bims-numges 2021-12-19 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–12–19
25 papers selected by
Sean Rudd, Karolinska Institutet

Targeting ribonucleotide reductase induces synthetic lethality in PP2A-deficient uterine serous carcinoma.
Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH).
DTYMK is essential for genome integrity and neuronal survival.
Mitochondrial-Linked De Novo Pyrimidine Biosynthesis Dictates Human T-Cell Proliferation but Not Expression of Effector Molecules.
Cyclin E/CDK2: DNA Replication, Replication Stress and Genomic Instability.
Damsel in distress calling on her knights: Illuminating the pioneering role of E3 ubiquitin ligases in guarding the genome integrity.
5'-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A.
R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.
LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining.
The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.
Measurement of DNA Damage Using the Neutral Comet Assay in Cultured Cells.
Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.
DNA Damage Repair Inhibitors-Combination Therapies.
New Roles of Poly(ADP-Ribose) Polymerase Inhibitors in the Treatment of Breast Cancer.
Progress towards a clinically-successful ATR inhibitor for cancer therapy.
Targeting chemotherapy to de-condensed H3K27me3-marked chromatin of AML cells enhances leukemia suppression.
Integrated, Integral, and Exploratory Biomarkers in the Development of Poly(ADP-Ribose) Polymerase Inhibitors.
Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.
Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.
Metabolic drug survey highlights cancer cell dependencies and vulnerabilities.
Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.
NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis.
Identification of crucial genes of pyrimidine metabolism as biomarkers for gastric cancer prognosis.
A narrative review of genetic factors affecting fluoropyrimidine toxicity.
Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers.

Cancer Res. 2021 Dec 17. pii: canres.1987.2021. [Epub ahead of print]

Targeting ribonucleotide reductase induces synthetic lethality in PP2A-deficient uterine serous carcinoma.

Caitlin M O'Connor, Sarah E Taylor, Kathryn M Miller, Lauren Hurst, Terrance J Haanen, Tahra K Suhan, Kaitlin P Zawacki, Fallon K Noto, Jonida Trako, Arathi Mohan, Jaya Sangodkar, Dmitriy Zamarin, Analisa DiFeo, Goutham Narla.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein A-alpha, occur in 30-40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in A-alpha results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of A-alpha mutant uterine serous tumors were sensitive to Clofarabine, an RNR inhibitor. A-alpha mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild type cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized wild type USC cells to RNRi. Analysis of TCGA data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of USC patients harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second or later line therapy revealed a trend for improved outcomes in USC patients treated with RNRi gemcitabine compared to patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1987
Front Oncol. 2021 ;11 778925

Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH).

David M Wilson, Matthew A J Duncton, Caleb Chang, Christie Lee Luo, Taxiarchis M Georgiadis, Patricia Pellicena, Ashley M Deacon, Yang Gao, Debanu Das.

  Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme's ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

Keywords:  DNA damage response (DDR); POLH; Pol eta; X-ray crystallography; cancer therapeutics; fragment-based drug discovery (FBDD); polymerases; structure-based drug discovery (SBDD)

DOI:  https://doi.org/10.3389/fonc.2021.778925
Acta Neuropathol. 2021 Dec 17.

DTYMK is essential for genome integrity and neuronal survival.

Jo M Vanoevelen, Jörgen Bierau, Janine C Grashorn, Ellen Lambrichs, Erik-Jan Kamsteeg, Levinus A Bok, Ron A Wevers, Marjo S van der Knaap, Marianna Bugiani, Junmei Hu Frisk, Rita Colnaghi, Mark O'Driscoll, Debby M E I Hellebrekers, Richard Rodenburg, Carlos R Ferreira, Han G Brunner, Arthur van den Wijngaard, Ghada M H Abdel-Salam, Liya Wang, Constance T R M Stumpel.

  Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Keywords:  DTYMK; Genome instability; Nucleotide metabolism; Zebrafish; dTMPK

DOI:  https://doi.org/10.1007/s00401-021-02394-0
Front Immunol. 2021 ;12 718863

Mitochondrial-Linked De Novo Pyrimidine Biosynthesis Dictates Human T-Cell Proliferation but Not Expression of Effector Molecules.

