bims-numges 2020-10-18 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020–10–18
39 papers selected by
Sean Rudd, Karolinska Institutet

cGAS suppresses genomic instability as a decelerator of replication forks.
Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ.
The E3 ligase RFWD3 stabilizes ORC in a p53-dependent manner.
Regulation of ALT-associated homology-directed repair by polyADP-ribosylation.
p31comet promotes homologous recombination by inactivating REV7 through the TRIP13 ATPase.
Inhibition of Chk1 by miR-320c increases oxaliplatin responsiveness in triple-negative breast cancer.
The SWI/SNF ATPase BRG1 stimulates DNA end resection and homologous recombination by reducing nucleosome density at DNA double strand breaks and by promoting the recruitment of the CtIP nuclease.
Hydrogen peroxide and Helicobacter pylori extract treatment combined with APE1 knockdown induce DNA damage, G2/M arrest and cell death in gastric cancer cell line.
Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.
The ubiquitination machinery of the Fanconi Anemia DNA repair pathway.
Roles of OB-Fold Proteins in Replication Stress.
The DNA damaging revolution.
Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.
Dendrogenin A Enhances Anti-Leukemic Effect of Anthracycline in Acute Myeloid Leukemia.
Synthetic Lethal Interactions of RECQ Helicases.
DNA Damage Response and Immune Defense.
The CINs of Polo-Like Kinase 1 in Cancer.
Targeting DNA Repair, Cell Cycle, and Tumor Microenvironment in B Cell Lymphoma.
Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells.
Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.
Venetoclax and alvocidib are both cytotoxic to acute myeloid leukemia cells resistant to cytarabine and clofarabine.
ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies.
Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase epsilon holoenzyme.
Elevated DHODH expression promotes cell proliferation via stabilizing β-catenin in esophageal squamous cell carcinoma.
ASLAN003, a potent dihydroorotate dehydrogenase inhibitor for differentiation of acute myeloid leukemia.
Intracellular Pharmacokinetics of Pyrimidine Analogues used in Oncology and the Correlation with Drug Action.
PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.
Mechanism of efficient double-strand break repair by a long non-coding RNA.
Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells.
Post-translational modifications of fumarase regulate its enzyme activity and function in respiration and the DNA damage response.
PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer.
CDK12: a potential therapeutic target in cancer.
Evolution of DNA replication origin specification and gene silencing mechanisms.
Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1.
H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage.
A pilot clinical trial of the cytidine deaminase inhibitor tetrahydrouridine combined with decitabine to target DNMT1 in advanced, chemorefractory pancreatic cancer.
SLFN5 influences proliferation and apoptosis by upregulating PTEN transcription via ZEB1 and inhibits the purine metabolic pathway in breast cancer.
Synthesis of benzimidazole nucleosides and their anticancer activity.
The role of Nrf2/PIWIL2/purine metabolism axis in controlling radiation-induced lung fibrosis.

Sci Adv. 2020 Oct;pii: eabb8941. [Epub ahead of print]6(42):

cGAS suppresses genomic instability as a decelerator of replication forks.

Hao Chen, Hao Chen, Jiamin Zhang, Yumin Wang, Antoine Simoneau, Hui Yang, Arthur S Levine, Lee Zou, Zhijian Chen, Li Lan.

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding-dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.

DOI: https://doi.org/10.1126/sciadv.abb8941
Nat Commun. 2020 Oct 16. 11(1): 5239

Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ.

Wei Yu, Chloé Lescale, Loelia Babin, Marie Bedora-Faure, Hélène Lenden-Hasse, Ludivine Baron, Caroline Demangel, José Yelamos, Erika Brunet, Ludovic Deriano.

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.

DOI: https://doi.org/10.1038/s41467-020-19060-w
Cell Cycle. 2020 Oct 12. 1-12

The E3 ligase RFWD3 stabilizes ORC in a p53-dependent manner.

Rosaline Y C Hsu, Sumanprava Giri, Yating Wang, Yo-Chuen Lin, Dazhen Liu, Susan Wopat, Arindam Chakraborty, Kannanganattu V Prasanth, Supriya G Prasanth.

  RFWD3 is an E3 ubiquitin ligase that plays important roles in DNA damage response and DNA replication. We have previously demonstrated that the stabilization of RFWD3 by PCNA at the replication fork enables ubiquitination of the single-stranded binding protein, RPA and its subsequent degradation for replication progression. Here, we report that RFWD3 associates with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA/LRWD1), components of the pre-replicative complex required for the initiation of DNA replication. Overexpression of ORC/ORCA leads to the stabilization of RFWD3. Interestingly, RFWD3 seems to stabilize ORC/ORCA in cells expressing wild type p53, as the depletion of RFWD3 reduces the levels of ORC/ORCA. Further, the catalytic activity of RFWD3 is required for the stabilization of ORC. Our results indicate that the RFWD3 promotes the stability of ORC, enabling efficient pre-RC assembly.

Keywords:  ORC; ORCA/LRWD1; RFWD3; p53; replication; ubiquitination

DOI:  https://doi.org/10.1080/15384101.2020.1829823
Nat Struct Mol Biol. 2020 Oct 12.

Regulation of ALT-associated homology-directed repair by polyADP-ribosylation.

Song My Hoang, Nicole Kaminski, Ragini Bhargava, Jonathan Barroso-González, Michelle L Lynskey, Laura García-Expósito, Justin L Roncaioli, Anne R Wondisford, Callen T Wallace, Simon C Watkins, Dominic I James, Ian D Waddell, Donald Ogilvie, Kate M Smith, Felipe da Veiga Leprevost, Dattatreya Mellacharevu, Alexey I Nesvizhskii, Jianfeng Li, Dominique Ray-Gallet, Robert W Sobol, Genevieve Almouzni, Roderick J O'Sullivan.

The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.

DOI: https://doi.org/10.1038/s41594-020-0512-7
Proc Natl Acad Sci U S A. 2020 Oct 13. pii: 202008830. [Epub ahead of print]

p31comet promotes homologous recombination by inactivating REV7 through the TRIP13 ATPase.

