquez-Cuevas FG.
      Cancer is a complex expression of an altered state of cellular differentiation associated with severe clinical repercussions. The effort to characterize this pathological entity to understand its underlying mechanisms and visualize potential therapeutic strategies has been constant. In this context, some cellular (enhanced duplication, immunological evasion), metabolic (aerobic glycolysis, failure in DNA repair mechanisms) and physiological (circadian disruption) parameters have been considered as cancer hallmarks. The list of these hallmarks has been growing in recent years, since it has been demonstrated that various physiological systems misfunction in well-characterized ways upon the onset and establishment of the carcinogenic process. This is the case with the purinergic system, a signaling pathway formed by nucleotides/nucleosides (mainly adenosine triphosphate (ATP), adenosine (ADO) and uridine triphosphate (UTP)) with their corresponding membrane receptors and defined transduction mechanisms. The dynamic equilibrium between ATP and ADO, which is accomplished by the presence and regulation of a set of ectonucleotidases, defines the pro-carcinogenic or anti-cancerous final outline in tumors and cancer cell lines. So far, the purinergic system has been recognized as a potential therapeutic target in cancerous and tumoral ailments.
    Keywords:  ATP; adenosine; cancer; ectonucleotidase; immune evasion in cancer; purinergic receptors; purinergic signaling; tumor microenvironment
    DOI:  https://doi.org/10.3390/cells9071612
  35. Front Pharmacol. 2020 ;11 897
    De Mattia E, Roncato R, Palazzari E, Toffoli G, Cecchin E.
      Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the standard of care for patients with Locally Advanced Rectal Cancer (LARC). Current selection for nCRT is based on clinical criteria regardless of any molecular marker. Pharmacogenomics may be a useful strategy to personalize and optimize nCRT in LARC. This review aims to summarize the most recent and relevant findings about the role of germline and somatic pharmacogenomics in the prediction of nCRT outcome in patients with LARC, discussing the state of the art of their application in the clinical practice. A systematic literature search of the PubMed database was completed to identify relevant English-language papers published up to January 2020. The chemotherapeutic backbone of nCRT is represented by fluoropyrimidines, mainly metabolized by DPD (Dihydro-Pyrimidine Dehydrogenase, DPYD). The clinical impact of testing DPYD*2A, DPYD*13, c.2846A > T and c.1236G > A-HapB3 before a fluoropyrimidines administration to increase treatment safety is widely acknowledged. Other relevant target genes are TYMS (Thymidylate Synthase) and MTHFR (Methylene-Tetrahydro-Folate Reductase), whose polymorphisms were mainly studied as potential markers of treatment efficacy in LARC. A pivotal role of a TYMS polymorphism in the gene promoter region (rs34743033) was reported and was pioneeringly used to guide nCRT treatment in a phase II study. The pharmacogenomic analysis of other pathways mostly involved in the cellular response to radiation damage, as the DNA repair and the activation of the inflammatory cascade, provided less consistent results. A high rate of somatic mutation in genes belonging to PI3K (Phosphatidyl-Inositol 3-Kinase) and MAPK (Mitogen-Activated Protein Kinase) pathways, as BRAF (V-raf murine sarcoma viral oncogene homolog B1), KRAS (Kirsten Rat Sarcoma viral oncogene homolog), NRAS (Neuroblastoma RAS viral (v-ras) oncogene homolog), PIK3CA (Phosphatidyl-Inositol-4,5-bisphosphate 3-Kinase, Catalytic Subunit Alpha), as well as TP53 (Tumor Protein 53) was reported in LARC. Their pharmacogenomic role, already defined in colorectal cancer, is under investigation in LARC with promising results concerning specific somatic mutations in KRAS and TP53, as predictors of tumor response and prognosis. The availability of circulating tumor DNA in plasma may also represent an opportunity to monitor somatic mutations in course of therapy.
    Keywords:  germline; mutation; neo-adjuvant chemoradiotherapy; pharmacogenomics; polymorphism; rectal cancer; somatic
    DOI:  https://doi.org/10.3389/fphar.2020.00897
  36. Pharmazie. 2020 Jul 01. 75(7): 329-334
    Takano M, Higashi M, Baba S, Kawami M, Yumoto R.
