bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒12‒06
28 papers selected by
Sean Rudd
Karolinska Institutet


  1. Mol Cell. 2020 Dec 03. pii: S1097-2765(20)30692-4. [Epub ahead of print]80(5): 862-875.e6
    Blessing C, Mandemaker IK, Gonzalez-Leal C, Preisser J, Schomburg A, Ladurner AG.
      The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.
    Keywords:  ALC1; BRCA; DNA damage; PARP inhibitors; PARP trapping; PARP2; cancer target; chromatin remodeling; helicases; synthetic lethality
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.009
  2. Nat Commun. 2020 11 30. 11(1): 6118
    Clements KE, Schleicher EM, Thakar T, Hale A, Dhoonmoon A, Tolman NJ, Sharma A, Liang X, Imamura Kawasawa Y, Nicolae CM, Wang HG, De S, Moldovan GL.
      Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.
    DOI:  https://doi.org/10.1038/s41467-020-19961-w
  3. Nat Metab. 2020 Nov 30.
    Kim J, Lee HM, Cai F, Ko B, Yang C, Lieu EL, Muhammad N, Rhyne S, Li K, Haloul M, Gu W, Faubert B, Kaushik AK, Cai L, Kasiri S, Marriam U, Nham K, Girard L, Wang H, Sun X, Kim J, Minna JD, Unsal-Kacmaz K, DeBerardinis RJ.
      In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.
    DOI:  https://doi.org/10.1038/s42255-020-00316-0
  4. Nat Cell Biol. 2020 Dec;22(12): 1460-1470
    Lamm N, Read MN, Nobis M, Van Ly D, Page SG, Masamsetti VP, Timpson P, Biro M, Cesare AJ.
      Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.
    DOI:  https://doi.org/10.1038/s41556-020-00605-6
  5. Mol Cancer Res. 2020 Nov 30. pii: molcanres.0708.2020. [Epub ahead of print]
    Agostini LC, Jain A, Shupp A, Nevler A, McCarthy G, Bussard KM, Yeo CJ, Brody JR.
      The DNA damage response (DDR) pathway sets the stage for tumorigenesis and provides both an opportunity for drug efficacy and resistance. Therapeutic approaches to target the DDR pathway include aiming to increase the efficacy of cytotoxic chemotherapies and synergistic drug strategies to enhance DNA damage, and hence cell death. Here, we report the first preclinical evaluation of a novel synergistic approach by using both genetic and small molecule inhibition methods of silencing the DDR related protein, PARG (poly (ADP) ribose glycohydrolase) and the checkpoint kinase inhibitor, Wee1, in pancreatic ductal adenocarcinoma (PDAC) and colorectal carcinoma (CRC) cells in vitro and in vivo. Mechanistically, we demonstrate that co-inhibition of PARG and Wee1 synergistically decreased cell survival and increased DNA damage in an S-phase dependent manner. Implications: In pre-clinical models, we demonstrate the efficacy and mechanism of action of targeting both PARG and Wee1 in PDAC and CRC cells.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0708
  6. Int J Mol Sci. 2020 Dec 01. pii: E9176. [Epub ahead of print]21(23):
    Jiang Y, Yam JC, Tham CC, Pang CP, Chu WK.
      Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.
    Keywords:  CtIP; RB; classical non-homologous end joining; homologous recombination; micro-homology mediated end joining; resection
    DOI:  https://doi.org/10.3390/ijms21239176
  7. Mol Cancer Ther. 2020 Dec 02. pii: molcanther.0365.2020. [Epub ahead of print]
    Parsels LA, Engelke CG, Parsels J, Flanagan SA, Zhang Q, Tanska D, Wahl DR, Canman CE, Lawrence TS, Morgan MA.