Prabha Sarangi, Connor S Clairmont, Lucas D Galli, Lisa A Moreau, Alan D D'Andrea.

  The repair of DNA double strand breaks (DSBs) that arise from external mutagenic agents and routine cellular processes is essential for life. DSBs are repaired by two major pathways, homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). DSB repair pathway choice is largely dictated at the step of 5'-3' DNA end resection, which is promoted during S phase, in part by BRCA1. Opposing end resection is the 53BP1 protein, which recruits the ssDNA-binding REV7-Shieldin complex to favor C-NHEJ repair. We recently identified TRIP13 as a proresection factor that remodels REV7, causing its dissociation from the Shieldin subunit SHLD3. Here, we identify p31comet, a negative regulator of MAD2 and the spindle assembly checkpoint, as an important mediator of the TRIP13-REV7 interaction. p31comet binds to the REV7-Shieldin complex in cells, promotes REV7 inactivation, and causes PARP inhibitor resistance. p31comet also participates in the extraction of REV7 from the chromatin. Furthermore, p31comet can counteract REV7 function in translesion synthesis (TLS) by releasing it from REV3 in the Pol ζ complex. Finally, p31comet, like TRIP13, is overexpressed in many cancers and this correlates with poor prognosis. Thus, we reveal a key player in the regulation of HR and TLS with significant clinical implications.

Keywords:  Fanconi anemia; PARP inhibitor; REV7; homologous recombination; translesion synthesis

DOI:  https://doi.org/10.1073/pnas.2008830117
Oncogenesis. 2020 Oct 11. 9(10): 91

Inhibition of Chk1 by miR-320c increases oxaliplatin responsiveness in triple-negative breast cancer.

Sera Lim, Yesol Kim, Soo-Been Lee, Hyeok-Gu Kang, Da-Hyun Kim, Jee Won Park, Daeun Chung, Hyunkyung Kong, Kyung Hyun Yoo, Yonghwan Kim, Wonshik Han, Kyung-Hee Chun, Jong Hoon Park.

Checkpoint kinase 1 (Chk1) expression is enhanced in most cancers owing to oncogenic activation and constant replicative stress. Chk1 inactivation is a promising cancer therapy, as its inactivation leads to genomic instability, chromosomal catastrophe, and cancer cell death. Herein, we observed that miR-320c, downregulated in triple-negative breast cancer (TNBC) patients, can target Chk1. In addition, downregulated miR-320c expression was associated with poor overall survival in TNBC patients. As Chk1 was associated with the DNA damage response (DDR), we investigated the effect of miR-320c on DDR in TNBC cells. To induce DNA damage, we used platinum-based drugs, especially oxaliplatin, which is most effective with miR-320c. We observed that overexpression of miR-320c in TNBC regulated the oxaliplatin responsiveness by mediating DNA damage repair through the negative regulation of Chk1 in vitro. Furthermore, using a xenograft model, a combination of miR-320c mimic and oxaliplatin effectively inhibited tumor progression. These investigations indicate the potential of miR-320c as a marker of oxaliplatin responsiveness and a therapeutic target to increase the efficacy of chemotherapy in TNBC.

DOI: https://doi.org/10.1038/s41389-020-00275-x
Cell Cycle. 2020 Oct 12. 1-19

The SWI/SNF ATPase BRG1 stimulates DNA end resection and homologous recombination by reducing nucleosome density at DNA double strand breaks and by promoting the recruitment of the CtIP nuclease.

Emily Hays, Elizabeth Nettleton, Caitlin Carter, Mariangel Morales, Lynn Vo, Max Passo, Renier Vélez-Cruz.

  DNA double strand breaks (DSBs) are among the most toxic DNA lesions and can be repaired accurately through homologous recombination (HR). HR requires processing of the DNA ends by nucleases (DNA end resection) in order to generate the required single-stranded DNA (ssDNA) regions. The SWI/SNF chromatin remodelers are 10-15 subunit complexes that contain one ATPase (BRG1 or BRM). Multiple subunits of these complexes have recently been identified as a novel family of tumor suppressors. These complexes are capable of remodeling chromatin by pushing nucleosomes along the DNA. More recent studies have identified these chromatin remodelers as important factors in DNA repair. Using the DR-U2OS reporter system, we show that the down regulation of BRG1 significantly reduces HR efficiency, while BRM has a minor effect. Inactivation of BRG1 impairs DSB repair and results in a defect in DNA end resection, as measured by the amount of BrdU-containing ssDNA generated after DNA damage. Inactivation of BRG1 also impairs the activation of the ATR kinase, reduces the levels of chromatin-bound RPA, and reduces the number of RPA and RAD51 foci after DNA damage. This defect in DNA end resection is explained by the defective recruitment of GFP-CtIP to laser-induced DSBs in the absence of BRG1. Importantly, we show that BRG1 reduces nucleosome density at DSBs. Finally, inactivation of BRG1 renders cells sensitive to anti-cancer drugs that induce DSBs. This study identifies BRG1 as an important factor for HR, which suggests that BRG1-mutated cancers have a DNA repair vulnerability that can be exploited therapeutically.

Keywords:  DNA end resection; DSB repair; SWI/SNF; chromatin remodelers; genomic instability; homologous recombination

DOI:  https://doi.org/10.1080/15384101.2020.1831256
DNA Repair (Amst). 2020 Sep 28. pii: S1568-7864(20)30225-1. [Epub ahead of print]96 102976

Hydrogen peroxide and Helicobacter pylori extract treatment combined with APE1 knockdown induce DNA damage, G2/M arrest and cell death in gastric cancer cell line.

Fernanda S Manoel-Caetano, Ana Flávia T Rossi, Marcelo Lima Ribeiro, Janesly Prates, Sonia Maria Oliani, Ana Elizabete Silva.