      The anticancer effect of ribavirin, a purine nucleoside analogue, has been studied using cultured cancer cells such as the human myelogenous leukemia cell line K562. In order to exert its pharmacological effect, ribavirin has to enter cancer cells. However, there is little information concerning the transport mechanism of ribavirin into K562 cells. In this study, therefore, we examined the uptake mechanism of ribavirin in K562 cells. The uptake of ribavirin in K562 cells was time- and temperature-dependent, and was saturable with a Km value of 1.5 mM. Ribavirin uptake was inhibited by nucleosides such as adenosine and uridine, and by inhibitors of equilibrative nucleoside transporter 1 (ENT1) such as S-(4-nitrobenzyl)-6-thioinosine and dipyridamole in a concentration-dependent manner. In addition, the expression of ENT1 mRNA in K562 cells was confirmed by real-time PCR. On the other hand, Na+-dependence of ribavirin uptake was not observed, suggesting the involvement of ENT1, but not Na+-dependent concentrative nucleoside transporters, in ribavirin uptake in K562 cells. Treatment of K562 cells with sodium butyrate induced erythroid differentiation, but ribavirin uptake activity and sensitivity of the uptake to various inhibitors were not different between native and differentiated K562 cells. These results suggest that ribavirin uptake into K562 cells is mainly mediated by ENT1, which may have a pivotal role in anticancer effect of ribavirin.
    DOI:  https://doi.org/10.1691/ph.2020.0440
  37. Leukemia. 2020 Jul 10.
    Neuse CJ, Lomas OC, Schliemann C, Shen YJ, Manier S, Bustoros M, Ghobrial IM.
      Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by clonal proliferation of plasma cells and a heterogenous genomic landscape. Copy number and structural changes due to chromosomal instability (CIN) are common features of MM. In this review, we describe how primary and secondary genetic events caused by CIN can contribute to increased instability across the genome of malignant plasma cells; with a focus on specific driver genomic events, and how they interfere with cell-cycle checkpoints, to prompt accelerated proliferation. We also provide insight into other forms of CIN, such as chromothripsis and chromoplexy. We evaluate how the tumor microenvironment can contribute to a further increase in chromosomal instability in myeloma cells. Lastly, we highlight the role of certain mutational signatures in leading to high mutation rate and genome instability in certain MM patients. We suggest that assessing CIN in MM and its precursors states may help improve predicting the risk of progression to symptomatic disease and relapse and identifying future therapeutic targets.
    DOI:  https://doi.org/10.1038/s41375-020-0921-y
  38. Elife. 2020 Jul 10. pii: e56782. [Epub ahead of print]9
    Lau AN, Li Z, Danai LV, Westermark AM, Darnell AM, Ferreira R, Gocheva V, Sivanand S, Lien EC, Sapp KM, Mayers JR, Biffi G, Chin CR, Davidson SM, Tuveson DA, Jacks T, Matheson NJ, Yilmaz O, Vander Heiden MG.
      Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.
    Keywords:  cancer biology; mouse
    DOI:  https://doi.org/10.7554/eLife.56782
  39. Pharmaceutics. 2020 Jul 04. pii: E627. [Epub ahead of print]12(7):
    D'Errico S, Falanga AP, Capasso D, Di Gaetano S, Marzano M, Terracciano M, Roviello GN, Piccialli G, Oliviero G, Borbone N.
      Herein, we reported on the synthesis of a novel Pt(II) neutral complex having as ligand the nucleoside tubercidin, a potent anti-tumor agent extracted from the bacterium Streptomyces Tubercidicus. In detail, the chelation of the metal by a diamine linker installed at C6 purine position of tubercidin assured the introduction of a cisplatin-like unit in the molecular scaffold. The behavior of the synthesized complex with a double-strand DNA model was monitored by CD spectroscopy and compared with that of cisplatin and tubercidin. In addition, the cell viability was evaluated against HeLa, A375 and WM266 human cancer cell lines using the MTT test. Lastly, the results of the apoptotic assay (FITC Annexin V) performed on the HeLa cancer cell line are also reported.
