bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒10‒10
twenty-nine papers selected by
Sean Rudd
Karolinska Institutet
  1. Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.
  2. Preventing excess replication origin activation to ensure genome stability.
  3. POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.
  4. CDK2 phosphorylation of Werner protein (WRN) contributes to WRN's DNA double-strand break repair pathway choice.
  5. DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks.
  6. Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.
  7. Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.
  8. Control of structure-specific endonucleases during homologous recombination in eukaryotes.
  9. Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ.
  10. Beyond Base Excision Repair: An evolving picture of mitochondrial DNA repair.
  11. USP37 regulates DNA damage response through stabilizing and deubiquitinating BLM.
  12. Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair.
  13. The SWI/SNF complex, transcription-replication conflicts and cancer: a connection with high therapeutic potential.
  14. CRISPR screens guide the way for PARP and ATR inhibitors biomarker discovery.
  15. Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway.
  16. CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.
  17. Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma.
  18. CDKL5 kinase controls transcription-coupled responses to DNA damage.
  19. USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.
  20. NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.
  21. Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.
  22. BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model.
  23. SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.
  24. STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.
  25. The amazing cN-II, the enzyme that keeps us busy.
  26. Specific 8-oxo-dGTPase activity of MTH1 (NUDT1) protein as a quantitative marker and prognostic factor in human colorectal cancer.
  27. Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound.
  28. NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.
  29. Teriflunomide - The common drug with underestimated oxygen - Dependent anticancer potential.
  1. Mol Cell. 2021 Oct 07. pii: S1097-2765(21)00747-4. [Epub ahead of print]81(19): 4026-4040.e8
    Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.
    Stephanie Tirman, Annabel Quinet, Matthew Wood, Alice Meroni, Emily Cybulla, Jessica Jackson, Silvia Pegoraro, Antoine Simoneau, Lee Zou, Alessandro Vindigni.
      PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
    Keywords:  BRCA1; BRCA2; DNA damage tolerance; DNA replication; PRIMPOL; genome stability; post-replicative repair; replication stress; ssDNA gaps; translesion synthesis polymerases
    DOI:  https://doi.org/10.1016/j.molcel.2021.09.013
  2. Trends Genet. 2021 Oct 05. pii: S0168-9525(21)00269-9. [Epub ahead of print]
    Preventing excess replication origin activation to ensure genome stability.
    Bhushan L Thakur, Anagh Ray, Christophe E Redon, Mirit I Aladjem.
      Cells activate distinctive regulatory pathways that prevent excessive initiation of DNA replication to achieve timely and accurate genome duplication. Excess DNA synthesis is constrained by protein-DNA interactions that inhibit initiation at dormant origins. In parallel, specific modifications of pre-replication complexes prohibit post-replicative origin relicensing. Replication stress ensues when the controls that prevent excess replication are missing in cancer cells, which often harbor extrachromosomal DNA that can be further amplified by recombination-mediated processes to generate chromosomal translocations. The genomic instability that accompanies excess replication origin activation can provide a promising target for therapeutic intervention. Here we review molecular pathways that modulate replication origin dormancy, prevent excess origin activation, and detect, encapsulate, and eliminate persistent excess DNA.
    Keywords:  DNA damage; dormant origins; extrachromosomal DNA; extrachromosomal circular DNA; genomic instability; overreplication; re-replication; replication origins
    DOI:  https://doi.org/10.1016/j.tig.2021.09.008
  3. Nat Cell Biol. 2021 Oct 06.
    POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.
    Marta Llorens-Agost, Michael Ensminger, Hang Phuong Le, Anugrah Gawai, Jie Liu, Andrés Cruz-García, Sarita Bhetawal, Richard D Wood, Wolf-Dietrich Heyer, Markus Löbrich.
      BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.
    DOI:  https://doi.org/10.1038/s41556-021-00764-0
  4. Aging Cell. 2021 Oct 06. e13484
    CDK2 phosphorylation of Werner protein (WRN) contributes to WRN's DNA double-strand break repair pathway choice.
    Jong-Hyuk Lee, Raghavendra A Shamanna, Tomasz Kulikowicz, Nima Borhan Fakouri, Edward W Kim, Louise S Christiansen, Deborah L Croteau, Vilhelm A Bohr.
      Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser-induced DNA double-strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non-homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN's affinity for RPA, likely increasing its long-range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2-dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.