Marlies J W Peeters, Pia Aehnlich, Adriano Pizzella, Kasper Mølgaard, Tina Seremet, Özcan Met, Lene Juel Rasmussen, Per Thor Straten, Claus Desler.

  T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.

Keywords:  T-cell activation; T-cell metabolism; immunosenescence and exhaustion; mitochondrial respiration and oxidative respiration; pyrimidine de novo synthesis

DOI:  https://doi.org/10.3389/fimmu.2021.718863
Front Cell Dev Biol. 2021 ;9 774845

Cyclin E/CDK2: DNA Replication, Replication Stress and Genomic Instability.

Rafaela Fagundes, Leonardo K Teixeira.

  DNA replication must be precisely controlled in order to maintain genome stability. Transition through cell cycle phases is regulated by a family of Cyclin-Dependent Kinases (CDKs) in association with respective cyclin regulatory subunits. In normal cell cycles, E-type cyclins (Cyclin E1 and Cyclin E2, CCNE1 and CCNE2 genes) associate with CDK2 to promote G1/S transition. Cyclin E/CDK2 complex mostly controls cell cycle progression and DNA replication through phosphorylation of specific substrates. Oncogenic activation of Cyclin E/CDK2 complex impairs normal DNA replication, causing replication stress and DNA damage. As a consequence, Cyclin E/CDK2-induced replication stress leads to genomic instability and contributes to human carcinogenesis. In this review, we focus on the main functions of Cyclin E/CDK2 complex in normal DNA replication and the molecular mechanisms by which oncogenic activation of Cyclin E/CDK2 causes replication stress and genomic instability in human cancer.

Keywords:  CCNE; CDK2; DNA replication; cancer; cell cycle; cyclin E; genomic instability; replication stress

DOI:  https://doi.org/10.3389/fcell.2021.774845
DNA Repair (Amst). 2021 Dec 10. pii: S1568-7864(21)00217-2. [Epub ahead of print]109 103261

Damsel in distress calling on her knights: Illuminating the pioneering role of E3 ubiquitin ligases in guarding the genome integrity.

Osheen Sahay, Ganesh Kumar Barik, Tanisha Sharma, Ajay D Pillai, Srikanth Rapole, Manas Kumar Santra.

  The maintenance of genomic integrity is of utmost importance for the organisms to survive and to accurately inherit traits to their progenies. Any kind of DNA damage either due to defect in DNA duplication and/ or uncontrolled cell division or intracellular insults or environment radiation can result in gene mutation, chromosomal aberration and ultimately genomic instability, which may cause several diseases including cancers. Therefore, cells have evolved machineries for the surveillance of genomic integrity. Enormous exciting studies in the past indicate that ubiquitination (a posttranslational modification of proteins) plays a crucial role in maintaining the genomic integrity by diverse ways. In fact, various E3 ubiquitin ligases catalyse ubiquitination of key proteins to control their central role during cell cycle, DNA damage response (DDR) and DNA repair. Some E3 ligases promote genomic instability while others prevent it, deregulation of both of which leads to several malignancies. In this review, we consolidate the recent findings wherein the role of ubiquitination in conferring genome integrity is highlighted. We also discuss the latest discoveries on the mechanisms utilized by various E3 ligases to preserve genomic stability, with a focus on their actions during cell cycle progression and different types of DNA damage response as well as repair pathways.

Keywords:  Cancer; Cell cycle checkpoint; DNA damage response; DNA repair; E3 ubiquitin ligase; Genomic instability

DOI:  https://doi.org/10.1016/j.dnarep.2021.103261
ChemMedChem. 2021 Dec 14.

5'-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A.

Mark Berney, Manav T Manoj, Ellen Mary Fay, Joanna Francelle McGouran.

  Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

Keywords:  DNA repair; SNM1A; nuclease inhibitor; nucleotide; zinc-binding group

DOI:  https://doi.org/10.1002/cmdc.202100603
Nat Commun. 2021 Dec 16. 12(1): 7314

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability.