    Keywords:  CD spectroscopy; DNA; ESI-MS stability studies; apoptosis; cancer; cell viability assay; cisplatin; nucleosides; platinum complexes; tubercidin
    DOI:  https://doi.org/10.3390/pharmaceutics12070627
  40. Nature. 2020 Jul 08.
    Martínez-Reyes I, Cardona LR, Kong H, Vasan K, McElroy GS, Werner M, Kihshen H, Reczek CR, Weinberg SE, Gao P, Steinert EM, Piseaux R, Budinger GRS, Chandel NS.
      The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.
    DOI:  https://doi.org/10.1038/s41586-020-2475-6
  41. Int J Cancer. 2020 Jul 07.
    Roider T, Wang X, Hüttl K, Müller-Tidow C, Klapper W, Rosenwald A, Stewart JP, Gonzalez de Castro D, Dreger P, Hermine O, Kluin-Nelemans HC, Grabe N, Dreyling M, Pott C, Ott G, Hoster E, Dietrich S.
      The sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) has been demonstrated to predict the response to high-dose cytarabine consolidation treatment in acute myeloid leukemia patients. Here, we evaluated SAMHD1 as potential biomarker for the response to high-dose cytarabine in mantle cell lymphoma (MCL) patients. We quantified SAMHD1 protein expression and determined the mutation status in patients of the MCL Younger and Elderly trials (n = 189), who had received high-dose cytarabine- or fludarabine-based polychemotherapy. Additionally, we quantified SAMHD1 expression in B cell lymphoma cell lines and exposed them to cytarabine, fludarabine, and clinically relevant combinations. Across both trials investigated, SAMHD1 mutations had a frequency of 7.1 % (n = 13) and did not significantly affect the failure-free survival (FFS, p = 0.47). In patients treated with high-dose cytarabine- or fludarabine-containing regimes, SAMHD1 expression was not significantly associated with FFS or complete remission rate. SAMHD1 expression in B cell lymphoma cell lines, however, inversely correlated with their in vitro response to cytarabine as single agent (R = 0.65, p = 0.0065). This correlation could be reversed by combining cytarabine with other chemotherapeutics, such as oxaliplatin and vincristine, similar to the treatment regime of the MCL Younger trial. We conclude that this might explain why we did not observe a significant association between SAMHD1 protein expression and the outcome of MCL patients upon cytarabine-based treatment. This article is protected by copyright. All rights reserved.
    Keywords:  B cell lymphoma; Mantle cell lymphoma; SAMHD1; cytarabine; resistance
    DOI:  https://doi.org/10.1002/ijc.33202
  42. Cell Metab. 2020 Jul 02. pii: S1550-4131(20)30311-9. [Epub ahead of print]
    Kelly B, Pearce EL.
      Amino acids are fundamental building blocks supporting life. Their role in protein synthesis is well defined, but they contribute to a host of other intracellular metabolic pathways, including ATP generation, nucleotide synthesis, and redox balance, to support cellular and organismal function. Immune cells critically depend on such pathways to acquire energy and biomass and to reprogram their metabolism upon activation to support growth, proliferation, and effector functions. Amino acid metabolism plays a key role in this metabolic rewiring, and it supports various immune cell functions beyond increased protein synthesis. Here, we review the mechanisms by which amino acid metabolism promotes immune cell function, and how these processes could be targeted to improve immunity in pathological conditions.
    Keywords:  ▪▪▪
    DOI:  https://doi.org/10.1016/j.cmet.2020.06.010
  43. Int J Med Sci. 2020 ;17(10): 1375-1384
    Xu L, Jiao J, Sun X, Sang W, Gao X, Yang P, Yan D, Song X, Sun C, Liu M, Qin Y, Tian Y, Zhu F, Zeng L, Li Z, Xu K.