    Keywords:  DNA double strand break; DNA repair; Werner Syndrome; aging; phosphorylation
    DOI:  https://doi.org/10.1111/acel.13484
  5. Elife. 2021 Oct 05. pii: e70787. [Epub ahead of print]10
    DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks.
    Shalini Aricthota, Devyani Haldar.
      In eukaryotes, paused replication forks are prone to collapse, which leads to genomic instability, a hallmark of cancer. Dbf4 Dependent Kinase (DDK)/Hsk1Cdc7 is a conserved replication initiator kinase with conflicting roles in replication stress response. Here, we show that fission yeast DDK/Hsk1 phosphorylates sirtuin, Hst4 upon replication stress at C-terminal serine residues. Phosphorylation of Hst4 by DDK marks it for degradation via the ubiquitin ligase SCFpof3. Phosphorylation defective hst4 mutant (4SA-hst4) displays defective recovery from replication stress, faulty fork restart, slow S-phase progression and decreased viability. The highly conserved Fork Protection Complex (FPC) stabilizes stalled replication forks. We found that the recruitment of FPC components, Swi1 and Mcl1 to the chromatin is compromised in the 4SA-hst4 mutant, although whole cell levels increased. These defects are dependent upon H3K56ac and independent of intra S-phase checkpoint activation. Finally, we show conservation of H3K56ac dependent regulation of Timeless, Tipin and And-1 in human cells. We propose that degradation of Hst4 via DDK increases H3K56ac, changing the chromatin state in the vicinity of stalled forks facilitating recruitment and function of FPC. Overall, this study identified a crucial role of DDK and FPC in the regulation of replication stress response with implications in cancer therapeutics.
    Keywords:  S. pombe; chromosomes; gene expression
    DOI:  https://doi.org/10.7554/eLife.70787
  6. J Cell Biol. 2021 Dec 06. pii: e202106116. [Epub ahead of print]220(12):
    Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.
    Delisa E Clay, Heidi S Bretscher, Erin A Jezuit, Korie B Bush, Donald T Fox.
      Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.
    DOI:  https://doi.org/10.1083/jcb.202106116
  7. Front Oncol. 2021 ;11 733700
    Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.
    Arafat Siddiqui, Manuela Tumiati, Alia Joko, Jouko Sandholm, Pia Roering, Sofia Aakko, Reetta Vainionpää, Katja Kaipio, Kaisa Huhtinen, Liisa Kauppi, Johanna Tuomela, Sakari Hietanen.
      Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.
    Keywords:  DNA repair; c-Abl; cell cycle arrest; imatinib; mitoxantrone
    DOI:  https://doi.org/10.3389/fonc.2021.733700
  8. Curr Opin Genet Dev. 2021 Oct 05. pii: S0959-437X(21)00111-8. [Epub ahead of print]71 195-205
    Control of structure-specific endonucleases during homologous recombination in eukaryotes.
    Cédric Giaccherini, Pierre-Henri L Gaillard.
      Structure-Specific Endonucleases (SSE) are specialized DNA endonucleases that recognize and process DNA secondary structures without any strict dependency on the nucleotide sequence context. This enables them to act virtually anywhere in the genome and to make key contributions to the maintenance of genome stability by removing DNA structures that may stall essential cellular processes such as DNA replication, transcription, repair and chromosome segregation. During repair of double strand breaks by homologous recombination mechanisms, DNA secondary structures are formed and processed in a timely manner. Their homeostasis relies on the combined action of helicases, SSE and topoisomerases. In this review, we focus on how SSE contribute to DNA end resection, single-strand annealing and double-strand break repair, with an emphasis on how their action is fine-tuned in those processes.
    DOI:  https://doi.org/10.1016/j.gde.2021.09.005
  9. Mitochondrion. 2021 Oct 04. pii: S1567-7249(21)00136-7. [Epub ahead of print]
    Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ.
    Elena J Ciesielska, Shalom Kim, Hyacintha-Ghislaine M Bisimwa, Cody Grier, Md Mostafijur Rahman, Carolyn K J Young, Matthew J Young, Marcos T Oliveira, Grzegorz L Ciesielski.
      The COVID-19 pandemic prompted the FDA to authorize a new nucleoside analogue, remdesivir, for emergency use in affected individuals. We examined the effects of its active metabolite, remdesivir triphosphate (RTP), on the activity of the replicative mitochondrial DNA polymerase, Pol γ. We found that while RTP is not incorporated by Pol γ into a nascent DNA strand, it remains associated with the enzyme impeding its synthetic activity and stimulating exonucleolysis. In spite of that, we found no evidence for deleterious effects of remdesivir treatment on the integrity of the mitochondrial genome in human cells in culture.