Thorsten Mosler, Francesca Conte, Gabriel M C Longo, Ivan Mikicic, Nastasja Kreim, Martin M Möckel, Giuseppe Petrosino, Johanna Flach, Joan Barau, Brian Luke, Vassilis Roukos, Petra Beli.

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

DOI: https://doi.org/10.1038/s41467-021-27530-y
Front Mol Biosci. 2021 ;8 787709

LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining.

Benhur Amare, Anthea Mo, Noorisah Khan, Dana J Sowa, Monica M Warner, Andriana Tetenych, Sara N Andres.

  DNA double-strand breaks are the most lethal form of damage for living organisms. The non-homologous end joining (NHEJ) pathway can repair these breaks without the use of a DNA template, making it a critical repair mechanism when DNA is not replicating, but also a threat to genome integrity. NHEJ requires proteins to anchor the DNA double-strand break, recruit additional repair proteins, and then depending on the damage at the DNA ends, fill in nucleotide gaps or add or remove phosphate groups before final ligation. In eukaryotes, NHEJ uses a multitude of proteins to carry out processing and ligation of the DNA double-strand break. Bacterial NHEJ, though, accomplishes repair primarily with only two proteins-Ku and LigD. While Ku binds the initial break and recruits LigD, it is LigD that is the primary DNA end processing machinery. Up to three enzymatic domains reside within LigD, dependent on the bacterial species. These domains are a polymerase domain, to fill in nucleotide gaps with a preference for ribonucleotide addition; a phosphoesterase domain, to generate a 3'-hydroxyl DNA end; and the ligase domain, to seal the phosphodiester backbone. To date, there are no experimental structures of wild-type LigD, but there are x-ray and nuclear magnetic resonance structures of the individual enzymatic domains from different bacteria and archaea, along with structural predictions of wild-type LigD via AlphaFold. In this review, we will examine the structures of the independent domains of LigD from different bacterial species and the contributions these structures have made to understanding the NHEJ repair mechanism. We will then examine how the experimental structures of the individual LigD enzymatic domains combine with structural predictions of LigD from different bacterial species and postulate how LigD coordinates multiple enzymatic activities to carry out DNA double-strand break repair in bacteria.

Keywords:  DNA double-strand break; LigD; ligase; non-homologous end joining; phosphoesterase; polymerase; protein structure and function

DOI:  https://doi.org/10.3389/fmolb.2021.787709
Mol Cell. 2021 Dec 16. pii: S1097-2765(21)00994-1. [Epub ahead of print]81(24): 4994-5006.e5

The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.

Johannes Rudolph, Uma M Muthurajan, Megan Palacio, Jyothi Mahadevan, Genevieve Roberts, Annette H Erbse, Pamela N Dyer, Karolin Luger.

  PARP1 is a key player in the response to DNA damage and is the target of clinical inhibitors for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation wherein PARP1 uses NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. We show that intact DNA makes contacts with the PARP1 BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the "monkey-bar mechanism" that mediates DNA transfer of PARP1.

Keywords:  BRCT; DNA repair; PARP1; cryoEM; intrastrand transfer; nucleosome

DOI:  https://doi.org/10.1016/j.molcel.2021.11.014
Bio Protoc. 2021 Nov 20. 11(22): e4226

Measurement of DNA Damage Using the Neutral Comet Assay in Cultured Cells.

Elena Clementi, Zuzana Garajova, Enni Markkanen.

  Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair for disease pathogenesis, measuring them is of considerable interest, and the comet assay is a widely used method for this. Cells treated with DNA damaging agents are embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates towards the anode depending on its degree of fragmentation, creating shapes resembling comets, which can be visualized and analysed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, and both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the neutral comet assay to assess double-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by ionizing radiation or present endogenously in the cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and cost-effective.

Keywords:  DNA damage; DNA repair; DNA repair kinetics; Genotoxic agents; Oxidative stress; Reactive oxygen species

DOI:  https://doi.org/10.21769/BioProtoc.4226
Development. 2021 Dec 15. pii: dev.199800. [Epub ahead of print]

Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.