      PARP inhibitor monotherapy (olaparib) was recently FDA-approved for the treatment of BRCA1/2 mutant, HR (homologous recombination repair)-deficient pancreatic cancer. Most pancreatic cancers, however, are HR-proficient and thus resistant to PARP inhibitor monotherapy. We tested the hypothesis that combined therapy with radiation and ATR inhibitor (AZD6738) would extend the therapeutic indication of olaparib to HR-proficient pancreatic cancers. We show that olaparib combined with AZD6738 significantly reduced radiation survival relative to either agent alone, regardless of HR status. While catalytic inhibition of PARP with low concentrations of olaparib radiosensitized HR-deficient models, maximal sensitization in HR-proficient models required concentrations of olaparib that induce formation of PARP1-DNA complexes. Furthermore, CRISPR-Cas9-mediated PARP1 deletion failed to recapitulate the effects of olaparib on radiosensitivity and negated the combinatorial efficacy of olaparib and AZD6738 on radiosensitization, suggesting that PARP1-DNA complexes, rather than PARP catalytic inhibition, were responsible for radiosensitization. Mechanistically, therapeutic concentrations of olaparib in combination with radiation and AZD6738 increased DNA double strand breaks. DNA fiber combing revealed that high concentrations of olaparib did not stall replication forks but instead accelerated replication fork progression in association with an ATR-mediated replication stress response that was antagonized by AZD6738. Finally, in HR-proficient tumor xenografts, the combination of olaparib, radiation and AZD6738 significantly delayed tumor growth compared to all other treatments. These findings suggest that PARP1-DNA complexes are required for the therapeutic activity of olaparib combined with radiation and ATR inhibitor in HR-proficient pancreatic cancer and support the clinical development of this combination for tumors intrinsically resistant to PARP inhibitors.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-20-0365
  8. Int J Mol Sci. 2020 Nov 27. pii: E9034. [Epub ahead of print]21(23):
    Nieto-Jimenez C, Alcaraz-Sanabria A, Martinez-Canales S, Corrales-Sanchez V, Montero JC, Burgos M, Nuncia-Cantarero M, Pandiella A, Galan-Moya EM, Ocaña A.
      Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.
    Keywords:  CHK1 inhibitors; DNA damage response (DDR); DNA-damaging agents; breast cancer; chemotherapy resistance; platinum compounds; standard-of-care chemotherapies; synthetic lethality
    DOI:  https://doi.org/10.3390/ijms21239034
  9. Sci Rep. 2020 Dec 03. 10(1): 21146
    Hanasaki M, Yaku K, Yamauchi M, Nakagawa T, Masumoto H.
      Cellular metabolism is directly or indirectly associated with various cellular processes by producing a variety of metabolites. Metabolic alterations may cause adverse effects on cell viability. However, some alterations potentiate the rescue of the malfunction of the cell system. Here, we found that the alteration of glucose metabolism suppressed genome instability caused by the impairment of chromatin structure. Deletion of the TDH2 gene, which encodes glyceraldehyde 3-phospho dehydrogenase and is essential for glycolysis/gluconeogenesis, partially suppressed DNA damage sensitivity due to chromatin structure, which was persistently acetylated histone H3 on lysine 56 in cells with deletions of both HST3 and HST4, encoding NAD+-dependent deacetylases. tdh2 deletion also restored the short replicative lifespan of cells with deletion of sir2, another NAD+-dependent deacetylase, by suppressing intrachromosomal recombination in rDNA repeats increased by the unacetylated histone H4 on lysine 16. tdh2 deletion also suppressed recombination between direct repeats in hst3∆ hst4∆ cells by suppressing the replication fork instability that leads to both DNA deletions among repeats. We focused on quinolinic acid (QUIN), a metabolic intermediate in the de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway, which accumulated in the tdh2 deletion cells and was a candidate metabolite to suppress DNA replication fork instability. Deletion of QPT1, quinolinate phosphoribosyl transferase, elevated intracellular QUIN levels and partially suppressed the DNA damage sensitivity of hst3∆ hst4∆ cells as well as tdh2∆ cells. qpt1 deletion restored the short replicative lifespan of sir2∆ cells by suppressing intrachromosomal recombination among rDNA repeats. In addition, qpt1 deletion could suppress replication fork slippage between direct repeats. These findings suggest a connection between glucose metabolism and genomic stability.
    DOI:  https://doi.org/10.1038/s41598-020-78302-5
  10. Cell Cycle. 2020 Dec 03. 1-16
    Ackerson SM, Gable CI, Stewart JA.
      CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like complex that binds to single-stranded DNA (ssDNA) and functions in the replication of telomeric and non-telomeric DNA. Previous studies demonstrated that deletion of CTC1 results in decreased cell proliferation and telomere DNA damage signaling. However, a detailed analysis of the consequences of conditional CTC1 knockout (KO) has not been fully elucidated. Here, we investigated the effects of CTC1 KO on cell cycle progression, genome-wide replication and activation of the DNA damage response. Consistent with previous findings, we demonstrate that CTC1 KO results in decreased cell proliferation, G2 arrest and RPA-bound telomeric ssDNA. However, despite the increased levels of telomeric RPA-ssDNA, global ATR-dependent CHK1 and p53 phosphorylation was not detected in CTC1 KO cells. Nevertheless, we show that RPA-ssDNA does activate ATR, leading to the phosphorylation of RPA and autophosphorylation of ATR. Further analysis determined that inactivation of ATR, but not CHK1 or ATM, suppressed the accumulation of G2 arrested cells and phosphorylated RPA following CTC1 removal. These results suggest that ATR is localized and active at telomeres but is unable to elicit a global checkpoint response through CHK1. Furthermore, CTC1 KO inhibited CHK1 phosphorylation following hydroxyurea-induced replication stress. Additional studies revealed that this suppression of CHK1 phosphorylation, following replication stress, is caused by decreased levels of the ATR activator TopBP1. Overall, our results identify CST as a novel regulator of the ATR-CHK1 pathway.
