bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒06‒21
forty-six papers selected by
Sean Rudd
Karolinska Institutet

  1. Nucleic Acids Res. 2020 Jun 16. pii: gkaa524. [Epub ahead of print]
    Falquet B, Ölmezer G, Enkner F, Klein D, Challa K, Appanah R, Gasser SM, Rass U.
      DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
  2. Nucleic Acids Res. 2020 Jun 15. pii: gkaa508. [Epub ahead of print]
    Dhoonmoon A, Schleicher EM, Clements KE, Nicolae CM, Moldovan GL.
      The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
  3. FASEB J. 2020 Jun 15.
    Ci S, Xia W, Liang W, Qin L, Zhang Y, Dianov GL, Wang M, Zhao X, Wu C, Alagamuthu KK, Hu Z, He L, Pan F, Guo Z.
      Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase β (Pol β) to function in DNA repair. Nuclear GAPDH promotes Pol β polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.
    Keywords:  DNA damage; GAPDH; Pol β; Src; base excision repair; chemotherapy
  4. Cell Res. 2020 Jun 19.
    Macheret M, Bhowmick R, Sobkowiak K, Padayachy L, Mailler J, Hickson ID, Halazonetis TD.
      DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.
  5. Cell Res. 2020 Jun 19.
    Ji F, Liao H, Pan S, Ouyang L, Jia F, Fu Z, Zhang F, Geng X, Wang X, Li T, Liu S, Syeda MZ, Chen H, Li W, Chen Z, Shen H, Ying S.
      Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. They are hotspots for chromosome rearrangements and structural variations, which have been extensively implicated in carcinogenesis, aging, and other pathological processes. Although many CFSs were identified decades ago, a consensus is still lacking for why they are particularly unstable and sensitive to replication perturbations. This is in part due to the lack of high-resolution mapping data for the vast majority of the CFSs, which has hindered mechanistic interrogations. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of the known CFSs, and comprehensively analyzed their sequence characteristics and genomic features. Our data on MDSs tallied well with long-standing hypotheses to explain CFS fragility while highlighting the contributions of late replication timing and large transcription units. Notably, the MDSs also encompassed most of the recurrent double-strand break clusters previously identified in mouse neural stem/progenitor cells, thus bridging evolutionarily conserved break points across species. Moreover, MiDAseq provides an important resource that can stimulate future research on CFSs to further unravel the mechanisms and biological relevance underlying these labile genomic regions.
  6. Genes Dev. 2020 Jun 19.
    Björkman A, Johansen SL, Lin L, Schertzer M, Kanellis DC, Katsori AM, Christensen ST, Luo Y, Andersen JS, Elsässer SJ, Londono-Vallejo A, Bartek J, Schou KB.
      RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.
    Keywords:  DNA damage response; DNA repair; DNA:RNA hybrid; Hoyeral-Hreiderson syndrome; POLδ; Poldip3; R-loop; RTEL1; dyskeratosis congenita; helicase; polymerase δ; telomere maintenance
  7. Elife. 2020 Jun 15. pii: e55828. [Epub ahead of print]9
    Meng F, Qian M, Peng B, Peng L, Wang X, Zheng K, Liu Z, Tang X, Zhang S, Sun S, Cao X, Pang Q, Zhao B, Ma W, Songyang Z, Xu B, Zhu WG, Xu X, Liu B.
      The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR in human and mouse cells. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair in humans and mice.
    Keywords:  biochemistry; chemical biology; human; mouse
  8. Int J Mol Sci. 2020 Jun 15. pii: E4245. [Epub ahead of print]21(12):
    Dang TT, Morales JC.
      Cellular survival is dependent on the efficient replication and transmission of genomic information. DNA damage can be introduced into the genome by several different methods, one being the act of DNA replication. Replication is a potent source of DNA damage and genomic instability, especially through the formation of DNA double strand breaks (DSBs). DNA polymerase alpha is responsible for replication initiation. One subunit of the DNA polymerase alpha replication machinery is POLA2. Given the connection between replication and genomic instability, we decided to examine the role of POLA2 in DSB repair, as little is known about this topic. We found that loss of POLA2 leads to an increase in spontaneous DSB formation. Loss of POLA2 also slows DSB repair kinetics after treatment with etoposide and inhibits both of the major double strand break repair pathways: non-homologous end-joining and homologous recombination. In addition, loss of POLA2 leads to increased sensitivity to ionizing radiation and PARP1 inhibition. Lastly, POLA2 expression is elevated in glioblastoma multiforme tumors and correlates with poor overall patient survival. These data demonstrate a role for POLA2 in DSB repair and resistance to genotoxic stress.
    Keywords:  POLA2; homologous recombination (HR); non-homologous end-joining (NHEJ)
  9. Sci Rep. 2020 Jun 18. 10(1): 9930
    Ha DH, Min A, Kim S, Jang H, Kim SH, Kim HJ, Ryu HS, Ku JL, Lee KH, Im SA.
      Due to its regulation of CDK1/2 phosphorylation, WEE1 plays essentially roles in the regulations of G2/M checkpoint and DNA damage response (DDR). WEE1 inhibition can increase genomic instability by inducing replication stress and G2/M checkpoint inactivation, which result in increased cellular sensitivity to DNA damaging agents. We considered an increase in genomic instability induced by WEE1 inhibition might be used to augment the effects of drugs targeting DNA repair protein. Typically, PARP inhibitors are effective in germline BRCA 1/2 mutated breast and ovarian cancer, but their applicabilities in triple-negative breast cancer (TNBC) are limited. This study was conducted to investigate the anti-tumor effects of the WEE1 inhibitor, AZD1775, and the mechanism responsible for its potentiation of sensitivity to olaparib (a PARP inhibitor) via the modulation of DDR in TNBC cells. Our results suggest that AZD1775 could be used to broaden the application range of olaparib in TNBC and provide a rationale for a clinical trial of combined olaparib and AZD1775 therapy.