  Chronic inflammation resulting from Helicobacter pylori (H. pylori) infection, the major risk factor for gastric cancer, results in increased release of reactive oxygen species (ROS), promoting oxidative stress and DNA damage. APE1 endonuclease, a key component of the base excision repair (BER) pathway, is responsible for the repair of damage induced by ROS. However, the APE1 gene and other DNA damage response (DDR) genes are still poorly understood in gastric cancer. Thus, we aimed to investigate whether the silencing of APE1 by shRNA can interfere with the survival of AGS gastric cancer cells after treatment with hydrogen peroxide (H2O2) and/or H. pylori extract (HPE) and its relation with the expression of DDR genes (ATM, ATR, and H2AX) and miRNAs that target DDR genes. In the AGS cells expressing APE1, isolated or combined treatment with H2O2 and HPE promoted a slight increase in the cell proliferation and increased the levels of intracellular ROS and DNA double strand breaks (DSBs) indicated by ©H2AX foci, a reduction in the proportion of cells in the G0/G1 phase and an increase in the initial apoptosis rate. Moreover, upregulation of APE1, ATR, miR-15a, miR-21, miR-24 and miR-421 and downregulation of ATM and H2AX was observed. In silenced AGS cells after treatment with H2O2 alone or combined with HPE, we observed an increase in the cell proliferation rate and the levels of intracellular ROS and DSBs and a reduction in the proportion of cells in S and G2/M phase arrest, leading to late apoptosis. APE1 knockdown also caused a reduction in the expression of ATM and miR-421, while ATR expression was increased. Based on our results, APE1 knockdown may promote changes in cellular processes by increasing genomic instability, leading to G2/M arrest and cell apoptosis, so it may be a promising strategy for controlling tumor progression.

Keywords:  AGS cell line; APE1 knockdown; DNA damage; DNA repair; Helicobacter pylori; Hydrogen peroxide

DOI:  https://doi.org/10.1016/j.dnarep.2020.102976
Nucleic Acids Res. 2020 Oct 12. pii: gkaa820. [Epub ahead of print]

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

Ikenna Obi, Matilda Rentoft, Vandana Singh, Jan Jamroskovic, Karam Chand, Erik Chorell, Fredrik Westerlund, Nasim Sabouri.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

DOI: https://doi.org/10.1093/nar/gkaa820
Prog Biophys Mol Biol. 2020 Oct 12. pii: S0079-6107(20)30098-5. [Epub ahead of print]

The ubiquitination machinery of the Fanconi Anemia DNA repair pathway.

Winnie Tan, Andrew J Deans.

  The Fanconi Anemia (FA) pathway maintains genome stability by preventing DNA damage from occurring when replication is blocked. Central to the FA pathway is the monoubiquitination of FANCI-FANCD2 mediated by a ubiquitin RING-E3 ligase complex called the FA core complex. Genetic mutation in any component of the FA core complex results in defective FANCI-FANCD2 monoubiquitination and phenotypes of DNA damage sensitivity, birth defects, early-onset bone marrow failure and cancer. Here, we discuss the mechanisms of the FA core complex and FANCI-FANCD2 monoubiquitination at sites of blocked replication and review our current understanding of the biological functions of these proteins in replication fork protection.

Keywords:  DNA repair; FA core Complex; FANCI-FANCD2; Fanconi anemia; Interstrand crosslink repair; Ubiquitination

DOI:  https://doi.org/10.1016/j.pbiomolbio.2020.09.009
Front Cell Dev Biol. 2020 ;8 574466

Roles of OB-Fold Proteins in Replication Stress.

Dinh-Duc Nguyen, Eugene Y Kim, Pau Biak Sang, Weihang Chai.

  Accurate DNA replication is essential for maintaining genome stability. However, this stability becomes vulnerable when replication fork progression is stalled or slowed - a condition known as replication stress. Prolonged fork stalling can cause DNA damage, leading to genome instabilities. Thus, cells have developed several pathways and a complex set of proteins to overcome the challenge at stalled replication forks. Oligonucleotide/oligosaccharide binding (OB)-fold containing proteins are a group of proteins that play a crucial role in fork protection and fork restart. These proteins bind to single-stranded DNA with high affinity and prevent premature annealing and unwanted nuclease digestion. Among these OB-fold containing proteins, the best studied in eukaryotic cells are replication protein A (RPA) and breast cancer susceptibility protein 2 (BRCA2). Recently, another RPA-like protein complex CTC1-STN1-TEN1 (CST) complex has been found to counter replication perturbation. In this review, we discuss the latest findings on how these OB-fold containing proteins (RPA, BRCA2, CST) cooperate to safeguard DNA replication and maintain genome stability.

Keywords:  BRCA2; CST; OB-fold protein; RPA; genome stability; replication fork; replication stress; single strand DNA-binding protein

DOI:  https://doi.org/10.3389/fcell.2020.574466
Crit Rev Oncol Hematol. 2020 Oct 03. pii: S1040-8428(20)30253-5. [Epub ahead of print]156 103117

The DNA damaging revolution.

Bulent Cetin, Chiara A Wabl, Ozge Gumusay.

  Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that plays a critical role in the repair of single-strand DNA damage via the base excision repair pathway. PARP inhibitors have substantial single-agent antitumor activity by inducing synthetic lethality. They have also emerged as promising anticancer targeted therapies, especially in tumors harboring deleterious germline or somatic breast cancer susceptibility gene (BRCA) mutations. PARP inhibition produces single-strand DNA breaks, which may be repaired by homologous recombination, a process partially dependent on BRCA1 and BRCA2. The PARP inhibitors olaparib, veliparib, talazoparib, niraparib, and rucaparib have predominantly been studied in patients with breast or ovarian cancers associated with deleterious germline mutations in BRCA1 and BRCA2. Ongoing clinical trials are evaluating the role of PARP inhibitors alone and in combination with other therapies, including selective inhibitors against key targets involved in the DNA damage response. In this review we summarize the use of PARP inhibitors in various tumor types, as well as possible approaches for overcoming resistance to PARP inhibitors.