Wei-Ting Yueh, Vijay Pratap Singh, Jennifer L Gerton.

  Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this critical period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome in this window of mammalian development is unknown. We discovered that although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the 2-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the 2-cell stage. Smc3 is a maternal gene with essential functions in repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

Keywords:  Chromosome segregation; Cohesin; Developmental competence; Juvenile; Maternal effect gene; Micronuclei; Mouse; SMC3; Spontaneous DNA damage; Zygote

DOI:  https://doi.org/10.1242/dev.199800
Cancer J. 2021 Nov-Dec 01;27(6):27(6): 501-505

DNA Damage Repair Inhibitors-Combination Therapies.

Gabriella Smith, Zachary Alholm, Robert L Coleman, Bradley J Monk.

ABSTRACT: DNA damage response and repair (DDR) is responsible for ensuring genomic integrity. It is composed of intricate, complex pathways that detect various DNA insults and then activate pathways to restore DNA fidelity. Mutations in this network are implicated in many malignancies but can also be exploited for cancer therapies. The advent of inhibitors of poly(ADP-ribose) polymerase has led to the investigation of other DDR inhibitors and combinations to address high unmet needs in cancer therapeutics. Specifically, regimens, often in combination with chemotherapy, radiation, or other DDR inhibitors, are being investigated. This review will focus on 4 main DDR pathways-ATR/CHK1, ATM/CHK2, DNA-PKcs, and polymerase θ-and the current state of clinical research and use of the inhibitors of these pathways with other DDR inhibitors.

DOI: https://doi.org/10.1097/PPO.0000000000000561
Cancer J. 2021 Nov-Dec 01;27(6):27(6): 441-456

New Roles of Poly(ADP-Ribose) Polymerase Inhibitors in the Treatment of Breast Cancer.

Elizabeth Harvey-Jones, Gemma Vinas Villaro, Andrew Tutt.

ABSTRACT: Since the proof of concept of synthetic lethality between poly(ADP-ribose) polymerase inhibition and loss of BRCA1/2 homologous recombination (HR) function in preclinical models and early phase clinical trials, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasing part of standard-of-care treatment for advanced breast cancers with BRCA gene mutations. The field has also recently seen benefits for PARPi in early breast cancer in those with germline BRCA1 and BRCA2 pathogenic mutations, and signals that synthetic lethal affects may occur in tumors with deficiencies in HR caused by germline, somatic, or epigenetic dysregulation of a number of HR genes. Despite the evidence of the synthetic lethal effects of PARPi, they are not always effective in HR defective cancers, and as they become part of standard of care in breast cancer, the study of prevalence of distinct mechanisms of resistance to PARPi and cross-resistance with other DNA-damaging agents such as platinum in breast cancer will be important and may inform therapy choices.

DOI: https://doi.org/10.1097/PPO.0000000000000559
Curr Res Pharmacol Drug Discov. 2021 ;2 100017

Progress towards a clinically-successful ATR inhibitor for cancer therapy.

Francis M Barnieh, Paul M Loadman, Robert A Falconer.

  The DNA damage response (DDR) is now known to play an important role in both cancer development and its treatment. Targeting proteins such as ATR (Ataxia telangiectasia mutated and Rad3-related) kinase, a major regulator of DDR, has demonstrated significant therapeutic potential in cancer treatment, with ATR inhibitors having shown anti-tumour activity not just as monotherapies, but also in potentiating the effects of conventional chemotherapy, radiotherapy, and immunotherapy. This review focuses on the biology of ATR, its functional role in cancer development and treatment, and the rationale behind inhibition of this target as a therapeutic approach, including evaluation of the progress and current status of development of potent and specific ATR inhibitors that have emerged in recent decades. The current applications of these inhibitors both in preclinical and clinical studies either as single agents or in combinations with chemotherapy, radiotherapy and immunotherapy are also extensively discussed. This review concludes with some insights into the various concerns raised or observed with ATR inhibition in both the preclinical and clinical settings, with some suggested solutions.