      Cladribine is a purine nucleoside analog used to treat B-cell chronic lymphocytic leukemia and hairy cell leukemia, also functions as an inhibitor of DNA synthesis to block the repair of the damaged DNA. The therapeutic role of cladribine against diffuse large B-cell lymphoma cells (DLBCL) is still undefined. In the present study, we demonstrated that cladribine inhibited cell proliferation and induced G1 phase arrest in human DLBCL cells. Furthermore, we showed that cladribine induced apoptosis by decreasing the expression of c-FLIPL and increasing the expression of DR4 and the cleaved form of caspase8. Cladribine also upregulated the expression of Bax, and downregulated the expression of Mcl-1 and Bcl-2 in a dose-dependent manner. It also activated endoplasmic reticulum (ER) stress, and ATF4 expression was required for cladribine induced apoptosis. Also, we showed that suberoylanilide hydroxamic acid (SAHA) enhanced the pro-apoptotic role of cladribine. Collectively, cladribine activated extrinsic and intrinsic apoptotic signaling pathways via stimulating ER stress signaling pathway and eliciting synergistic effect with SAHA in DLBCL cells.
    Keywords:  ATF4; DLBCL; SAHA; apoptosis; cladribine
    DOI:  https://doi.org/10.7150/ijms.41793
  44. Int J Mol Sci. 2020 Jul 08. pii: E4824. [Epub ahead of print]21(14):
    Delos Santos K, Kwon E, Moon NS.
      While a plethora of genetic techniques have been developed over the past century, modifying specific sequences of the fruit fly genome has been a difficult, if not impossible task. clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 truly redefined molecular genetics and provided new tools to model human diseases in Drosophila melanogaster. This is particularly true for genes whose protein sequences are highly conserved. Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism whose missense mutations are found in several neurological disorders, including Arts syndrome. In addition, PRPS is deregulated in cancer, particularly those that become resistant to cancer therapy. Notably, Drosophila PRPS shares about 90% protein sequence identity with its human orthologs, making it an ideal gene to study via CRISPR/Cas9. In this review, we will summarize recent findings on PRPS mutations in human diseases including cancer and on the molecular mechanisms by which PRPS activity is regulated. We will also discuss potential applications of Drosophila CRISPR/Cas9 to model PRPS-dependent disorders and other metabolic diseases that are associated with nucleotide metabolism.
    Keywords:  Drosophila CRISPR; PRPS-associated disease; metabolic disorders; neurological disorders
    DOI:  https://doi.org/10.3390/ijms21144824
  45. Oral Dis. 2020 Jul 08.
    Wang S, Wang XL, Wu ZZ, Yang QC, Xiong HG, Xiao Y, Li H, Sun ZJ.
      OBJECTIVES: Ribonucleotide reductase M2 (RRM2) is a rate-limiting enzyme involved in DNA repair and synthesis. This study aims to investigate the expression level, clinicopathological significance and prognostic value of RRM2 in oral squamous cell carcinoma (OSCC).MATERIALS AND METHODS: Human OSCC tissue microarrays were used to detect the expression of RRM2, cancer stem cell (CSCs) markers CD44 and aldehyde dehydrogenase 1 (ALDH1), and the epithelial-mesenchymal transition (EMT) marker Slug. The correlation of RRM2 expression with clinicopathological parameters was evaluated. The effects of RRM2 on cell proliferation, migration and apoptosis were investigated.
    RESULTS: Compared with normal and dysplastic tissues, the expression of RRM2 in human primary OSCC was significantly increased, and its overexpression was correlated with advanced pathological grade. The overall survival rate of patients with high RRM2 expression was lower than that of patients with low RRM2 expression. The overexpression of RRM2 was significantly associated with OSCC recurrence, and its overexpression was correlated with the CSCs markers CD44 and ALDH1 and the EMT marker Slug. The expression of RRM2 promotes the proliferation and migration of human OSCC cells and inhibits apoptosis.
    CONCLUSION: RRM2 may be a novel target in the diagnosis, prognosis, and therapy of OSCC.