    Keywords:  Antiviral nucleoside analogues; COVID-19; DNA polymerase gamma; Mitochondrial DNA; Remdesivir
    DOI:  https://doi.org/10.1016/j.mito.2021.09.010
  10. Biosci Rep. 2021 Oct 05. pii: BSR20211320. [Epub ahead of print]
    Beyond Base Excision Repair: An evolving picture of mitochondrial DNA repair.
    Kathrin Allkanjari, Robert A Baldock.
      Mitochondria are highly specialised organelles required for cellular processes including ATP-production through cellular respiration and controlling apoptosis. Mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function - deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular aging and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.
    Keywords:  DNA replication and recombination; DNA synthesis and repair; genome integrity; mtDNA
    DOI:  https://doi.org/10.1042/BSR20211320
  11. Nucleic Acids Res. 2021 Oct 04. pii: gkab842. [Epub ahead of print]
    USP37 regulates DNA damage response through stabilizing and deubiquitinating BLM.
    Chenming Wu, Yiming Chang, Junliang Chen, Yang Su, Lei Li, Yuping Chen, Yunhui Li, Jinhuan Wu, Jinzhou Huang, Fei Zhao, Wenrui Wang, Hui Yin, Shunli Wang, Mingpeng Jin, Zhenkun Lou, Wei-Guo Zhu, Kuntian Luo, Jie Zhang, Jian Yuan.
      The human RecQ helicase BLM is involved in the DNA damage response, DNA metabolism, and genetic stability. Loss of function mutations in BLM cause the genetic instability/cancer predisposition syndrome Bloom syndrome. However, the molecular mechanism underlying the regulation of BLM in cancers remains largely elusive. Here, we demonstrate that the deubiquitinating enzyme USP37 interacts with BLM and that USP37 deubiquitinates and stabilizes BLM, thereby sustaining the DNA damage response (DDR). Mechanistically, DNA double-strand breaks (DSB) promotes ATM phosphorylation of USP37 and enhances the binding between USP37 and BLM. Moreover, knockdown of USP37 increases BLM polyubiquitination, accelerates its proteolysis, and impairs its function in DNA damage response. This leads to enhanced DNA damage and sensitizes breast cancer cells to DNA-damaging agents in both cell culture and in vivo mouse models. Collectively, our results establish a novel molecular mechanism for the USP37-BLM axis in regulating DSB repair with an important role in chemotherapy and radiotherapy response in human cancers.
    DOI:  https://doi.org/10.1093/nar/gkab842
  12. Life Sci Alliance. 2021 Dec;pii: e202101144. [Epub ahead of print]4(12):
    Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair.
    Jingyi Tang, Patrick J Casey, Mei Wang.
      DNA damage is a double-edged sword for cancer cells. On the one hand, DNA damage-induced genomic instability contributes to cancer development; on the other hand, accumulating damage compromises proliferation and survival of cancer cells. Understanding the key regulators of DNA damage repair machinery would benefit the development of cancer therapies that induce DNA damage and apoptosis. In this study, we found that isoprenylcysteine carboxylmethyltransferase (ICMT), a posttranslational modification enzyme, plays an important role in DNA damage repair. We found that ICMT suppression consistently reduces the activity of MAPK signaling, which compromises the expression of key proteins in the DNA damage repair machinery. The ensuing accumulation of DNA damage leads to cell cycle arrest and apoptosis in multiple breast cancer cells. Interestingly, these observations are more pronounced in cells grown under anchorage-independent conditions or grown in vivo. Consistent with the negative impact on DNA repair, ICMT inhibition transforms the cancer cells into a "BRCA-like" state, hence sensitizing cancer cells to the treatment of PARP inhibitor and other DNA damage-inducing agents.
    DOI:  https://doi.org/10.26508/lsa.202101144
  13. Mol Cell Oncol. 2021 ;8(4): 1976582
    The SWI/SNF complex, transcription-replication conflicts and cancer: a connection with high therapeutic potential.
    Aleix Bayona-Feliu, Andrés Aguilera.
      Genome instability is a hallmark of cancer. ATP-dependent chromatin remodelers are frequently altered in cancer. We have recently reported that the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protects the genome by limiting R-loop-mediated genome instability, mainly that caused by transcription-replication conflicts. Here we discuss the significance and biomedical applications of this finding.