Keywords:  BRCA mutation; Combination therapy; DNA damage response; PARP inhibitorsl

DOI:  https://doi.org/10.1016/j.critrevonc.2020.103117
Gastroenterology. 2020 Oct 08. pii: S0016-5085(20)35229-X. [Epub ahead of print]

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

Glasgow Precision Oncology Laboratory

   BACKGROUND AND AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC.
METHODS: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids.
RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency.
CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.

Keywords:  DNA damage response; Pancreatic cancer; personalized medicine; replication stress

DOI:  https://doi.org/10.1053/j.gastro.2020.09.043
Cancers (Basel). 2020 Oct 12. pii: E2933. [Epub ahead of print]12(10):

Dendrogenin A Enhances Anti-Leukemic Effect of Anthracycline in Acute Myeloid Leukemia.

Pierre-Luc Mouchel, Nizar Serhan, Rémy Betous, Thomas Farge, Estelle Saland, Philippe De Medina, Jean-Sébastien Hoffmann, Jean-Emmanuel Sarry, Marc Poirot, Sandrine Silvente-Poirot, Christian Récher.

  Dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor suppressor properties, has recently been shown to exhibit strong anti-leukemic activity in acute myeloid leukemia (AML) cells by triggering lethal autophagy. Here, we demonstrated that DDA synergistically enhanced the toxicity of anthracyclines in AML cells but not in normal hematopoietic cells. Combination index of DDA treatment with either daunorubicin or idarubicin indicated a strong synergism in KG1a, KG1 and MV4-11 cell lines. This was confirmed in vivo using immunodeficient mice engrafted with MOLM-14 cells as well as in a panel of 20 genetically diverse AML patient samples. This effect was dependent on Liver X Receptor β, a major target of DDA. Furthermore, DDA plus idarubicin strongly increased p53BP1 expression and the number of DNA strand breaks in alkaline comet assays as compared to idarubicin alone, whereas DDA alone was non-genotoxic. Mechanistically, DDA induced JNK phosphorylation and the inhibition of AKT phosphorylation, thereby maximizing DNA damage induced by idarubicin and decreasing DNA repair. This activated autophagic cell death machinery in AML cells. Overall, this study shows that the combination of DDA and idarubicin is highly promising and supports clinical trials of dendrogenin A in AML patients.

Keywords:  AML; CLDX; DDA; DNA damage; LXR; anthracycline; autophagy; dendrogenin A; primary sample; synergy

DOI:  https://doi.org/10.3390/cancers12102933
Trends Cancer. 2020 Oct 08. pii: S2405-8033(20)30255-7. [Epub ahead of print]

Synthetic Lethal Interactions of RECQ Helicases.

Arindam Datta, Srijita Dhar, Sanket Awate, Robert M Brosh.

  DNA helicases have risen to the forefront as genome caretakers. Their prominent roles in chromosomal stability are demonstrated by the linkage of mutations in helicase genes to hereditary disorders with defects in DNA repair, the replication stress response, and/or transcriptional activation. Conversely, accumulating evidence suggests that DNA helicases in cancer cells have a network of pathway interactions such that codeficiency of some helicases and their genetically interacting proteins results in synthetic lethality (SL). Such genetic interactions may potentially be exploited for cancer therapies. We discuss the roles of RECQ DNA helicases in cancer, emphasizing some of the more recent developments in SL.

Keywords:  Bloom’s syndrome; RECQ; Rothmund–Thomson Syndrome; Werner syndrome; cancer; genetic disease; genomic stability; helicase; synthetic lethality

DOI:  https://doi.org/10.1016/j.trecan.2020.09.001
Int J Mol Sci. 2020 Oct 12. pii: E7504. [Epub ahead of print]21(20):

DNA Damage Response and Immune Defense.

Claudia Nastasi, Laura Mannarino, Maurizio D'Incalci.

  DNA damage is the cause of numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. The DNA damage response (DDR), in turn, coordinates DNA damage checkpoint activation and promotes the removal of DNA lesions. In recent years, several studies have shown how the DDR and the immune system are tightly connected, revealing an important crosstalk between the two of them. This interesting interplay has opened up new perspectives in clinical studies for immunological diseases as well as for cancer treatment. In this review, we provide an overview, from cellular to molecular pathways, on how DDR and the immune system communicate and share the crucial commitment of maintaining the genomic fitness.

Keywords:  DNA damage response; DNA repair; cancer; immune defense; immune signalling; innate immunity

DOI:  https://doi.org/10.3390/ijms21207504
Cancers (Basel). 2020 Oct 13. pii: E2953. [Epub ahead of print]12(10):

The CINs of Polo-Like Kinase 1 in Cancer.

Chelsea E Cunningham, Mackenzie J MacAuley, Frederick S Vizeacoumar, Omar Abuhussein, Andrew Freywald, Franco J Vizeacoumar.

  Polo-like kinase 1 (PLK1) is overexpressed near ubiquitously across all cancer types and dysregulation of this enzyme is closely tied to increased chromosomal instability and tumor heterogeneity. PLK1 is a mitotic kinase with a critical role in maintaining chromosomal integrity through its function in processes ranging from the mitotic checkpoint, centrosome biogenesis, bipolar spindle formation, chromosome segregation, DNA replication licensing, DNA damage repair, and cytokinesis. The relation between dysregulated PLK1 and chromosomal instability (CIN) makes it an attractive target for cancer therapy. However, clinical trials with PLK1 inhibitors as cancer drugs have generally displayed poor responses or adverse side-effects. This is in part because targeting CIN regulators, including PLK1, can elevate CIN to lethal levels in normal cells, affecting normal physiology. Nevertheless, aiming at related genetic interactions, such as synthetic dosage lethal (SDL) interactions of PLK1 instead of PLK1 itself, can help to avoid the detrimental side effects associated with increased levels of CIN. Since PLK1 overexpression contributes to tumor heterogeneity, targeting SDL interactions may also provide an effective strategy to suppressing this malignant phenotype in a personalized fashion.

Keywords:  DNA damage repair; Polo-like kinase 1; chromosomal instability; synthetic dosage lethality

DOI:  https://doi.org/10.3390/cancers12102953
Cells. 2020 Oct 14. pii: E2287. [Epub ahead of print]9(10):

Targeting DNA Repair, Cell Cycle, and Tumor Microenvironment in B Cell Lymphoma.