  10. Cancers (Basel). 2020 Jun 17. pii: E1607. [Epub ahead of print]12(6):
    McMullen M, Karakasis K, Madariaga A, Oza AM.
      Platinum chemotherapy remains the cornerstone of treatment for epithelial ovarian cancer (OC) and Poly (ADP-ribose) polymerase inhibitors (PARPi) now have an established role as maintenance therapy. The mechanisms of action of these agents is, in many ways, complementary, and crucially reliant on the intracellular DNA Damage Repair (DDR) response. Here, we review mechanisms of primary and acquired resistance to treatment with platinum and PARPi, examining the interplay between both classes of agents. A key resistance mechanism appears to be the restoration of the Homologous Recombination (HR) repair pathway, through BRCA reversion mutations and epigenetic upregulation of BRCA1. Alterations in non-homologous end-joint (NHEJ) repair, replication fork protection, upregulation of cellular drug efflux pumps, reduction in PARP1 activity and alterations to the tumour microenvironment have also been described. These resistance mechanisms reveal molecular vulnerabilities, which may be targeted to re-sensitise OC to platinum or PARPi treatment. Promising therapeutic strategies include ATR inhibition, epigenetic re-sensitisation through DNMT inhibition, cell cycle checkpoint inhibition, combination with anti-angiogenic therapy, BET inhibition and G-quadruplex stabilisation. Translational studies to elucidate mechanisms of treatment resistance should be incorporated into future clinical trials, as understanding these biologic mechanisms is crucial to developing new and effective therapeutic approaches in advanced OC.
    Keywords:  PARP; homologous recombination; ovarian cancer; platinum; resistance mechanisms
  11. Mol Cell Biol. 2020 Jun 15. pii: MCB.00145-20. [Epub ahead of print]
    Ahamad N, Khan S, Xu YJ.
      Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect and heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.
  12. Front Cell Dev Biol. 2020 ;8 416
    Kafer GR, Cesare AJ.
      Murine development demands that pluripotent epiblast stem cells in the peri-implantation embryo increase from approximately 120 to 14,000 cells between embryonic days (E) 4.5 and E7.5. This is possible because epiblast stem cells can complete cell cycles in under 3 h in vivo. To ensure conceptus fitness, epiblast cells must undertake this proliferative feat while maintaining genome integrity. How epiblast cells maintain genome health under such an immense proliferation demand remains unclear. To illuminate the contribution of genome stability pathways to early mammalian development we systematically reviewed knockout mouse data from 347 DDR and repair associated genes. Cumulatively, the data indicate that while many DNA repair functions are dispensable in embryogenesis, genes encoding replication stress response and homology directed repair factors are essential specifically during the peri-implantation stage of early development. We discuss the significance of these findings in the context of the unique proliferative demands placed on pluripotent epiblast stem cells.
    Keywords:  DNA damage response; DNA repair; DNA replication; early development; embryology; pluripotency; replication stress response
  13. Nucleic Acids Res. 2020 Jun 15. pii: gkaa503. [Epub ahead of print]
    Awad A, Glousker G, Lamm N, Tawil S, Hourvitz N, Smoom R, Revy P, Tzfati Y.
      Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and diverse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While diverse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3' overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.
  14. Biomolecules. 2020 Jun 13. pii: E902. [Epub ahead of print]10(6):
    Rodriguez-Alvarez M, Kim D, Khobta A.
      The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed "transcriptional mutagenesis" (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.
    Keywords:  DNA damage; DNA damage tolerance; damage bypass; enhanced green fluorescent protein (EGFP); host cell reactivation (HCR); mutation assay; reporter assay; transcriptional mutagenesis; translesion synthesis (TLS)
  15. Cells. 2020 Jun 15. pii: E1466. [Epub ahead of print]9(6):
    Borsos BN, Majoros H, Pankotai T.
      Nucleotide excision repair (NER) is a versatile DNA repair pathway which can be activated in response to a broad spectrum of UV-induced DNA damage, such as bulky adducts, including cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Based on the genomic position of the lesion, two sub-pathways can be defined: (I) global genomic NER (GG-NER), involved in the ablation of damage throughout the whole genome regardless of the transcription activity of the damaged DNA locus, and (II) transcription-coupled NER (TC-NER), activated at DNA regions where RNAPII-mediated transcription takes place. These processes are tightly regulated by coordinated mechanisms, including post-translational modifications (PTMs). The fine-tuning modulation of the balance between the proteins, responsible for PTMs, is essential to maintain genome integrity and to prevent tumorigenesis. In this review, apart from the other substantial PTMs (SUMOylation, PARylation) related to NER, we principally focus on reversible ubiquitylation, which involves E3 ubiquitin ligase and deubiquitylase (DUB) enzymes responsible for the spatiotemporally precise regulation of NER.
    Keywords:  DUBs; E3 ligases; GG-NER; K48 chains; K63 chains; TC-NER; ubiquitylation
  16. Nucleic Acids Res. 2020 Jun 15. pii: gkaa496. [Epub ahead of print]
    Hoitsma NM, Whitaker AM, Beckwitt EC, Jang S, Agarwal PK, Van Houten B, Freudenthal BD.
      Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.
  17. Nat Commun. 2020 Jun 18. 11(1): 3088
    Daley JM, Tomimatsu N, Hooks G, Wang W, Miller AS, Xue X, Nguyen KA, Kaur H, Williamson E, Mukherjee B, Hromas R, Burma S, Sung P.
      DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.
  18. Nucleic Acids Res. 2020 Jun 15. pii: gkaa501. [Epub ahead of print]
    Kim S, Kang N, Park SH, Wells J, Hwang T, Ryu E, Kim BG, Hwang S, Kim SJ, Kang S, Lee S, Stirling P, Myung K, Lee KY.
      R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.
  19. Nutrients. 2020 Jun 17. pii: E1792. [Epub ahead of print]12(6):
    AlGhamdi AA, Mohammed MRS, Zamzami MA, Al-Malki AL, Qari MH, Khan MI, Choudhry H.
      Thymoquinone (TQ), a naturally occurring anticancer compound extracted from Nigella sativa oil, has been extensively reported to possess potent anti-cancer properties. Experimental studies showed the anti-proliferative, pro-apoptotic, and anti-metastatic effects of TQ on different cancer cells. One of the possible mechanisms underlying these effects includes alteration in key metabolic pathways that are critical for cancer cell survival. However, an extensive landscape of the metabolites altered by TQ in cancer cells remains elusive. Here, we performed an untargeted metabolomics study using leukemic cancer cell lines during treatment with TQ and found alteration in approximately 335 metabolites. Pathway analysis showed alteration in key metabolic pathways like TCA cycle, amino acid metabolism, sphingolipid metabolism and nucleotide metabolism, which are critical for leukemic cell survival and death. We found a dramatic increase in metabolites like thymine glycol in TQ-treated cancer cells, a metabolite known to induce DNA damage and apoptosis. Similarly, we observed a sharp decline in cellular guanine levels, important for leukemic cancer cell survival. Overall, we provided an extensive metabolic landscape of leukemic cancer cells and identified the key metabolites and pathways altered, which could be critical and responsible for the anti-proliferative function of TQ.
    Keywords:  DNA damage; LC-MS/MS; leukemia; metabolism; metabolites; thymoquinone
  20. J Biol Chem. 2020 Jun 15. pii: jbc.RA120.012962. [Epub ahead of print]
    Abdisalaam S, Bhattacharya S, Mukherjee S, Sinha D, Srinivasan K, Zhu M, Akbay EA, Sadek HA, Shay JW, Aroumougame A.
      Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double-strand breaks specifically in the telomeres, chromatin immunoprecipitation, telomere immunofluorescence, fluorescence in situ hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.
    Keywords:  CRISPR/Cas; DNA damage; DNA damage response; chromatin; cyclic GMP-AMP synthase (cGAS); genome maintenance; micronuclei; senescence; telomere
  21. Genes (Basel). 2020 Jun 11. pii: E643. [Epub ahead of print]11(6):
    Betlej G, Bator E, Pyrkosz A, Kwiatkowska A.
      Monocytes, which play a crucial role in the immune system, are characterized by an enormous sensitivity to oxidative stress. As they lack four key proteins responsible for DNA damage response (DDR) pathways, they are especially prone to reactive oxygen species (ROS) exposure leading to oxidative DNA lesions and, consequently, ROS-driven apoptosis. Although such a phenomenon is of important biological significance in the regulation of monocyte/macrophage/dendritic cells' balance, it also a challenge for monocytic mechanisms that have to provide and maintain genetic stability of its own DNA. Interestingly, apurinic/apyrimidinic endonuclease 1 (APE1), which is one of the key proteins in two DDR mechanisms, base excision repair (BER) and non-homologous end joining (NHEJ) pathways, operates in monocytic cells, although both BER and NHEJ are impaired in these cells. Thus, on the one hand, APE1 endonucleolytic activity leads to enhanced levels of both single- and double-strand DNA breaks (SSDs and DSBs, respectively) in monocytic DNA that remain unrepaired because of the impaired BER and NHEJ. On the other hand, there is some experimental evidence suggesting that APE1 is a crucial player in monocytic genome maintenance and stability through different molecular mechanisms, including induction of cytoprotective and antioxidant genes. Here, the dual face of APE1 is discussed.
    Keywords:  APE1; TNFα; chromosomal instability; genetic stability; monocytes; tissue homeostasis
  22. Oncogene. 2020 Jun 19.
    Haider N, Dutt P, van de Kooij B, Ho J, Palomero L, Pujana MA, Yaffe M, Stambolic V.
      In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10's unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.
  23. Future Oncol. 2020 Jun 15.
    Lamberti G, Andrini E, Sisi M, Federico AD, Ricciuti B.
      DNA damage response and repair (DDR) genes play a central role in the life of actively replicating cells, cooperating to maintenance of genomic integrity. However, exogenous or endogenous factors, including deficiency in DDR genes, can cause different degrees of DNA damage that profoundly impacts the tumor immunogenicity and enhance antitumor immune response through neoantigen-dependent and neoantigen-independent mechanisms. Inhibition of DDRs is already an effective therapeutic strategy in different cancer types. In addition, because DDR inhibition can also induce and amplify DNA damage in cancer cells, with a deep impact on antitumor immune responses, combining DDR inhibitors with immune checkpoint inhibitors represent an attractive therapeutic strategy to potentially improve the clinical outcomes of patients with metastatic cancer. In this review, we provide an overview of the rational and potential of combining DDR and immune checkpoint inhibition to exploit the enhanced antitumor immune response induced by DNA damage.