Keywords:  ATM; ATR; ATR inhibitors; Chemo- and radiosensitisers; DDR

DOI:  https://doi.org/10.1016/j.crphar.2021.100017
Cancer Res. 2021 Dec 13. pii: canres.1297.2021. [Epub ahead of print]

Targeting chemotherapy to de-condensed H3K27me3-marked chromatin of AML cells enhances leukemia suppression.

Patrizia Porazzi, Svetlana Petruk, Luca Pagliaroli, Marco De Dominici, David Deming, Matthew V Puccetti, Saul Kushinsky, Gaurav Kumar, Valentina Minieri, Elisa Barbieri, Sandra Deliard, Alexis Grande, Marco Trizzino, Alessandro Gardini, Eli Canaani, Neil Palmisiano, Pierluigi Porcu, Adam Ertel, Paolo M Fortina, Christine M Eischen, Alexander Mazo, Bruno Calabretta.

Despite treatment with intensive chemotherapy, acute myeloid leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to de-condense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin de-condensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin co-treatment induced transcriptional reprogramming that was, in part, dependent on de-repression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes. In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in AML patients. This strategy might allow for a lowering of chemotherapy doses with a consequent reduction of treatment-related side effects in elderly AML patients or those with significant comorbidities.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1297
Cancer J. 2021 Nov-Dec 01;27(6):27(6): 482-490

Integrated, Integral, and Exploratory Biomarkers in the Development of Poly(ADP-Ribose) Polymerase Inhibitors.

Gottfried E Konecny, Cinthiya Chander, Liying Zhang.

ABSTRACT: In this article, we highlight biomarkers for poly(ADP-ribose) polymerase inhibitor (PARPi) sensitivity and resistance and discuss their implications for the clinic. We review the predictive role of a range of DNA repair genes, genomic scars, mutational signatures, and functional assays available or in development. The biomarkers used for patient selection in the specific Food and Drug Administration-approved indications for breast, ovarian, prostate, and pancreatic cancer vary across tumor type and likely depend on disease-specific DNA repair deficiencies but also the specifics of the individual clinical trials that were conducted. Mutations in genes involved in homologous recombination and/or replication fork protection are synthetic lethal with PARPi. Cancers with homologous recombination deficiency exhibit high genomic instability, characterized by genome-wide loss of heterozygosity, among other genomic aberrations. Next-generation sequencing can identify multiple patterns of genomic changes including copy number variations, single-nucleotide variations, insertions/deletions, and structural variations rearrangements characteristic of homologous recombination deficiency. Clinical trial evidence supports the use of BRCA mutation testing for patient selection, and for ovarian cancer, there are 3 commercial assays available that additionally incorporate genomic instability for identifying subgroups of patients that derive different magnitudes of benefit from PARPi therapy. Finally, we summarize new strategies for extending the benefit of PARPi therapy toward broader populations of patients through the use of novel biomarkers. Ultimately, design of a composite biomarker test combining multiple mutational signatures or development of a dynamic assay for functional assessments of homologous recombination may help improve the test accuracy for future patient stratification.

DOI: https://doi.org/10.1097/PPO.0000000000000564
J Med Chem. 2021 Dec 14.

Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.

Chungen Li, Xiaowei Yang, Yuan Luo, Huan Liu, Xi Zhong, Xia Zhou, Ting Zeng, Lei Tao, Yue Zhou, Kun Gou, Xinyu Yang, Xiaocong Liu, Qiang Chen, Yinglan Zhao, Youfu Luo.

Human dihydroorotate dehydrogenase (hDHODH), as the fourth and rate-limiting enzyme of the de novo pyrimidine synthesis pathway, is regarded as an attractive target for malignancy therapy. In the present study, a novel series of teriflunomide derivatives were designed, synthesized, and evaluated as hDHODH inhibitors. 13t was the optimal compound with promising enzymatic activity (IC50 = 16.0 nM), potent antiproliferative activity against human lymphoma Raji cells (IC50 = 7.7 nM), and excellent aqueous solubility (20.1 mg/mL). Mechanistically, 13t directly inhibited hDHODH and induced cell cycle S-phase arrest in Raji cells. The acute toxicity assay indicated a favorable safety profile of 13t. Notably, 13t displayed significant tumor growth inhibition activity with a tumor growth inhibition (TGI) rate of 81.4% at 30 mg/kg in a Raji xenograft model. Together, 13t is a promising inhibitor of hDHODH and a preclinical candidate for antitumor therapy, especially for lymphoma.