Paul J Bröckelmann, Mathilde R W de Jong, Ron D Jachimowicz.

  The DNA double-strand break (DSB) is the most cytotoxic lesion and compromises genome stability. In an attempt to efficiently repair DSBs, cells activate ATM kinase, which orchestrates the DNA damage response (DDR) by activating cell cycle checkpoints and initiating DSB repair pathways. In physiological B cell development, however, programmed DSBs are generated as intermediates for effective immune responses and the maintenance of genomic integrity. Disturbances of these pathways are at the heart of B cell lymphomagenesis. Here, we review the role of DNA repair and cell cycle control on B cell development and lymphomagenesis. In addition, we highlight the intricate relationship between the DDR and the tumor microenvironment (TME). Lastly, we provide a clinical perspective by highlighting treatment possibilities of defective DDR signaling and the TME in mantle cell lymphoma, which serves as a blueprint for B cell lymphomas.

Keywords:  ATM; B cell development; DNA damage response; DSB repair; STING; cell cycle; cyclin D1; immunotherapy; mantle cell lymphoma; tumor microenvironment

DOI:  https://doi.org/10.3390/cells9102287
Int J Mol Sci. 2020 Oct 13. pii: E7564. [Epub ahead of print]21(20):

Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells.

Dipon Das, Molly L Bristol, Pietro Pichierri, Iain M Morgan.

  Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20-50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein-protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1-E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

Keywords:  DNA damage; DNA lesion; DNA replication; E1 and E2; cervical cancer; head and neck cancer; human papillomaviruses; model system; replication and repair

DOI:  https://doi.org/10.3390/ijms21207564
Nucleic Acids Res. 2020 Oct 14. pii: gkaa859. [Epub ahead of print]

Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.

Tanja Kaufmann, Sébastien Herbert, Benjamin Hackl, Johanna Maria Besold, Christopher Schramek, Josef Gotzmann, Kareem Elsayad, Dea Slade.

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.

DOI: https://doi.org/10.1093/nar/gkaa859
BMC Cancer. 2020 Oct 12. 20(1): 984

Venetoclax and alvocidib are both cytotoxic to acute myeloid leukemia cells resistant to cytarabine and clofarabine.

Rie Nishi, Hiroko Shigemi, Eiju Negoro, Miyuki Okura, Naoko Hosono, Takahiro Yamauchi.

   BACKGROUND: Cytarabine (ara-C) is the major drug for the treatment of acute myeloid leukemia (AML), but cellular resistance to ara-C is a major obstacle to therapeutic success. The present study examined enhanced anti-apoptosis identified in 3 newly established nucleoside analogue-resistant leukemic cell line variants and approaches to overcoming this resistance.
METHODS: HL-60 human AML cells were used to develop the ara-C- or clofarabine (CAFdA)-resistant variants. The Bcl-2 inhibitor venetoclax and the Mcl-1 inhibitor alvocidib were tested to determine whether they could reverse these cells' resistance.
RESULTS: A 10-fold ara-C-resistant HL-60 variant, a 4-fold CAFdA-resistant HL-60 variant, and a 30-fold CAFdA-resistant HL-60 variant were newly established. The variants demonstrated reduced deoxycytidine kinase and deoxyguanosine kinase expression, but intact expression of surface transporters (hENT1, hENT2, hCNT3). The variants exhibited lower expression of intracellular nucleoside analogue triphosphates compared with non-variant HL-60 cells. The variants also overexpressed Bcl-2 and Mcl-1. Venetoclax as a single agent was not cytotoxic to the resistant variants. Nevertheless, venetoclax with nucleoside analogs demonstrated synergistic cytotoxicity against the variants. Alvocidib as a single agent was cytotoxic to the cells. However, alvocidib induced G1 arrest and suppressed the cytotoxicity of the co-administered nucleoside analogs.
CONCLUSIONS: Three new nucleoside analogue-resistant HL-60 cell variants exhibited reduced production of intracellular analogue triphosphates and enhanced Bcl-2 and Mcl-1 expressions. Venetoclax combined with nucleoside analogs showed synergistic anti-leukemic effects and overcame the drug resistance.

Keywords:  Alvocidib; Bcl-2; Clofarabine (CAFdA); Cytarabine (ara-C); Mcl-1; Venetoclax

DOI:  https://doi.org/10.1186/s12885-020-07469-x
Haematologica. 2019 Nov 14. 105(10): 2440-2447

ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies.

Oronza A Botrugno, Silvia Bianchessi, Desirée Zambroni, Michela Frenquelli, Daniela Belloni, Lucia Bongiovanni, Stefania Girlanda, Simona Di Terlizzi, Marina Ferrarini, Elisabetta Ferrero, Maurilio Ponzoni, Magda Marcatti, Giovanni Tonon.

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

DOI: https://doi.org/10.3324/haematol.2018.215210
J Biol Chem. 2020 Oct 13. pii: jbc.RA120.013903. [Epub ahead of print]

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase epsilon holoenzyme.

Walter J Zahurancik, Zucai Suo.

  In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, while DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the non-catalytic p59, p17, and p12 small subunits. So far, the contribution of the non-catalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared to the 140-kDa N-terminal catalytic fragment of p261 (p261N) which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to three orders of magnitude. Thus, the presence of p261C and the three non-catalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

Keywords:  DNA polymerase; DNA replication; enzyme mechanism; pre-steady-state kinetics; substrate specificity

DOI:  https://doi.org/10.1074/jbc.RA120.013903
Cell Death Dis. 2020 Oct 15. 11(10): 862

Elevated DHODH expression promotes cell proliferation via stabilizing β-catenin in esophageal squamous cell carcinoma.

Yu Qian, Xiao Liang, Pengzhou Kong, Yikun Cheng, Heyang Cui, Ting Yan, Jinghao Wang, Ling Zhang, Yiqian Liu, Shiping Guo, Xiaolong Cheng, Yongping Cui.