    Keywords:  DNA damage and repair; PARP inhibitor; PD-L1; STING; immune checkpoint inhibitors; mutational burden
  24. Cancer Lett. 2020 Jun 15. pii: S0304-3835(20)30303-7. [Epub ahead of print]
    Sakurai Y, Ichinoe M, Yoshida K, Nakazato Y, Saito S, Satoh M, Nakada N, Sanoyama I, Umezawa A, Numata Y, Shi-Xu J, Ichihara M, Takahashi M, Murakumo Y.
      REV7 is a multitasking protein involved in replication past DNA lesions, cell cycle regulation, and gene expression. REV7 is highly expressed in the adult testis and plays an essential role in primordial germ cell maintenance in mice. In this study, we analyzed whether REV7 can be a molecular target for the treatment of testicular germ cell tumors (TGCTs), in which acquired chemoresistance is a major cause of treatment failure. Strong expression of REV7 was detected in human TGCT tissues by immunohistochemistry. REV7 depletion in the TGCT cell lines suppressed cell proliferation and increased sensitivity to cisplatin and doxorubicin. cDNA microarray analysis revealed that REV7 depletion downregulated genes in the DNA repair gene set and upregulated genes in the apoptosis gene set. REV7 depletion-provoked chemosensitivity was associated with DNA double-strand break accumulation and apoptosis activation. In addition, inactivation of REV7 in cisplatin-resistant TGCT cells recovered chemosensitivity at almost equal levels as parental cells in vitro and in vivo. Our results indicate that inactivation of REV7 enhances chemosensitivity and overcomes chemoresistance in TGCT cells, suggesting REV7 as a potential therapeutic target in chemoresistant TGCTs.
    Keywords:  Apoptosis; Chemotherapy; Cisplatin; DNA damage; Translesion synthesis
  25. Proc Natl Acad Sci U S A. 2020 Jun 17. pii: 201922072. [Epub ahead of print]
    Qiao X, van der Zanden SY, Wander DPA, Borràs DM, Song JY, Li X, van Duikeren S, van Gils N, Rutten A, van Herwaarden T, van Tellingen O, Giacomelli E, Bellin M, Orlova V, Tertoolen LGJ, Gerhardt S, Akkermans JJ, Bakker JM, Zuur CL, Pang B, Smits AM, Mummery CL, Smit L, Arens R, Li J, Overkleeft HS, Neefjes J.
      The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.
    Keywords:  DNA damage; cardiotoxicity; chromatin damage; doxorubicin; therapy-related tumors
  26. Front Immunol. 2020 ;11 1084
    Stratigopoulou M, van Dam TP, Guikema JEJ.
      The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000-30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases.
    Keywords:  autoimmune diseases; base excision repair (BER); class switch recombination (CSR); germinal center (GC); lymphoma; somatic hypermutation (SHM)
  27. Chem Res Toxicol. 2020 Jun 19.
    Pande P, Rebello KR, Chatterjee A, Naldiga S, Basu AK.
      The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields two major adducts with 2'-deoxyguanosine (dG), one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding 5'-O-dimethoxytrityl 3'-phosphoramidite, which was used to synthesize a site-specifically adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS) efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%) inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%, respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ, hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8% in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs error-prone bypass.
  28. Redox Biol. 2020 Jun 08. pii: S2213-2317(20)30809-0. [Epub ahead of print]36 101604
    Xu L, Wu T, Lu S, Hao X, Qin J, Wang J, Zhang X, Liu Q, Kong B, Gong Y, Liu Z, Shao C.
      Ovarian cancer is the most lethal gynecological malignancy. Abnormal homologous recombination repair, high level of reactive oxygen species (ROS) and upregulation of antioxidant genes are characteristic features of ovarian cancer. However, the molecular mechanisms governing the redox homeostasis in ovarian cancer cells remain to be fully elucidated. We here demonstrated a critical role of RAD51, a protein essential for homologous recombination, in the maintenance of redox homeostasis. We found that RAD51 is overexpressed in high grade serous ovarian cancer and is associated with poor prognosis. Depletion or inhibition of RAD51 results in G2/M arrest, increased production of reactive oxygen species and accumulation of oxidative DNA damage. Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation caused by RAD51 depletion. We further demonstrated that RAD51 inhibition or depletion led to elevated production of mitochondrial superoxide and increased accumulation of mitochondria. Moreover, CHK1 activation is required for the G2/M arrest and the generation of mitochondrial stress in response to RAD51 depletion. Together, our results indicate that nuclear DNA damage caused by RAD51 depletion may trigger mitochondria-originated redox dysregulation. Our findings suggest that a vicious cycle of nuclear DNA damage, mitochondrial accumulation and oxidative stress may contribute to the tumor-suppressive effects of RAD51 depletion or inhibition.
    Keywords:  CHK1; G2/M arrest; Mitochondria stress; Ovarian cancer; RAD51; Redox homeostasis
  29. Blood. 2020 Jun 15. pii: blood.2019001279. [Epub ahead of print]
    Numata A, Kwok HS, Zhou QL, Li J, Tirado-Magallanes R, Espinosa Angarcia V, Hannah RL, Park J, Wang CQ, Krishnan V, Rajagopalan D, Zhang Y, Zhou S, Welner R, Osato M, Jha S, Bohlander SK, Göttgens B, Yang H, Benoukraf T, Lough JW, Bararia D, Tenen DG.