    Keywords:  RRM2; TCGA; clinicopathologic significance; oral squamous cell carcinoma; overall survival; tissue microarray
    DOI:  https://doi.org/10.1111/odi.13540
  46. Angew Chem Int Ed Engl. 2020 Jul 08.
    Zhu L, Guo Y, Qian Q, Yan D, Li Y, Zhu X, Zhang C.
      Combinatorial antitumor therapies using different chemodrug combo or chemo/gene agent combo emerge as promising ways to overcome the drug resistance, a major cause for the failure of cancer treatment. However, dramatic pharmacokinetic differences of adopted drugs greatly impede the combinatorial antitumor practices, raising the great demand of developing appropriate drug delivery systems (DDSs) for tumor treatment. By employing fluorescent dithiomaleimide (DTM) as a linker, herein, we conjugate two paclitaxel (PTX) molecules with a floxuridine (FdU)-integrated antisense oligonucleotide (termed as chemogene) to form a precise macromolecular drug-chemogene conjugate. This PTX-chemogene conjugate can self-assemble into spherical nucleic acid (SNA)-like micellular nanoparticle as a carrier-free DDS, which effectively knocks down the expression of P-glycoprotein first and subsequently releases the FdU and PTX to exert a synergistic antitumor effect and greatly inhibit the tumor growth. Our carrier-free DDS bearing drug/drug synergy and chemo/gene synergy may shed light on reversing multi-drug resistance of cancer.
    Keywords:  Chemotherapy; chemogene; drug resistance; gene therapy; nucleoside analogue
    DOI:  https://doi.org/10.1002/anie.202006895
  47. Purinergic Signal. 2020 Jul 08.
    Wang H, Wang X, Xu L, Zhang J, Cao H.
      Reprogramming of metabolism is described in many types of cancer and is associated with the clinical outcomes. However, the prognostic significance of pyrimidine metabolism signaling pathway in lung adenocarcinoma (LUAD) is unclear. Using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets, we found that the pyrimidine metabolism signaling pathway was significantly enriched in LUAD. Compared with normal lung tissues, the pyrimidine metabolic rate-limiting enzymes were highly expressed in lung tumor tissues. The high expression levels of pyrimidine metabolic-rate limiting enzymes were associated with unfavorable prognosis. However, purinergic receptors P2RX1, P2RX7, P2RY12, P2RY13, and P2RY14 were relatively downregulated in lung cancer tissues and were associated with favorable prognosis. Moreover, we found that hypo-DNA methylation, DNA amplification, and TP53 mutation were contributing to the high expression levels of pyrimidine metabolic rate-limiting enzymes in lung cancer cells. Furthermore, combined pyrimidine metabolic rate-limiting enzymes had significant prognostic effects in LUAD. Comprehensively, the pyrimidine metabolic rate-limiting enzymes were highly expressed in bladder cancer, breast cancer, colon cancer, liver cancer, and stomach cancer. And the high expression levels of pyrimidine metabolic rate-limiting enzymes were associated with unfavorable prognosis in liver cancer. Overall, our results suggested the mRNA levels of pyrimidine metabolic rate-limiting enzymes CAD, DTYMK, RRM1, RRM2, TK1, TYMS, UCK2, NR5C2, and TK2 were predictive of lung cancer as well as other cancers.
    Keywords:  Gene expression omnibus; Lung adenocarcinoma; Purinergic receptors; Pyrimidine metabolic rate–limiting enzymes; The Cancer genome atlas
    DOI:  https://doi.org/10.1007/s11302-020-09711-4
  48. J Am Chem Soc. 2020 Jul 06.
    Reinhardt CR, Li P, Kang G, Stubbe J, Drennan CL, Hammes-Schiffer S.
      Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of E. coli Ia RNR is composed of two homodimers that form the active α2β2 complex. Catalysis is initiated by long-range radical translocation over a ~32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface. Resolving the PCET pathway at the α/β interface has been a long-standing challenge due to the lack of structural data. Herein molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α2β2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.
    DOI:  https://doi.org/10.1021/jacs.0c04325