As a key enzyme in de novo pyrimidine biosynthesis, the expression level of dihydroorotate dehydrogenase (DHODH) has been reported to be elevated in various types of malignant tumors and its tumor-promoting effect was considered to relate to its pyrimidine synthesis function. Here, we revealed one intriguing potential mechanism that DHODH modulated β-catenin signaling in esophageal squamous cell carcinoma (ESCC). We demonstrated that DHODH directly bound to the NH2 terminal of β-catenin, thereby, interrupting the interaction of GSK3β with β-catenin and leading to the abrogation of β-catenin degradation and accumulation of β-catenin in the nucleus, which in turn, resulted in the activation of β-catenin downstream genes, including CCND1, E2F3, Nanog, and OCT4. We further demonstrated that the regulation of β-catenin by DHODH was independent of DHODH catalyzing activity. Univariate and multivariate analyses suggested that DHODH expression might be an independent prognostic factor for ESCC patients. Collectively, our study highlights the pivotal role of DHODH mediated β-catenin signaling and indicates that DHODH may act as a multi-functional switcher from catalyzing pyrimidine metabolism to regulating tumor-related signaling pathways in ESCC.

DOI: https://doi.org/10.1038/s41419-020-03044-1
Haematologica. 2019 Nov 07. 105(9): 2286-2297

ASLAN003, a potent dihydroorotate dehydrogenase inhibitor for differentiation of acute myeloid leukemia.

Jianbiao Zhou, Jessie Yiying Quah, Yvonne Ng, Jing-Yuan Chooi, Sabrina Hui-Min Toh, Baohong Lin, Tuan Zea Tan, Hiroki Hosoi, Motomi Osato, Qihui Seet, A G Lisa Ooi, Bertil Lindmark, Mark McHale, Wee-Joo Chng.

Differentiation therapies achieve remarkable success in acute promyelocytic leukemia, a subtype of acute myeloid leukemia. However, excluding acute promyelocytic leukemia, clinical benefits of differentiation therapies are negligible in acute myeloid leukemia except for mutant isocitrate dehydrogenase 1/2. Dihydroorotate dehydrogenase catalyses the fourth step of the de novo pyrimidine synthesis pathway. ASLAN003 is a highly potent dihydroorotate dehydrogenase inhibitor that induces differentiation, as well as reduces cell proliferation and viability, of acute myeloid leukemia cell lines and primary acute myeloid leukemia blasts including in chemo-resistant cells. Apoptotic pathways are triggered by ASLAN003, and it also significantly inhibits protein synthesis and activates AP-1 transcription, contributing to its differentiation promoting capacity. Finally, ASLAN003 substantially reduces leukemic burden and prolongs survival in acute myeloid leukemia xenograft mice and acute myeloid leukemia patient-derived xenograft models. Notably, the drug has no evident effect on normal hematopoietic cells and exhibits excellent safety profiles in mice, even after a prolonged period of administration. Our results, therefore, suggest that ASLAN003 is an agent targeting dihydroorotate dehydrogenase with potential in the treatment of acute myeloid leukemia. ASLAN003 is currently being evaluated in phase 2a clinical trial in acute myeloid leukemia patients.

DOI: https://doi.org/10.3324/haematol.2019.230482
Clin Pharmacokinet. 2020 Oct 16.

Intracellular Pharmacokinetics of Pyrimidine Analogues used in Oncology and the Correlation with Drug Action.

Ellen J B Derissen, Jos H Beijnen.

Pyrimidine analogues can be considered as prodrugs, like their natural counterparts, they have to be activated within the cell. The intracellular activation involves several metabolic steps including sequential phosphorylation to its monophosphate, diphosphate and triphosphate. The intracellularly formed nucleotides are responsible for the pharmacological effects. This review provides a comprehensive overview of the clinical studies that measured the intracellular nucleotide concentrations of pyrimidine analogues in patients with cancer. The objective was to gain more insight into the parallels between the different pyrimidine analogues considering their intracellular pharmacokinetics. For cytarabine and gemcitabine, the intracellular pharmacokinetics have been extensively studied over the years. However, for 5-fluorouracil, capecitabine, azacitidine and decitabine, the intracellular pharmacokinetics was only very minimally investigated. This is probably owing to the fact that there were no suitable bioanalytical assays for a long time. Since the advent of suitable assays, the first exploratory studies indicate that the intracellular 5-fluorouracil, azacitidine and decitabine nucleotide concentrations are very low compared with the intracellular nucleotide concentrations obtained during treatment with cytarabine or gemcitabine. Based on their pharmacology, the intracellular accumulation of nucleotides appears critical to the cytotoxicity of pyrimidine analogues. However, not many clinical studies have actually investigated the relationship between the intracellular nucleotide concentrations in patients with cancer and the anti-tumour effect. Only for cytarabine, a relationship was demonstrated between the intracellular triphosphate concentrations in leukaemic cells and the response rate in patients with AML. Future clinical studies should show, for the other pyrimidine analogues, whether there is a relationship between the intracellular nucleotide concentrations and the clinical outcome of patients. Research that examined the intracellular pharmacokinetics of cytarabine and gemcitabine focused primarily on the saturation aspect of the intracellular triphosphate formation. Attempts to improve the dosing regimen of gemcitabine were aimed at maximising the intracellular gemcitabine triphosphate concentrations. However, this strategy does not make sense, as efficient administration also means that less gemcitabine can be administered before dose-limiting toxicities are achieved. For all pyrimidine analogues, a linear relationship was found between the dose and the plasma concentration. However, no correlation was found between the plasma concentration and the intracellular nucleotide concentration. The concentration-time curves for the intracellular nucleotides showed considerable inter-individual variation. Therefore, the question arises whether pyrimidine analogue therapy should be more individualised. Future research should show which intracellular nucleotide concentrations are worth pursuing and whether dose individualisation is useful to achieve these concentrations.

DOI: https://doi.org/10.1007/s40262-020-00934-7
Genes Dev. 2020 Oct 15.