      Hematopoietic stem cells (HSC) have the potential to replenish the blood system for the lifetime of the organism. Their two defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Here, using conditional gene knockout models, we demonstrate a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance both in the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 co-localizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell-cycle and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin and Tip60 deletion induced a robust reduction in the acH2A.Z / H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance at least in part through Tip60 dependent H2A.Z acetylation to activate Myc target genes.
  30. Oncogenesis. 2020 Jun 15. 9(6): 60
    Matsui M, Sakasai R, Abe M, Kimura Y, Kajita S, Torii W, Katsuki Y, Ishiai M, Iwabuchi K, Takata M, Nishi R.
      The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localization to nuclear speckles is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. In conclusion, our data propose a model in which USP42 facilitates BRCA1 loading to DSB sites, resolution of DSB-induced R-loop and preferential DSB repair by HR, indicating the importance of nuclear speckle-mediated regulation of DSB repair.
  31. J Biol Chem. 2020 06 16. pii: jbc.REV120.012784. [Epub ahead of print]
    Ghosh S, Marsh ENG.
      Viperin plays an important and multifaceted role in the innate immune response to viral infection.  Viperin is also notable as one of very few radical SAM-dependent enzymes present in higher animals; however, the enzyme appears broadly conserved across all kingdoms of life, which suggests that it represents an ancient defense mechanism against viral infections.  Although viperin was discovered some 20 years ago, only recently was the enzyme's structure determined and its catalytic activity elucidated.  The enzyme converts CTP to 3',4'-didehydro-3'-deoxy-CTP, which functions as novel chain-terminating antiviral nucleotide when misincorporated by viral RNA-dependent RNA polymerases.  Moreover, in higher animals, viperin interacts with numerous other host and viral proteins, and it is apparent that this complex network of interactions constitutes another important aspect of the protein's antiviral activity.  An emerging theme is that viperin appears to facilitate ubiquitin-dependent proteasomal degradation of some of the proteins it interacts with.  Viperin- targeted protein degradation contributes to the antiviral response either by down-regulating various metabolic pathways important for viral replication or by directly targeting viral proteins for degradation. Here, we review recent advances in our understanding of the structure and catalytic activity of viperin, together with studies investigating the interactions between viperin and its target proteins.  These studies have provided detailed insights into the biochemical processes underpinning this unusual enzyme's wide-ranging antiviral activity. We also highlight recent intriguing reports that implicate a broader role for viperin in regulating non-pathological cellular processes, including thermogenesis and protein secretion.
    Keywords:  antiviral agent; enzyme mechanism; innate immunity; lipid metabolism; protein degradation; radical SAM enzymes; thermogenesis; ubiquitylation (ubiquitination); viral replication
  32. PLoS Genet. 2020 Jun 15. 16(6): e1008740
    Odermatt DC, Lee WTC, Wild S, Jozwiakowski SK, Rothenberg E, Gari K.
      FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.
  33. Curr Genet. 2020 Jun 17.
    Kumar K, Moirangthem R, Kaur R.
      Histone proteins regulate cellular factors' accessibility to DNA, and histone dosage has previously been linked with DNA damage susceptibility and efficiency of DNA repair pathways. Surplus histones are known to impede the DNA repair process by interfering with the homologous recombination-mediated DNA repair in Saccharomyces cerevisiae. Here, we discuss the recent finding of association of methyl methanesulfonate (MMS) resistance with the reduced histone H4 gene dosage in the pathogenic yeast Candida glabrata. We have earlier shown that while the low histone H3 gene dosage led to MMS susceptibility, the lack of two H4-encoding ORFs, CgHHF1 and CgHHF2, led to resistance to MMS-induced DNA damage. This resistance was linked with a higher rate of homologous recombination (HR). Taking these findings further, we review the interactome analysis of histones H3 and H4 in C. glabrata. We also report that the arginine residue present at the 95th position in the C-terminal tail of histone H4 protein is required for complementation of the MMS resistance in the Cghhf1Δhhf2Δ mutant, thereby pointing out a probable role of this residue in association with HR factors. Additionally, we present evidence that reduction in H4 protein levels may constitute an important part of varied stress responses in C. glabrata. Altogether, we present an overview of histone H4 dosage, HR-mediated repair of damaged DNA and stress resistance in this opportunistic human fungal pathogen.
    Keywords:  Chromatin; Genome integrity; Histones; Homologous recombination; Human fungal pathogens; Methyl methanesulfonate (MMS); Stress resistance
  34. Proc Natl Acad Sci U S A. 2020 Jun 17. pii: 201920049. [Epub ahead of print]
    Lex K, Maia Gil M, Lopes-Bastos B, Figueira M, Marzullo M, Giannetti K, Carvalho T, Ferreira MG.
      Cancer incidence increases exponentially with age when human telomeres are shorter. Similarly, telomerase reverse transcriptase (tert) mutant zebrafish have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a noncell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when wild-type (WT) tumors are generated in tert mutant zebrafish. Tissues adjacent to melanoma lesions (skin) and distant organs (intestine) in tert mutants exhibited higher levels of senescence and inflammation. In addition, we transferred second generation (G2) tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced increased tumor necrosis factor1-α (TNF1-α) expression and senescence in larval tissues in a noncell autonomous manner, creating an inflammatory environment. Considering that inflammation is protumorigenic, we transplanted melanoma-derived cells into G2 tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased tumor development. To test if inflammation was necessary for this effect, we treated melanoma transplants with nonsteroid anti-inflammatory drugs and show that higher melanoma dissemination can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence.