    Keywords:  ATR; CST; CTC1; DNA repair; TopBP1; telomere
    DOI:  https://doi.org/10.1080/15384101.2020.1849979
  11. Cell Rep. 2020 Dec 01. pii: S2211-1247(20)31458-3. [Epub ahead of print]33(9): 108469
    Landsverk HB, Sandquist LE, Bay LTE, Steurer B, Campsteijn C, Landsverk OJB, Marteijn JA, Petermann E, Trinkle-Mulcahy L, Syljuåsen RG.
      Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.
    Keywords:  CDC73; CTD phosphorylation; Cancer; PNUTS; PP1 phosphatase; Proteasome-mediated degradation; RNA polymerase II; Replication stress; Transcription-replication conflicts; WDR82
    DOI:  https://doi.org/10.1016/j.celrep.2020.108469
  12. PLoS One. 2020 ;15(11): e0235998
    Li Z, Yu DS, Doetsch PW, Werner E.
      In contrast to the vast majority of research that has focused on the immediate effects of ionizing radiation, this work concentrates on the molecular mechanism driving delayed effects that emerge in the progeny of the exposed cells. We employed functional protein arrays to identify molecular changes induced in a human bronchial epithelial cell line (HBEC3-KT) and osteosarcoma cell line (U2OS) and evaluated their impact on outcomes associated with radiation induced genomic instability (RIGI) at day 5 and 7 post-exposure to a 2Gy X-ray dose, which revealed replication stress in the context of increased FOXM1b expression. Irradiated cells had reduced DNA replication rate detected by the DNA fiber assay and increased DNA resection detected by RPA foci and phosphorylation. Irradiated cells increased utilization of homologous recombination-dependent repair detected by a gene conversion assay and DNA damage at mitosis reflected by RPA positive chromosomal bridges, micronuclei formation and 53BP1 positive bodies in G1, all known outcomes of replication stress. Interference with the function of FOXM1, a transcription factor widely expressed in cancer, employing an aptamer, decreased radiation-induced micronuclei formation and cell transformation while plasmid-driven overexpression of FOXM1b was sufficient to induce replication stress, micronuclei formation and cell transformation.
    DOI:  https://doi.org/10.1371/journal.pone.0235998
  13. DNA Repair (Amst). 2020 Nov 12. pii: S1568-7864(20)30285-8. [Epub ahead of print]97 103025
    Bishara LA, Machour FE, Awwad SW, Ayoub N.
      The negative elongation factor (NELF) is a four-subunit protein complex (NELF-E, NELF-A, NELF-B and NELF-C/D) that negatively regulates transcription elongation of RNA polymerase II (Pol II). Interestingly, upregulation of NELF-E subunit promotes hepatocellular carcinoma (HCC) and pancreatic cancer. In addition, we have previously shown that NELF complex fosters double-strand break (DSB)-induced transcription silencing and promotes homology-directed repair (HDR). However, the mechanisms underlying NELF-E regulation of HDR of DSBs remain unknown. Here, we show that NELF-E interacts with BRCA1 and promotes its recruitment to laser-microirradiated sites and facilitates ionizing radiation-induced foci (IRIF) of BRCA1 in HCC cells (Hep3B). The reduction in BRCA1 IRIF is accompanied by decreased RAD51 IRIF. A corollary to this, NELF-E-deficient Hep3B cells exhibit defective HDR of chromosomal DSBs induced by CRISPR-Cas9 system. Consequently, the disruption of NELF complex integrity, by NELF-E downregulation, sensitizes Hep3B cells to PARP inhibition. Altogether, our results suggest that NELF promotes HDR by facilitating BRCA1 and RAD51 IRIF formation and identify NELF complex as a novel synthetic lethal partner of PARP1.