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

Scottish Genomes Partnership

  DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Keywords:  DNA replication; genome stability; growth disorders; human genetics; rare disease

DOI:  https://doi.org/10.1101/gad.340190.120
Nucleic Acids Res. 2020 Oct 12. pii: gkaa784. [Epub ahead of print]

Mechanism of efficient double-strand break repair by a long non-coding RNA.

Roopa Thapar, Jing L Wang, Michal Hammel, Ruiqiong Ye, Ke Liang, Chengcao Sun, Ales Hnizda, Shikang Liang, Su S Maw, Linda Lee, Heather Villarreal, Isaac Forrester, Shujuan Fang, Miaw-Sheue Tsai, Tom L Blundell, Anthony J Davis, Chunru Lin, Susan P Lees-Miller, Terence R Strick, John A Tainer.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

DOI: https://doi.org/10.1093/nar/gkaa784
Cancers (Basel). 2020 Oct 11. pii: E2919. [Epub ahead of print]12(10):

Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells.

Sylwia K Król, Agnieszka Kaczmarczyk, Kamil Wojnicki, Bartosz Wojtas, Bartłomiej Gielniewski, Wieslawa Grajkowska, Katarzyna Kotulska, Cezary Szczylik, Ryszard Czepko, Mariusz Banach, Wojciech Kaspera, Wojciech Szopa, Andrzej Marchel, Tomasz Czernicki, Bozena Kaminska.

  Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics.

Keywords:  RecQ helicases; drug sensitivity; gene knockdown; gliomas; proliferation

DOI:  https://doi.org/10.3390/cancers12102919
J Mol Biol. 2020 Oct 12. pii: S0022-2836(20)30572-6. [Epub ahead of print]

Post-translational modifications of fumarase regulate its enzyme activity and function in respiration and the DNA damage response.

Suqing Wang, Dharanidharan Ramamurthy, Jasper Tan, Jingyan Liu, Joyce Yip, Andrea Chua, Zhang Yu, Teck Kwang Lim, Qingsong Lin, Ophry Pines, Norbert Lehming.

  The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.

Keywords:  DNA repair; Fumarase; TCA cycle; enzymatic activity; post-translational modification

DOI:  https://doi.org/10.1016/j.jmb.2020.09.021
Cancers (Basel). 2020 Oct 10. pii: E2907. [Epub ahead of print]12(10):

PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer.

Pablo Iglesias, Marcos Seoane, Irene Golán, Isabel Castro-Piedras, Máximo Fraga, Víctor M Arce, Jose A Costoya.

  In recent years, poly (ADP-ribose) polymerase (PARP) inhibitors have been evaluated for treating homologous recombination-deficient tumours, taking advantage of synthetic lethality. However, increasing evidence indicates that PARP1 exert several cellular functions unrelated with their role on DNA repair, including function as a co-activator of transcription through protein-protein interaction with E2F1. Since the RB/E2F1 pathway is among the most frequently mutated in many tumour types, we investigated whether the absence of PARP activity could counteract the consequences of E2F1 hyperactivation. Our results demonstrate that genetic ablation of Parp1 extends the survival of Rb-null embryos, while genetic inactivation of Parp1 results in reduced development of pRb-dependent tumours. Our results demonstrate that PARP1 plays a key role as a transcriptional co-activator of the transcription factor E2F1, an important component of the cell cycle regulation. Considering that most oncogenic processes are associated with cell cycle deregulation, the disruption of this PARP1-E2F1 interaction could provide a new therapeutic target of great interest and a wide spectrum of indications.

Keywords:  E2F1 transcription factor; Poly (ADP-Ribose) Polymerase-1; animal disease models; cell cycle; glioma; neoplasm

DOI:  https://doi.org/10.3390/cancers12102907
Drug Discov Today. 2020 Oct 07. pii: S1359-6446(20)30395-0. [Epub ahead of print]

CDK12: a potential therapeutic target in cancer.

Fatemeh Emadi, Theodosia Teo, Muhammed H Rahaman, Shudong Wang.

Cyclin-dependent kinase (CDK) 12 engages in diversified biological functions, from transcription, post-transcriptional modification, cell cycle, and translation to cellular proliferation. Moreover, it regulates the expression of cancer-related genes involved in DNA damage response (DDR) and replication, which are responsible for maintaining genomic stability. CDK12 emerges as an oncogene or tumor suppressor in different cellular contexts, where its dysregulation results in tumorigenesis. Current CDK12 inhibitors are nonselective, which impedes the process of pharmacological target validation and drug development. Herein, we discuss the latest understanding of the biological roles of CDK12 in cancers and provide molecular analyses of CDK12 inhibitors to guide the rational design of selective inhibitors.

DOI: https://doi.org/10.1016/j.drudis.2020.09.035
Nat Commun. 2020 10 14. 11(1): 5175

Evolution of DNA replication origin specification and gene silencing mechanisms.

Y Hu, A Tareen, Y-J Sheu, W T Ireland, C Speck, H Li, L Joshua-Tor, J B Kinney, B Stillman.

DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

DOI: https://doi.org/10.1038/s41467-020-18964-x
DNA Repair (Amst). 2020 Oct 02. pii: S1568-7864(20)30226-3. [Epub ahead of print]96 102977

Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1.

Kostantin Kiianitsa, Yinbo Zhang, Nancy Maizels.

  The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

Keywords:  5-methylcytosine; CG island; DNA methylation; DNA-protein crosslink; RADAR enrichment; decitabine

DOI:  https://doi.org/10.1016/j.dnarep.2020.102977
Nat Struct Mol Biol. 2020 Oct 12.

H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage.

Siyao Wang, David H Meyer, Björn Schumacher.

DNA damage causes cancer, impairs development and accelerates aging. Transcription-blocking lesions and transcription-coupled repair defects lead to developmental failure and premature aging in humans. Following DNA repair, homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that, in Caenorhabditis elegans, removal of the WRAD complex of the MLL/COMPASS H3K4 methyltransferase exacerbates developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylases SPR-5 and AMX-1 promotes developmental growth and extends lifespan amid ultraviolet-induced damage. We demonstrate that DNA-damage-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis and protein homeostasis and regulates the recovery of protein biosynthesis that ensures survival following genotoxic stress. Our study uncovers a role for H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis required for developmental growth and longevity after DNA damage.