    Keywords:  aging; cancer; inflammation; telomerase; telomeres
  35. ACS Med Chem Lett. 2020 Jun 11. 11(6): 1118-1124
    Gavande NS, VanderVere-Carozza PS, Pawelczak KS, Vernon TL, Jordan MR, Turchi JJ.
      Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights, and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility, and excellent cellular up-take. Among a series of analogues, compounds 43, 44, 45, and 46 hold promise for further development of novel anticancer agents.
  36. JCI Insight. 2020 Jun 18. pii: 134885. [Epub ahead of print]5(12):
    Magkouta SF, Pappas AG, Vaitsi PC, Agioutantis PC, Pateras IS, Moschos CA, Iliopoulou MP, Kosti CN, Loutrari HV, Gorgoulis VG, Kalomenidis IT.
      Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor's response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.
    Keywords:  Angiogenesis; Cancer; DNA repair; NF-kappaB; Oncology
  37. Cell Res. 2020 Jun 15.
    Yum S, Li M, Chen ZJ.
      The discovery of cancer immune surveillance and immunotherapy has opened up a new era of cancer treatment. Immunotherapies modulate a patient's immune system to specifically eliminate cancer cells; thus, it is considered a very different approach from classic cancer therapies that usually induce DNA damage to cause cell death in a cell-intrinsic manner. However, recent studies have revealed that classic cancer therapies such as radiotherapy and chemotherapy also elicit antitumor immunity, which plays an essential role in their therapeutic efficacy. The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and the downstream effector Stimulator of Interferon Genes (STING) have been determined to be critical for this interplay. Here, we review the antitumor roles of the cGAS-STING pathway during tumorigenesis, cancer immune surveillance, and cancer therapies. We also highlight classic cancer therapies that elicit antitumor immune responses through cGAS activation.
  38. Chem Res Toxicol. 2020 Jun 19.
    Gajewski S, Hartwig A.
      PARP1 and p53 are key players in maintaining genomic stability, but their interplay is still not fully understood. We investigated the impact of PARP1 knockout on the DNA damage response after ionizing radiation (IR) by comparing a U2OS based PARP1-knockout cell line, established by using the genome-editing system CRISPR/Cas9, with its wild-type counterpart. We intended to gain more insight into the impact of PARP1 on the transcriptional level under basal conditions, after low dose (1 Gy) and after high dose (10 Gy) DNA damage induced by IR, aiming to reveal the potential connections between the involved pathways. In the absence of additionally induced DNA damage, lacking PARP1 led to an increased up-regulation of CDKN1A (p21), which caused a G1 arrest and slightly diminished cell proliferation. While a small but comparable transcriptional DNA damage response was observed upon 1 Gy IR in both cell lines, a pronounced transcriptional induction of p53 target genes was evident after treatment with 10 Gy IR exclusively in PARP1-proficient cells, suggesting that PARP1 facilitates the p53 signaling response after IR. Additionally, PARP1 appeared to be required for the ATM-dependent activation of PLK3, which in turn activates p53, leading to its transcriptional damage response. Our results support the involvement of PARP1 activation among the first steps in IR induced DNA damage response.
  39. Sci Rep. 2020 Jun 17. 10(1): 9804
    Carlisle SM, Trainor PJ, Hong KU, Doll MA, Hein DW.
      Human arylamine N-acetyltransferase 1 (NAT1), present in all tissues, is classically described as a phase-II xenobiotic metabolizing enzyme but can also catalyze the hydrolysis of acetyl-Coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a cofactor. NAT1 activity varies inter-individually and has been shown to be overexpressed in estrogen receptor-positive (ER+) breast cancers. NAT1 has also been implicated in breast cancer progression however the exact role of NAT1 remains unknown. The objective of this study was to evaluate the effect of varying levels of NAT1 N-acetylation activity in MDA-MB-231 breast cancer cells on global cellular metabolism and to probe for unknown endogenous NAT1 substrates. Global, untargeted metabolomics was conducted via ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on MDA-MB-231 breast cancer cell lines constructed with siRNA and CRISPR/Cas9 technologies to vary only in NAT1 N-acetylation activity. Many metabolites were differentially abundant in NAT1-modified cell lines compared to the Scrambled parental cell line. N-acetylasparagine and N-acetylputrescine abundances were strongly positively correlated (r = 0.986 and r = 0.944, respectively) with NAT1 N-acetylation activity whereas saccharopine abundance was strongly inversely correlated (r = -0.876). Two of the most striking observations were a reduction in de novo pyrimidine biosynthesis and defective β-oxidation of fatty acids in the absence of NAT1. We have shown that NAT1 expression differentially affects cellular metabolism dependent on the level of expression. Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism.
  40. Mol Oncol. 2020 Jun 13.
    Pan CH, Otsuka Y, Sridharan B, Woo M, Leiton CV, Babu S, Torrente Gonçalves M, Kawalerski RR, Bai JDK, Chang DK, Biankin A, Scampavia L, Spicer T, Escobar-Hoyos LF, Shroyer KR.
      Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17 expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to Gemcitabine (Gem). In multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells, and orthotopic xenografts, we determined that the expression of K17 results in a more than two-fold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that Podophyllotoxin (PPT), a microtubule inhibitor, showed significantly higher sensitivity in K17-positive compared to K17-negative PDAC cell lines and animal models. In the clinic, another microtubule inhibitor, Paclitaxel (PTX), is used in combination with Gem as a first line chemotherapeutic regimen for PDAC. Surprisingly, we found that when combined with Gem, PPT but not PTX, was synergistic in inhibiting the viability of K17 expressing PDAC cells. Importantly, in pre-clinical models, PPT in combination with Gem effectively decreased tumor growth and enhanced the survival of mice bearing K17 expressing tumors. This provides evidence that PPT, and its derivatives could potentially be combined with Gem to enhance treatment efficacy for the approximately 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.
    Keywords:  Chemoresistance; Combined therapy; Drug screen; Keratin 17; Pancreatic ductal adenocarcinoma; Predictive biomarker
  41. Leukemia. 2020 Jun 16.
    Issa GC, Kantarjian HM, Xiao L, Ning J, Alvarado Y, Borthakur G, Daver N, DiNardo CD, Jabbour E, Bose P, Jain N, Kadia TM, Naqvi K, Pemmaraju N, Takahashi K, Verstovsek S, Andreeff M, Kornblau SM, Estrov Z, Ferrajoli A, Garcia-Manero G, Ohanian M, Wierda WG, Ravandi F, Cortes JE.
      CPX-351 is a liposomal formulation of cytarabine/daunorubicin with a 5:1 fixed molar ratio. We investigated the safety and efficacy of escalating doses of CPX-351 in patients with acute myeloid leukemia (AML) at high risk of induction mortality with standard chemotherapy determined through assessment of leukemia and patient-related risk factors for intensive chemotherapy in an open-label, phase II trial. Patients were randomized to receive 50 or 75 units/m2 on days 1, 3, and 5. Once safety was established, a 100 units/m2 arm was opened. Fifty-six patients were enrolled, 16, 24, and 16 in the 50, 75, and 100 units/m2 arms, respectively. The composite complete remission rate (complete remission + complete remission with incomplete blood count recovery) was lowest with 50 units/m2 (19%) compared with 75 units/m2 (38%) and 100 units/m2 (44%) (P = 0.35). The 50 units/m2 arm had a median OS of 4.3 months, compared with 8.6 and 6.2 months for the 75 and 100 units/m2 respectively (P = 0.04). Nonhematologic grade 3/4 treatment-emergent adverse events included febrile neutropenia (34%), pneumonia (23%), and sepsis (16%). CPX-351 at 75 units/m2 has favorable safety and efficacy for AML patients at high risk of induction mortality with some tolerating the standard dose of 100 units/m2.
  42. J Inherit Metab Dis. 2020 Jun 18.
    Ramond F, Rio M, Héron B, Imbard A, Marie S, Billemaz K, Denommé-Pichon AS, Kuentz P, Ceballos I, Piraud M, Vincent MF, Touraine R.
      5-amino-4-imidazolecarboxamide-ribosiduria (AICA)-ribosiduria is an exceedingly rare autosomal recessive condition resulting from the disruption of the bifunctional purine biosynthesis protein PURH (ATIC), which catalyzes the last two steps of de novo purine synthesis. It is characterized biochemically by the accumulation of AICA-riboside in urine. AICA-ribosiduria had been reported in only one individual, 15 years ago. In this article, we report three novel cases of AICA-ribosiduria from two independent families, with two novel pathogenic variants in ATIC. We also provide a clinical update on the first patient. Based on the phenotypic features shared by these four patients, we define AICA-ribosiduria as the syndromic association of severe-to-profound global neurodevelopemental impairment, severe visual impairment due to chorioretinal atrophy, ante-postnatal growth impairment, and severe scoliosis. Dysmorphic features were observed in all four cases, especially neonatal/infancy coarse facies with upturned nose. Early-onset epilepsy is frequent and can be pharmacoresistant. Less frequently observed features are aortic coarctation, chronic hepatic cytolysis, minor genital malformations and nephrocalcinosis. Alteration of the transformylase activity of ATIC might result in a more severe impairment than the alteration of the cyclohydrolase activity. Data from literature points towards a cytotoxic mechanism of the accumulated AICA-riboside. This article is protected by copyright. All rights reserved.
    Keywords:  AICA-riboside; AICA-ribosiduria; AICAR; ATIC; clinical genetics; de novo purine biosynthesis; metabolic disease; rare disease
  43. Macromol Biosci. 2020 Jun 17. e2000083
    Zhang F, Yin J, Zhang C, Han M, Wang X, Fu S, Du J, Zhang H, Li W.
      Affibody-conjugated RALA (affi-RA) are designed for delivering oligomeric 5-fluorodeoxyuridine (FUdR, metabolite of 5-FU) strand to raise the selectivity of 5-fluorouracil (5-FU), decrease its toxicity and improve its suboptimal therapeutic efficacy. The nanodrugs, FUdR@affi-RA, are spontaneously assembled by electrostatic interaction between positively charged affi-RA and negatively charged FUdR15 -strands (15 consecutive FUdR). FUdR@affi-RA exhibits excellent stability under simulated physiological conditions. Compared with free FUdR, FUdR@affi-RA shows excellent targeting and higher cytotoxicity in human epidermal growth factor receptor 2 (HER2) overexpressing gastric cancer N87 cells. Moreover, the anticancer mechanism studies reveal that FUdR@affi-RA enhances the expression and activity of apoptosis-associated proteins in the Bcl-2/Bax-caspase 8,9-caspase 3 apoptotic pathway induced by FUdR. This study indicates that the fusion vector, affi-RA, presents a promising delivery system platform for nucleoside analogue drugs and provides a new strategy for the development of therapeutics of cancer treatment.