    Keywords:  BRCA1; Double-strand break (DSB); Hepatocellular carcinoma (HCC); Homology-directed repair (HDR); NELF-E; Negative elongation factor (NELF); RAD51
    DOI:  https://doi.org/10.1016/j.dnarep.2020.103025
  14. Cancers (Basel). 2020 Nov 24. pii: E3489. [Epub ahead of print]12(12):
    Pavlov YI, Zhuk AS, Stepchenkova EI.
      Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named "division of labor," remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants' effects on cancer.
    Keywords:  DNA polymerases; cancer predisposition; mutation rates; proofreading exonucleases; replication fidelity
    DOI:  https://doi.org/10.3390/cancers12123489
  15. EMBO J. 2020 Dec 02. e104542
    Chen Q, Bian C, Wang X, Liu X, Ahmad Kassab M, Yu Y, Yu X.
      Optimal DNA damage response is associated with ADP-ribosylation of histones. However, the underlying molecular mechanism of DNA damage-induced histone ADP-ribosylation remains elusive. Herein, using unbiased mass spectrometry, we identify that glutamate residue 141 (E141) of variant histone H2AX is ADP-ribosylated following oxidative DNA damage. In-depth studies performed with wild-type H2AX and the ADP-ribosylation-deficient E141A mutant suggest that H2AX ADP-ribosylation plays a critical role in base excision repair (BER). Mechanistically, ADP-ribosylation on E141 mediates the recruitment of Neil3 glycosylase to the sites of DNA damage for BER. Moreover, loss of this ADP-ribosylation enhances serine-139 phosphorylation of H2AX (γH2AX) upon oxidative DNA damage and erroneously causes the accumulation of DNA double-strand break (DSB) response factors. Taken together, these results reveal that H2AX ADP-ribosylation not only facilitates BER repair, but also suppresses the γH2AX-mediated DSB response.
    Keywords:  ADP-ribosylation; H2AX; PARP1; base excision repair
    DOI:  https://doi.org/10.15252/embj.2020104542
  16. Clin Cancer Res. 2020 Nov 30. pii: clincanres.3358.2020. [Epub ahead of print]
    van Bijsterveldt L, Durley S, Maughan TS, Humphrey T.
      Pre-clinical models of cancer have demonstrated enhanced efficacy of cell cycle checkpoint kinase inhibitors when used in combination with genotoxic agents. This combination therapy is predicted to be exquisitely toxic to cells with a deficient G1/S checkpoint or cells with a genetic predisposition leading to intrinsic DNA replication stress, as these cancer cells become fully dependent on the intra-S and G2/M checkpoints for DNA repair and cellular survival. Therefore, abolishing remaining cell cycle checkpoints after damage leads to increased cell death in a tumour cell-specific fashion. However, the preclinical success of these drug combinations is not consistently replicated in clinical trials. Here, we provide a perspective on the translation of pre-clinical studies into rationally designed clinical studies. We will discuss successes and failures of current treatment combinations and drug regimens and provide a detailed overview of all clinical trials using ATR, CHK1, or WEE1 inhibitors in combination with genotoxic agents. This highlights the need for revised patient stratification and the use of appropriate pharmacodynamic biomarkers to improve the success rate of clinical trials.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-3358
  17. Proc Natl Acad Sci U S A. 2020 Nov 30. pii: 202017734. [Epub ahead of print]
    Nogalski MT, Shenk T.
      Pericentromeric human satellite II (HSATII) repeats are normally silent but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected fibroblasts, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with DNA-damaging agents, etoposide or zeocin, induces HSATII RNA expression, and a kinase-independent function of ATM is required for the induction. Additionally, various breast cancer cell lines growing in adherent, two-dimensional cell culture express HSATII RNA at different levels, and levels are markedly increased when cells are infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlate with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduces cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a noncanonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement.
    Keywords:  DNA damage response; HSATII; breast cancer cells; human cytomegalovirus; human satellite II
    DOI:  https://doi.org/10.1073/pnas.2017734117
  18. BMC Cancer. 2020 Nov 30. 20(1): 1171
    Waters T, Goss KL, Koppenhafer SL, Terry WW, Gordon DJ.
      BACKGROUND: The treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities.METHODS: We analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug.
    RESULTS: We found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known "off-target" effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress.
    CONCLUSIONS: Ewing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.
    Keywords:  Eltrombopag; Ewing sarcoma; Iron chelation
    DOI:  https://doi.org/10.1186/s12885-020-07668-6
  19. Nat Commun. 2020 12 02. 11(1): 6178
    Sanders JT, Freeman TF, Xu Y, Golloshi R, Stallard MA, Hill AM, San Martin R, Balajee AS, McCord RP.