DOI: https://doi.org/10.1038/s41594-020-00513-1
Am J Cancer Res. 2020 ;10(9): 3047-3060

A pilot clinical trial of the cytidine deaminase inhibitor tetrahydrouridine combined with decitabine to target DNMT1 in advanced, chemorefractory pancreatic cancer.

Davendra Sohal, Smitha Krishnamurthi, Rita Tohme, Xiaorong Gu, Daniel Lindner, Terry H Landowski, John Pink, Tomas Radivoyevitch, Sherry Fada, Zhenghong Lee, Dale Shepard, Alok Khorana, Yogen Saunthararajah.

DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic vs resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.

Keywords: Pancreatic cancer; clinical trial; deaminase inhibitor
Am J Cancer Res. 2020 ;10(9): 2832-2850

SLFN5 influences proliferation and apoptosis by upregulating PTEN transcription via ZEB1 and inhibits the purine metabolic pathway in breast cancer.

Xuefeng Gu, Guoqing Wan, Yue Yang, Yihao Liu, Xintong Yang, Yanjun Zheng, Liying Jiang, Peng Zhang, Dingsheng Liu, Weiming Zhao, Gang Huang, Changlian Lu.

Human Schlafen-5 (SLFN5) is aberrantly involved in tumorigenesis in several types of cancer. However, its implications in breast cancer (BRCA) are unknown. Herein, we demonstrated that SLFN5 expression is negatively associated with the tumour growth of human BRCA using GEO database analysis and clinical sample immunostaining. Lentiviral overexpression of SLFN5 in BRCA cell lines suppressed tumourigenicity in nude mice. Knockdown and overexpression of SLFN5 in BRCA cell lines proved that SLFN5 can inhibit cell proliferation and colony formation and promote apoptosis by upregulating the transcription of a known cancer suppressor gene (the phosphatase and tensin homologue on chromosome 10, PTEN), resulting in molecular changes in the downstream AKT pathway and in proliferation/apoptosis. Lentiviral knockdown and overexpression of ZEB1 blocked the changes in the PTEN and AKT pathways and in the colony formation ability caused by SLFN5 knockdown and overexpression, respectively. Luciferase reporter assays demonstrated that ZEB1 can inhibit the PTEN promoter activity in MCF7 cells by binding to a motif in the PTEN promoter. Metabonomics analysis showed that SLFN5 influences many metabolic pathways and especially decreases purine metabolites, including inosine, xanthine, and hypoxanthine. In conclusion, our findings suggest that SLFN5 may be an important protective factor against BRCA, as it regulates PTEN transcription, the AKT pathway, and proliferation/apoptosis via ZEB1 mediation and inhibits the purine metabolic pathway. Thus, SLFN5 may be a potential therapeutic target for BRCA.

Keywords: PTEN; SLFN5; apoptosis; breast cancer; purine metabolism
Carbohydr Res. 2020 Oct 08. pii: S0008-6215(20)30549-8. [Epub ahead of print]498 108178

Synthesis of benzimidazole nucleosides and their anticancer activity.

Vaishali S Shinde, Pravin P Lawande, Vyankat A Sontakke, Ayesha Khan.

  An efficient route for the synthesis of benzimidazole nucleosides 1-8 from readily available d-glucose via 3,5-dihydroxy-1,2-O-isopropylidene-α-d-ribofuranose and 3-azido-3-deoxy-1,2-O-isopropylidene-α-d-xylofuranose intermediates has been adopted. Ribofuranosyl nucleosides 1-4 with different benzimidazole bases, and 3'-deoxy-3'-azido-ribofuranosyl nucleosides 5-8, as another series, were obtained. All these newly synthesized analogs were evaluated for anticancer activity using MDA-MB-231 cell line. Among the differently substituted derivatives, 3'-azide substituted nucleosides (5-8) are more potent compared to ribofuranosyl analogs 1-4. The C-3'-azido analog 8 having anthryl group at 2-position of nucleobase show almost similar potency as that of standard etoposide.

Keywords:  Anticancer activity; Benzimidazole; Sugar-modified nucleosides; Vorbrüggen glycosylation; d-Glucose

DOI:  https://doi.org/10.1016/j.carres.2020.108178
Am J Cancer Res. 2020 ;10(9): 2752-2767

The role of Nrf2/PIWIL2/purine metabolism axis in controlling radiation-induced lung fibrosis.

Guan-Lian Zou, Xiao-Ran Zhang, Yan-Li Ma, Qing Lu, Ren Zhao, Yong-Zhao Zhu, Yan-Yang Wang.

NF-E2-related factor 2 (Nrf2) is a key transcription factor recently implicated in the control of radiation-induced lung fibrosis (RILF). However, the molecular mechanism of Nrf2 in the pathogenesis of RILF is still unclear. The purpose of this study was to evaluate the regulatory effect and mechanism of Nrf2 in the pathogenesis of RILF. The effects of different Nrf2 expression levels on RILF were explored in vitro and in vivo. The RILF model of Nrf2 knockout mice was established for in vivo study. In the study of the mechanism of action, ChIP-seq assay and metabolomics analysis were performed. The discovered mechanism of Nrf2-mediated RILF alleviation was further validated in vitro and in vivo. We found that overexpression of Nrf2 significantly alleviated the fibrosis caused by irradiation in vivo and in vitro. Conversely, Nrf2 silencing strongly aggravated the development of RILF. Mechanistically, Nrf2 signaling increased the expression of piwi-like RNA-mediated gene silencing 2 (PIWIL2), leading to the alteration of purine metabolism and contributing to the relief of RILF. These results suggest that Nrf2 promotes the attenuation of RILF in vivo and in vitro by directly targeting PIWIL2 and activating purine metabolism.

Keywords: Nrf2; PIWIL2; Radiation-induced lung fibrosis; purine metabolism