    Keywords:  5-fluorodeoxyuridine; HER2; RALA; affibody; targeting therapies
  44. BMC Cancer. 2020 Jun 16. 20(1): 560
    Negarandeh R, Salehifar E, Saghafi F, Jalali H, Janbabaei G, Abdhaghighi MJ, Nosrati A.
      BACKGROUND: 5-Fluorouracil (5-FU) and capecitabine are fluoropyrimidine derivatives that mainly metabolized with dihydropyrimidine dehydrogenase enzyme (DPD). The genetic polymorphism in the genes encoding this enzyme may result in a decrease or loss of enzyme activity which may lead to the accumulation of medicines, their metabolites and potential toxicity.METHOD: This cross-sectional study was conducted on 88 participants with colorectal cancer (CRC). After DNA extraction, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the DPD gene (DPYD) polymorphisms including IVS 14 + 1 G > A, 2846 A > T and 2194 G > A. Chemotherapy-induced side effects were evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE Version 5.0).
    RESULT: Data were collected from 227 chemotherapy cycles of 88 patients with CRC. In a comparison of FOLFOX and FOLFIRI regimens, there was no significant difference in the occurrence of chemotherapy-induced diarrhea, nausea, vomiting and oral mucositis. However, the peripheral neuropathy was more frequent in patients who were treated with FOLFOX (P <  0.001) and hair loss was more common in patients who received FOLFIRI regimen (P = 0.048). Incidence of the DPD IVS14 + 1 G > A polymorphism was observed in four patients (5.5%). There was no association between IVS14 + 1 G > A polymorphism and the occurrence of adverse reactions.
    CONCLUSION: FOLFOX and FOLFIRI were the most common regimens in CRC patients and their toxicity profile was different in some adverse reactions. Prevalence of IVS14 + 1G > A variant was relatively higher than other similar studies.
    TRIAL REGISTRATION: Approval code; IR.MAZUMS.REC.95.2480.
    Keywords:  5-fluorouracil; Colorectal cancer; Dihydropyrimidine dehydrogenase; Polymorphism; Side effects
  45. Oncogene. 2020 Jun 16.
    Zhang YH, Shi WN, Wu SH, Miao RR, Sun SY, Luo DD, Wan SB, Guo ZK, Wang WY, Yu XF, Cui SX, Qu XJ.
      Aberrant sphingolipid metabolism has been implicated in chemoresistance, but the underlying mechanisms are still poorly understood. Herein we revealed a previously unrecognized mechanism of 5-fluorouracil (5-FU) resistance contributed by high SphK2-upregulated dihydropyrimidine dehydrogenase (DPD) in colorectal cancer (CRC), which is evidenced from human CRC specimens, animal models, and cancer cell lines. TMA samples from randomly selected 60 CRC specimens firstly identified the clinical correlation between high SphK2 and increased DPD (p < 0.001). Then the regulatory mechanism was explored in CRC models of villin-SphK2 Tg mice, SphK2-/-mice, and human CRC cells xenografted nude mice. Assays of ChIP-Seq and luciferase reporter gene demonstrated that high SphK2 upregulated DPD through promoting the HDAC1-mediated H3K56ac, leading to the degradation of intracellular 5-FU into inactive α-fluoro-β-alanine (FBAL). Lastly, inhibition of SphK2 by SLR080811 exhibited excellent inhibition on DPD expression and potently reversed 5-FU resistance in colorectal tumors of villin-SphK2 Tg mice. Overall, this study manifests that SphK2high conferred 5-FU resistance through upregulating tumoral DPD, which highlights the strategies of blocking SphK2 to overcome 5-FU resistance in CRC.
  46. Cancers (Basel). 2020 Jun 17. pii: E1602. [Epub ahead of print]12(6):
    Ludwig N, Gillespie DG, Reichert TE, Jackson EK, Whiteside TL.
      Body fluids of patients with head and neck squamous cell carcinoma (HNSCC) are enriched in exosomes that reflect properties of the tumor. The aim of this study was to determine whether purine metabolites are carried by exosomes and evaluate their role as potential contributors to tumor immune escape. The gene expression levels of the purine synthesis pathway were studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer database. Exosomes were isolated from supernatants of UMSCC47 cells and from the plasma of HNSCC patients (n = 26) or normal donors (NDs; n = 5) using size exclusion chromatography. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to assess levels of 19 purine metabolites carried by exosomes. In HNSCC tissues, expression levels of genes involved in the purinergic pathway were upregulated indicating an accelerated purine metabolism compared to normal tissues. Exosomes from supernatants of UMSCC47 cells contained several purine metabolites, predominantly adenosine and inosine. Purine metabolite levels were enriched in exosomes isolated from the plasma of HNSCC patients compared to those isolated from NDs and carried elevated levels of adenosine (p = 0.0223). Exosomes of patients with early-stage disease and no lymph node metastasis contained significantly elevated levels of adenosine and 5'-GMP (p = 0.0247 and p = 0.0229, respectively). The purine metabolite levels in exosomes decreased in patients with advanced cancer and nodal involvement. This report provides the first evidence that HNSCC cells shuttle purine metabolites in exosomes, with immunosuppressive adenosine being the most prominent purine. Changes in the content and levels of purine metabolites in circulating exosomes reflect disease progression in HNSCC patients.
    Keywords:  HNSCC; TEX; adenosine; exosomes; extracellular vesicles; head and neck cancer; purine metabolites; purinergic signaling