      The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.
    DOI:  https://doi.org/10.1038/s41467-020-20047-w
  20. Cell Death Dis. 2020 Nov 30. 11(11): 1019
    Constantin D, Widmann C.
      It is of clinical importance to identify biomarkers predicting the efficacy of DNA damaging drugs (genotoxins) so that nonresponders are not unduly exposed to the deleterious effects of otherwise inefficient drugs. Here, we initially focused on the bleomycin genotoxin because of the limited information about the genes implicated in the sensitivity or resistance to this compound. Using a whole-genome CRISPR/Cas9 gene knockout approach, we identified ASH2L, a core component of the H3K4 methyl transferase complex, as a protein required for bleomycin sensitivity in L1236 Hodgkin lymphoma. Knocking down ASH2L in these cells and in the NT2D1 testicular cancer cell line rendered them resistant to bleomycin, etoposide, and cisplatin but did not affect their sensitivity toward ATM or ATR inhibitors. ASH2L knockdown decreased cell proliferation and facilitated DNA repair via homologous recombination and nonhomologous end-joining mechanisms. Data from the Tumor Cancer Genome Atlas indicate that patients with testicular cancer carrying alterations in the ASH2L gene are more likely to relapse than patients with unaltered ASH2L genes. The cell models we have used are derived from cancers currently treated either partially (Hodgkin's lymphoma), or entirely (testicular cancer) with genotoxins. For such cancers, ASH2L levels could be used as a biomarker to predict the response to genotoxins. In situations where tumors are expressing low levels of ASH2L, which may allow them to resist genotoxic treatment, the use of ATR or ATM inhibitors may be more efficacious as our data indicate that ASH2L knockdown does not affect sensitivity to these inhibitors.
    DOI:  https://doi.org/10.1038/s41419-020-03231-0
  21. Subcell Biochem. 2021 ;96 233-258
    Yao NY, O'Donnell ME.
      In all cell types, a multi-protein machinery is required to accurately duplicate the large duplex DNA genome. This central life process requires five core replisome factors in all cellular life forms studied thus far. Unexpectedly, three of the five core replisome factors have no common ancestor between bacteria and eukaryotes. Accordingly, the replisome machines of bacteria and eukaryotes have important distinctions in the way that they are organized and function. This chapter outlines the major replication proteins that perform DNA duplication at replication forks, with particular attention to differences and similarities in the strategies used by eukaryotes and bacteria.
    Keywords:  Clamp loader; DNA polymerase; Helicase; Primase; Replisome; Sliding clamp
    DOI:  https://doi.org/10.1007/978-3-030-58971-4_5
  22. Nucleic Acids Res. 2020 Dec 04. pii: gkaa1145. [Epub ahead of print]
    Hanamshet K, Mazin AV.
      RAD52 is a member of the homologous recombination pathway that is important for survival of BRCA-deficient cells. Inhibition of RAD52 leads to lethality in BRCA-deficient cells. However, the exact mechanism of how RAD52 contributes to viability of BRCA-deficient cells remains unknown. Two major activities of RAD52 were previously identified: DNA or RNA pairing, which includes DNA/RNA annealing and strand exchange, and mediator, which is to assist RAD51 loading on RPA-covered ssDNA. Here, we report that the N-terminal domain (NTD) of RAD52 devoid of the potential mediator function is essential for maintaining viability of BRCA-deficient cells owing to its ability to promote DNA/RNA pairing. We show that RAD52 NTD forms nuclear foci upon DNA damage in BRCA-deficient human cells and promotes DNA double-strand break repair through two pathways: homology-directed repair (HDR) and single-strand annealing (SSA). Furthermore, we show that mutations in the RAD52 NTD that disrupt these activities fail to maintain viability of BRCA-deficient cells.
    DOI:  https://doi.org/10.1093/nar/gkaa1145
  23. Nat Commun. 2020 12 03. 11(1): 6182
    Sun X, Liu T, Zhao J, Xia H, Xie J, Guo Y, Zhong L, Li M, Yang Q, Peng C, Rouvet I, Belot A, Shu HB, Feng P, Zhang J.