    Keywords:  Cancer; R-loops; SWI/SNF; cBAF; chromatin; epigenetics; genome instability; transcription-replication conflicts
    DOI:  https://doi.org/10.1080/23723556.2021.1976582
  14. FEBS J. 2021 Oct 03.
    CRISPR screens guide the way for PARP and ATR inhibitors biomarker discovery.
    Emily M Schleicher, George-Lucian Moldovan.
      DNA repair pathways are heavily studied for their role in cancer initiation and progression. Due to the large amount of inherent DNA damage in cancer cells, tumor cells profoundly rely on proper DNA repair for efficient cell cycle progression. Several current chemotherapeutics promote excessive DNA damage in cancer cells, thus leading to cell death during cell cycle progression. However, if the tumor has efficient DNA repair mechanisms, DNA damaging therapeutics may not be as effective. Therefore, directly inhibiting DNA repair pathways alone and in combination with chemotherapeutics that cause DNA damage, may result in improved clinical outcomes. Nevertheless, tumors can acquire resistance to DNA repair inhibitors. It is essential to understand the genetic mechanisms underlying this resistance. Genome-wide CRISPR screening has emerged as a powerful tool to identify biomarkers of resistance or sensitivity to DNA repair inhibitors. CRISPR knockout and CRISPR activation screens can be designed to investigate how the loss or overexpression of any human gene impacts resistance or sensitivity to specific inhibitors. This review will address the role of CRISPR-screening in identifying biomarkers of resistance and sensitivity to DNA repair pathway inhibitors. We will focus on inhibitors targeting the PARP1 and ATR enzymes, and how the biomarkers identified from CRISPR screens can help inform the treatment plan for cancer patients.
    Keywords:  ATR; BRCA; CRISPR screens; DNA repair; PARP; genetic stability
    DOI:  https://doi.org/10.1111/febs.16217
  15. Cell Death Differ. 2021 Oct 05.
    Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway.
    Cristian Prieto-Garcia, Oliver Hartmann, Michaela Reissland, Thomas Fischer, Carina R Maier, Mathias Rosenfeldt, Christina Schülein-Völk, Kevin Klann, Reinhard Kalb, Ivan Dikic, Christian Münch, Markus E Diefenbacher.
      Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.
    DOI:  https://doi.org/10.1038/s41418-021-00875-z
  16. Cell Rep Med. 2021 Sep 21. 2(9): 100394
    CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models.
    Haineng Xu, Erin George, Yasuto Kinose, Hyoung Kim, Jennifer B Shah, Jasmine D Peake, Benjamin Ferman, Sergey Medvedev, Thomas Murtha, Carter J Barger, Kyle M Devins, Kurt D'Andrea, Bradley Wubbenhorst, Lauren E Schwartz, Wei-Ting Hwang, Gordon B Mills, Katherine L Nathanson, Adam R Karpf, Ronny Drapkin, Eric J Brown, Fiona Simpkins.
      CCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.
    Keywords:  ATR; CCNE1 copy number; WEE1; biomarker; ovarian and endometrial cancer
    DOI:  https://doi.org/10.1016/j.xcrm.2021.100394
  17. Blood Cancer J. 2021 Oct 08. 11(10): 166
    Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma.
    Srikanth Talluri, Mehmet K Samur, Leutz Buon, Subodh Kumar, Lakshmi B Potluri, Jialan Shi, Rao H Prabhala, Masood A Shammas, Nikhil C Munshi.
      Multiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.
    DOI:  https://doi.org/10.1038/s41408-021-00554-9
  18. EMBO J. 2021 Oct 04. e108271
    CDKL5 kinase controls transcription-coupled responses to DNA damage.
    Taran Khanam, Ivan Muñoz, Florian Weiland, Thomas Carroll, Michael Morgan, Barbara N Borsos, Vasiliki Pantazi, Meghan Slean, Miroslav Novak, Rachel Toth, Paul Appleton, Tibor Pankotai, Houjiang Zhou, John Rouse.
      Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.
    Keywords:  CDKL5 disorder; DNA damage response; kinase; poly(ADP-ribose); transcriptional regulation
    DOI:  https://doi.org/10.15252/embj.2021108271
  19. Nucleic Acids Res. 2021 Oct 06. pii: gkab892. [Epub ahead of print]
    USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.
    Jae Jin Kim, Seo Yun Lee, Yiseul Hwang, Soyeon Kim, Jee Min Chung, Sangwook Park, Junghyun Yoon, Hansol Yun, Jae-Hoon Ji, Sunyoung Chae, Hyeseong Cho, Chan Gil Kim, Ted M Dawson, Hongtae Kim, Valina L Dawson, Ho Chul Kang.
      Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.
    DOI:  https://doi.org/10.1093/nar/gkab892
  20. Mol Cell. 2021 Oct 07. pii: S1097-2765(21)00750-4. [Epub ahead of print]81(19): 4041-4058.e15
    NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.
    Hongshan Guo, Gabriel Golczer, Ben S Wittner, Adam Langenbucher, Marcus Zachariah, Taronish D Dubash, Xin Hong, Valentine Comaills, Risa Burr, Richard Y Ebright, Elad Horwitz, Joanna A Vuille, Soroush Hajizadeh, Devon F Wiley, Brittany A Reeves, Jia-Min Zhang, Kira L Niederhoffer, Chenyue Lu, Benjamin Wesley, Uyen Ho, Linda T Nieman, Mehmet Toner, Shobha Vasudevan, Lee Zou, Raul Mostoslavsky, Shyamala Maheswaran, Michael S Lawrence, Daniel A Haber.
      Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.
    Keywords:  CTCs; IEGs; R-loops; circulating tumor cells; genomic instability; immediate early genes; orphan nuclear receptor; replication stress; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molcel.2021.09.016
  21. Nucleic Acids Res. 2021 Oct 04. pii: gkab871. [Epub ahead of print]
    Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.
    Linlin Yang, Changxian Shen, Adriana Estrada-Bernal, Ryan Robb, Moumita Chatterjee, Nikhil Sebastian, Amy Webb, Xiaokui Mo, Wei Chen, Sunil Krishnan, Terence M Williams.
      KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.
    DOI:  https://doi.org/10.1093/nar/gkab871
  22. Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2108421118. [Epub ahead of print]118(41):
    BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model.
    Ye He, Joshua Rivera, Miklos Diossy, Haohui Duan, Christian Bowman-Colin, Rachel Reed, Rebecca Jennings, Jesse Novak, Stevenson V Tran, Elizabeth F Cohen, David Szuts, Anita Giobbie-Hurder, Roderick T Bronson, Adam J Bass, Sabina Signoretti, Zoltan Szallasi, David M Livingston, Shailja Pathania.
      BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.
    Keywords:  BRCA1; haploinsufficiency; mouse model; replication stress
    DOI:  https://doi.org/10.1073/pnas.2108421118
  23. Blood. 2020 Oct 08. pii: blood.2021012081. [Epub ahead of print]
    SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.
    Anne C Wilke, Carmen Doebele, Alena Zindel, Kwang Seok Lee, Sara A Rieke, Michele Ceribelli, Federico Comoglio, James D Phelan, James Q Wang, Yana Pikman, Dominique Jahn, Björn Häupl, Constanze Schneider, Sebastian Scheich, Frances A Tosto, Hanibal Bohnenberger, Philipp Stauder, Frank Schnuetgen, Mikolaj Slabicki, Zana A Coulibaly, Sebastian Wolf, Kamil Bojarczuk, Björn Chapuy, Christian H Brandts, Philipp Stroebel, Caroline A Lewis, Michael Engelke, Xincheng Xu, Hahn Kim, Thanh Hung Dang, Roland Schmitz, Daniel J Hodson, Kimberly Stegmaier, Henning Urlaub, Hubert Serve, Clemens A Schmitt, Fernando Kreuz, Gero Knittel, Joshua D Rabinowitz, Hans Christian Reinhardt, Matthew G Vander Heiden, Craig Thomas, Louis Michael Staudt, Thorsten Zenz, Thomas Oellerich.
      Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome of the majority of BL patients, chemotherapy-related toxicity and disease relapse remain as major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. As a consequence, this led to a collapse of tonic B-cell receptor signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we furthermore identified drugs such as methotrexate that synergized with SHMT inhibitors (SHMT2i). Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.
    DOI:  https://doi.org/10.1182/blood.2021012081
  24. Oncogene. 2021 Oct 08.
    STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.
    Laura Cheradame, Ida Chiara Guerrera, Julie Gaston, Alain Schmitt, Vincent Jung, Nicolas Goudin, Marion Pouillard, Nina Radosevic-Robin, Mauro Modesti, Jean-Gabriel Judde, Stefano Cairo, Vincent Goffin.
      STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance.