DOI: https://doi.org/10.1021/acs.jmedchem.1c01711
Int J Cancer. 2021 Dec 16.

Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.

Nao Yoshida-Sakai, Tatsuro Watanabe, Yuta Yamamoto, Hiroshi Ureshino, Kazuharu Kamachi, Yuki Kurahashi, Yuki Fukuda-Kurahashi, Shinya Kimura.

  Adult T-cell leukemia-lymphoma (ATL) is an aggressive neoplasm derived from T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Recently, we reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC), and a new DAC prodrug, OR-2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA-, DAC-, and OR21-resistant (AZA-R, DAC-R, and OR21-R, respectively) cells from the ATL cell line TL-Om1 and the HTLV-1-infected cell line MT-2 via long-term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1, and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC-R TL-Om1 and AZA-R TL-Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.

Keywords:  acquired resistance; adult T-cell leukemia-lymphoma; azacitidine; decitabine; pyrimidine metabolism

DOI:  https://doi.org/10.1002/ijc.33901
Nat Commun. 2021 Dec 14. 12(1): 7190

Metabolic drug survey highlights cancer cell dependencies and vulnerabilities.

Tea Pemovska, Johannes W Bigenzahn, Ismet Srndic, Alexander Lercher, Andreas Bergthaler, Adrián César-Razquin, Felix Kartnig, Christoph Kornauth, Peter Valent, Philipp B Staber, Giulio Superti-Furga.

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

DOI: https://doi.org/10.1038/s41467-021-27329-x
Nucleic Acids Res. 2021 Dec 14. pii: gkab1218. [Epub ahead of print]

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Klara Prochazkova, Andreas Finke, Eva Dvořák Tomaštíková, Jaroslav Filo, Heinrich Bente, Petr Dvořák, Miroslav Ovečka, Jozef Šamaj, Ales Pecinka.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

DOI: https://doi.org/10.1093/nar/gkab1218
Elife. 2021 Dec 17. pii: e70046. [Epub ahead of print]10

NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis.

Jiaqi Liu, Xianzun Tao, Yi Zhu, Chong Li, Kai Ruan, Zoraida Diaz-Perez, Priyamvada Rai, Hongbo Wang, R Grace Zhai.

  Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have had limited success due to insufficient mechanistic understanding. Here, we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth.

Keywords:  D. melanogaster; NAD; PARP; RAS; cancer biology; caspase; deacetylation; glial cell; neuroscience

DOI:  https://doi.org/10.7554/eLife.70046
Cancer Cell Int. 2021 Dec 14. 21(1): 668

Identification of crucial genes of pyrimidine metabolism as biomarkers for gastric cancer prognosis.

Zhengxin Wu, Jinshui Tan, Yifan Zhuang, Mengya Zhong, Yubo Xiong, Jingsong Ma, Yan Yang, Zhi Gao, Jiabao Zhao, Zhijian Ye, Huiwen Zhou, Yuekun Zhu, Haijie Lu, Xuehui Hong.