      Upon sensing cytosolic DNA, the enzyme cGAS induces innate immune responses that underpin anti-microbial defenses and certain autoimmune diseases. Missense mutations of PRKDC encoding the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) are associated with autoimmune diseases, yet how DNA-PK deficiency leads to increased immune responses remains poorly understood. In this study, we report that DNA-PK phosphorylates cGAS and suppresses its enzymatic activity. DNA-PK deficiency reduces cGAS phosphorylation and promotes antiviral innate immune responses, thereby potently restricting viral replication. Moreover, cells isolated from DNA-PKcs-deficient mice or patients carrying PRKDC missense mutations exhibit an inflammatory gene expression signature. This study provides a rational explanation for the autoimmunity of patients with missense mutations of PRKDC, and suggests that cGAS-mediated immune signaling is a potential target for therapeutic interventions.
    DOI:  https://doi.org/10.1038/s41467-020-19941-0
  24. Nucleic Acids Res. 2020 Dec 02. pii: gkaa1081. [Epub ahead of print]
    Stivison EA, Young KJ, Symington LS.
      Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.
    DOI:  https://doi.org/10.1093/nar/gkaa1081
  25. Biochem J. 2020 Dec 01. pii: BCJ20200800. [Epub ahead of print]
    Jung H, Hawkins M, Lee S.
      The exocyclic amines of nucleobases can undergo deamination by various DNA damaging agents such as reactive oxygen species, nitric oxide, and water. The deamination of guanine and adenine generates the promutagenic xanthine and hypoxanthine, respectively. The exocyclic amines of bases in DNA are hydrogen bond donors, while the carbonyl moiety generated by the base deamination acts as hydrogen bond acceptors, which can alter base pairing properties of the purines. Xanthine is known to base pair with both cytosine and thymine, while hypoxanthine predominantly pairs with cytosine to promote A to G mutations. Despite the known promutagenicity of the major deaminated purines, structures of DNA polymerase bypassing these lesions have not been reported. To gain insights into the deaminated-induced mutagenesis, we solved crystal structures of human DNA polymerase η (polη) catalyzing across xanthine and hypoxanthine. In the catalytic site of polη, the deaminated guanine (i.e., xanthine) forms three Watson-Crick-like hydrogen bonds with an incoming dCTP, indicating the O2-enol tautomer of xanthine involves in the base pairing. The formation of the enol tautomer appears to be promoted by the minor groove contact by Gln38 of polη. When hypoxanthine is at the templating position, the deaminated adenine uses its O6-keto tautomer to form two Watson-Crick hydrogen bonds with an incoming dCTP, providing the structural basis for the high promutagenicity of hypoxanthine.
    Keywords:  DNA damage; DNA polymerase; deamination; mutagenesis; tautomerization; translesion synthesis
    DOI:  https://doi.org/10.1042/BCJ20200800
  26. Int J Mol Sci. 2020 Nov 27. pii: E9027. [Epub ahead of print]21(23):
    Boldinova EO, Belousova EA, Gagarinskaya DI, Maltseva EA, Khodyreva SN, Lavrik OI, Makarova AV.
      Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.
    Keywords:  FEN1; PolDIP2; PrimPol; RPA; strand displacement
    DOI:  https://doi.org/10.3390/ijms21239027
  27. Cancers (Basel). 2020 Nov 24. pii: E3494. [Epub ahead of print]12(12):
    Curtin N, Bai P.
      The role of poly(ADP-ribose) polymerase-1 (PARP1) in DNA repair and as a potential target for anticancer therapy has been under investigation for more than 50 years [...].
    DOI:  https://doi.org/10.3390/cancers12123494
  28. Mol Pharmacol. 2020 Dec 01. pii: MOLPHARM-AR-2020-000169. [Epub ahead of print]
    Miller SR, Zhang X, Hau RK, Jilek JL, Jennings EQ, Galligan JJ, Foil DH, Zorn KM, Ekins S, Wright SH, Cherrington NJ.
      Equilibrative nucleoside transporters (ENT) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/Cas9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse transcriptase inhibitors and thirty-seven nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine if there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, while nevirapine and lexibulin were ENT1 and ENT2 non-transported inhibitors. Bayesian models generated using Assay Central® machine learning software yielded reasonably high internal validation performance (ROC > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training dataset correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates, and can thereby circumvent the BTB through this transepithelial transport pathway. Significance Statement This study is the first to predict drug interactions with ENT1 and ENT2 using Bayesian modeling. Novel CRISPR/Cas9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates, and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.
    Keywords:  Uptake transporters (OATP, OAT, OCT, PEPT, MCT, NTCP, ASBT, etc.)s; nucleosides
    DOI:  https://doi.org/10.1124/molpharm.120.000169