    DOI:  https://doi.org/10.1038/s41388-021-02037-4
  25. Nucleosides Nucleotides Nucleic Acids. 2021 Oct 06. 1-8
    The amazing cN-II, the enzyme that keeps us busy.
    Lars Petter Jordheim.
      cN-II is a cytosolic 5'-nucleotidase with preference for IMP and GMP over AMP. The enzyme has been extensively studied over the last 20-30 years both for its enzymatic activity, structure, role in nucleotide metabolism and in cell biology, as well as in diseases. With the aim of highlighting the complexity of the enzyme, I will, as during PP21, present work from our group and others working on cN-II and its various roles and not give an exhaustive overview of new data.
    Keywords:  5’-nucleotidase; Enzyme; diseases
    DOI:  https://doi.org/10.1080/15257770.2021.1983828
  26. Free Radic Biol Med. 2021 Oct 05. pii: S0891-5849(21)00752-8. [Epub ahead of print]
    Specific 8-oxo-dGTPase activity of MTH1 (NUDT1) protein as a quantitative marker and prognostic factor in human colorectal cancer.
    Karol Bialkowski, Anna Szpila.
      The MTH1 (NUDT1) gene, because it is frequently upregulated in many types of human cancers, has been considered a general marker of carcinogenesis for over two decades. The MTH1 protein hydrolyzes the oxidized mutagenic DNA precursor, 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), to the corresponding 5'-monophosphate and inorganic pyrophosphate. This prevents its incorporation into DNA by DNA polymerases and protects cells from the accumulation of 8-oxo-dGTP-induced point mutations. Elevated MTH1 mRNA and protein in many types of human cancer indicate a worse prognosis. However, the enzymatic activity of MTH1 has remained largely uninvestigated in this context. Therefore, we have set out to determine the specific 8-oxo-dGTPase activity of MTH1 in 57 pairs of human colorectal cancers (CRC) and adjacent cancer-free tissues (CFCF). The goal was to ascertain the potential for measuring this enzymatic activity as a way to differentiate cancerous from non-cancerous specimens of the intestine, as well as defining its capabilities as a prognostic value for disease-free survival. We found that 79% of CRC tumors exhibited a higher MTH1 activity than did CFCF, with a significant 1.6-fold increase in overall median value (p < 1E-6). The 8-oxo-dGTPase in both tissues was proportional to the corresponding levels of MTH1 protein, as assayed by Western blotting. Activity higher than the ROC-optimized threshold (AUC = 0.71) indicated cancerous tissue, with a 54% sensitivity and an 83% specificity. Postoperative fate followed for up to 100 months showed that higher 8-oxo-dGTPase, in either the CFCF or the CRC tumor, clearly lowered the probability of a relapse-free survival, although borderline statistical significance (p < 0.05) was crossed only for the CFCF.
    Keywords:  8-oxo-dGTPase activity; Antimutagenic enzyme; Cancer marker; Human colorectal cancer; MTH1; NUDT1; Prognostic marker
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.10.004
  27. Cell Death Dis. 2021 Oct 06. 12(10): 914
    Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound.
    Tatiana A Giovannucci, Florian A Salomons, Martin Haraldsson, Lotta H M Elfman, Malin Wickström, Patrick Young, Thomas Lundbäck, Jürgen Eirich, Mikael Altun, Rozbeh Jafari, Anna-Lena Gustavsson, John Inge Johnsen, Nico P Dantuma.
      Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.
    DOI:  https://doi.org/10.1038/s41419-021-04191-9
  28. Oncotarget. 2021 Sep 28. 12(20): 2068-2074
    NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.
    Adam S Kittai, Olga V Danilova, Vi Lam, Tingting Liu, Nur Bruss, Scott Best, Guang Fan, Alexey V Danilov.
      Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.
    Keywords:  T-cell lymphoma; anaphase catastrophe; chromosomal instability; pevonedistat
    DOI:  https://doi.org/10.18632/oncotarget.28063
  29. Biochem Biophys Rep. 2021 Dec;28 101141
    Teriflunomide - The common drug with underestimated oxygen - Dependent anticancer potential.
    Dagmara Otto-Ślusarczyk, Wojciech Graboń, Magdalena Mielczarek-Puta, Alicja Chrzanowska.
      Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect. The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21% oxygen level occurred in routine cell culture environment. The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels. We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced. Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.
    Keywords:  Colon cancer; DHODH; Hypoxia; Leflunomide; Teriflunomide; Tissue normoxia
    DOI:  https://doi.org/10.1016/j.bbrep.2021.101141
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