   BACKGROUND: Metabolic reprogramming has been reported in various kinds of cancers and is related to clinical prognosis, but the prognostic role of pyrimidine metabolism in gastric cancer (GC) remains unclear.
METHODS: Here, we employed DEG analysis to detect the differentially expressed genes (DEGs) in pyrimidine metabolic signaling pathway and used univariate Cox analysis, Lasso-penalizes Cox regression analysis, Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis to explore their prognostic roles in GC. The DEGs were experimentally validated in GC cells and clinical samples by quantitative real-time PCR.
RESULTS: Through DEG analysis, we found NT5E, DPYS and UPP1 these three genes are highly expressed in GC. This conclusion has also been verified in GC cells and clinical samples. A prognostic risk model was established according to these three DEGs by Univariate Cox analysis and Lasso-penalizes Cox regression analysis. Kaplan-Meier survival analysis suggested that patient cohorts with high risk score undertook a lower overall survival rate than those with low risk score. Stratified survival analysis, Univariate and multivariate Cox regression analysis of this model confirmed that it is a reliable and independent clinical factor. Therefore, we made nomograms to visually depict the survival rate of GC patients according to some important clinical factors including our risk model.
CONCLUSION: In a word, our research found that pyrimidine metabolism is dysregulated in GC and established a prognostic model of GC based on genes differentially expressed in pyrimidine metabolism.

Keywords:  Bioinformatics; Biomarker; GC; Prognosis risk model; Pyrimidine metabolism

DOI:  https://doi.org/10.1186/s12935-021-02385-x
Precis Cancer Med. 2021 Dec;pii: 38. [Epub ahead of print]4

A narrative review of genetic factors affecting fluoropyrimidine toxicity.

William H Gmeiner.

   Objective: Our objective is to document progress in developing personalized therapy with fluoropyrimidine drugs (FPs) to improve outcomes for cancer patients and to identify areas requiring further investigation.
Background: FPs including 5-fluorouracil (5-FU), are among the most widely used drugs for treating colorectal cancer (CRC) and other gastrointestinal (GI) malignancies. While FPs confer a survival benefit for CRC patients, serious systemic toxicities, including neutropenia, occur in ~30% of patients with lethality in 0.5-1% of patients. While serious systemic toxicities may occur in any patient, patients with polymorphisms in DPYD, which encodes the rate-limiting enzyme for pyrimidine degradation are at very high risk. Other genetic factors affecting risk for 5-FU toxicity, including miR-27a, are under investigation.
Methods: Literature used to inform the text of this article was selected from PubMed.gov from the National Library of Medicine while regulatory documents were identified via Google search.
Conclusions: Clinical studies to date have validated four DPYD polymorphisms (DPYD*2A, DPYD*13, c.2846A>T, HapB3) associated with serious toxicities in patients treated with 5-FU. Genetic screening for these is being implemented in the Netherlands and the UK and has been shown to be a cost-effective way to improve outcomes. Factors other than DPYD polymorphisms (e.g., miR-27a, TYMS, ENOSF1, p53) also affect 5-FU toxicity. Functional testing for deficient pyrimidine catabolism {defined as [U] >16 ng/mL or [UH2]:[U] <10} is being implemented in France and has demonstrated utility in identifying patients with elevated risk for 5-FU toxicity. Therapeutic drug monitoring (TDM) from plasma levels of 5-FU during first cycle treatment also is being used to improve outcomes and pharmacokinetic-based dosing is being used to increase the percent of patients within optimal area under the curve (AUC) (18-28 mg*h/L) values. Patients maintained in the optimal AUC range experienced significantly reduced systemic toxicities. As understanding the genetic basis for increased risk of 5-FU toxicity becomes more refined, the development of functional-based methods to optimize treatment is likely to become more widespread.

Keywords:  Fluoropyrimidine (FP); colorectal cancer (CRC); dihydropyrimidine dehydrogenase (DPD); therapeutic drug monitoring (TDM)

DOI:  https://doi.org/10.21037/pcm-21-17
Cancer Res. 2021 Dec 15. pii: canres.CAN-21-2745-E.2021. [Epub ahead of print]

Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers.

Chengheng Liao, Cherise Ryan Glodowski, Cheng Fan, Juan Liu, Kevin R Mott, Akash Kaushik, Hieu Vu, Jason W Locasale, Samuel K McBrayer, Ralph J DeBerardinis, Charles M Perou, Qing Zhang.

Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and estrogen receptor (ER)-positive breast cancer patient samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histological subtypes: a "Nucleotide/Carbohydrate-Enriched" group and a "Lipid/Fatty Acid-Enriched" group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared to those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof-of-principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2745