bims-numges 2021-05-02 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–05–02
35 papers selected by
Sean Rudd, Karolinska Institutet

Cellular pyrimidine imbalance triggers mitochondrial DNA-dependent innate immunity.
NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487.
Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress.
DNA2 in Chromosome Stability and Cell Survival-Is It All about Replication Forks?
A Link between Replicative Stress, Lamin Proteins, and Inflammation.
The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition.
Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches.
CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model.
Human ALKBH6 Is Required for Maintenance of Genomic Stability and Promoting Cell Survival During Exposure of Alkylating Agents in Pancreatic Cancer.
Critical DNA damaging pathways in tumorigenesis.
FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.
The molecular mechanism of nuclear signaling for degradation of cytoplasmic DNA: Importance in DNA damage response and cancer.
Inhibition of DNA replication initiation by silver nanoclusters.
Mouse Models for Deciphering the Impact of Homologous Recombination on Tumorigenesis.
The PARP Enzyme Family and the Hallmarks of Cancer Part 2: Hallmarks Related to Cancer Host Interactions.
RNA Editing of the Human DNA Glycosylase NEIL1 Alters Its Removal of 5-Hydroxyuracil Lesions in DNA.
RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle.
The PARP Enzyme Family and the Hallmarks of Cancer Part 1. Cell Intrinsic Hallmarks.
Functional Roles of PARP2 in Assembling Protein-Protein Complexes Involved in Base Excision DNA Repair.
DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.
The Multifaceted Roles of Ku70/80.
BBIT20 inhibits homologous DNA repair with disruption of the BRCA1-BARD1 interaction in breast and ovarian cancer.
Can small molecular inhibitors that stop de novo serine synthesis be used in cancer treatment?
FLYWCH1, a Multi-Functional Zinc Finger Protein Contributes to the DNA Repair Pathway.
Dual roles of SAMHD1 in tumor development and chemoresistance to anticancer drugs.
Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.
Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells.
Treatment Strategies for ARID1A-Deficient Ovarian Clear Cell Carcinoma.
Knockdown of RRM1 with Adenoviral shRNA Vectors to Inhibit Tumor Cell Viability and Increase Chemotherapeutic Sensitivity to Gemcitabine in Bladder Cancer Cells.
Inosine in Biology and Disease.
Characterization of deoxyribonucleoside transport mediated by concentrative nucleoside transporters.
Targeting the metabolic vulnerability of acute myeloid leukemia blasts with a combination of venetoclax and 8-chloro-adenosine.
Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration.
Issues and limitations of available biomarkers for fluoropyrimidine-based chemotherapy toxicity, a narrative review of the literature.
Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A.

Nat Metab. 2021 Apr 26.

Cellular pyrimidine imbalance triggers mitochondrial DNA-dependent innate immunity.

Hans-Georg Sprenger, Thomas MacVicar, Amir Bahat, Kai Uwe Fiedler, Steffen Hermans, Denise Ehrentraut, Katharina Ried, Dusanka Milenkovic, Nina Bonekamp, Nils-Göran Larsson, Hendrik Nolte, Patrick Giavalisco, Thomas Langer.

Cytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP-AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS-STING-TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS-STING-TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

DOI: https://doi.org/10.1038/s42255-021-00385-9
Int J Mol Sci. 2021 Apr 27. pii: 4542. [Epub ahead of print]22(9):

NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487.

Bishoy M F Hanna, Maurice Michel, Thomas Helleday, Oliver Mortusewicz.

  DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.

Keywords:  8-oxoguanine; DNA oxidative damage; NEIL1 glycosylase; NEIL2; OGG1 inhibitor; TH5487; backup pathway; base excision repair; chromatin binding dynamics; recruitment kinetics

DOI:  https://doi.org/10.3390/ijms22094542
Int J Mol Sci. 2021 Apr 10. pii: 3924. [Epub ahead of print]22(8):

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress.

Domenico Maiorano, Jana El Etri, Camille Franchet, Jean-Sébastien Hoffmann.

  DNA can experience "replication stress", an important source of genome instability, induced by various external or endogenous impediments that slow down or stall DNA synthesis. While genome instability is largely documented to favor both tumor formation and heterogeneity, as well as drug resistance, conversely, excessive instability appears to suppress tumorigenesis and is associated with improved prognosis. These findings support the view that karyotypic diversity, necessary to adapt to selective pressures, may be limited in tumors so as to reduce the risk of excessive instability. This review aims to highlight the contribution of specialized DNA polymerases in limiting extreme genetic instability by allowing DNA replication to occur even in the presence of DNA damage, to either avoid broken forks or favor their repair after collapse. These mechanisms and their key regulators Rad18 and Polθ not only offer diversity and evolutionary advantage by increasing mutagenic events, but also provide cancer cells with a way to escape anti-cancer therapies that target replication forks.

Keywords:  DSB repair; POLQ; Pol theta; TMEJ; genome instability; replicative stress; specialized DNA polymerases; translesion synthesis (TLS), Rad18

DOI:  https://doi.org/10.3390/ijms22083924
Int J Mol Sci. 2021 Apr 13. pii: 3984. [Epub ahead of print]22(8):

DNA2 in Chromosome Stability and Cell Survival-Is It All about Replication Forks?

Jessica J R Hudson, Ulrich Rass.

  The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

Keywords:  DNA end-resection; DNA replication stress; Okazaki fragment processing; anaphase bridge; chicken-foot structure; chromosome stability; chromosome underreplication; replication fork recovery; replication fork reversal; stalled replication fork

DOI:  https://doi.org/10.3390/ijms22083984
Genes (Basel). 2021 Apr 09. pii: 552. [Epub ahead of print]12(4):

A Link between Replicative Stress, Lamin Proteins, and Inflammation.

Simon Willaume, Emilie Rass, Paula Fontanilla-Ramirez, Angela Moussa, Paul Wanschoor, Pascale Bertrand.

  Double-stranded breaks (DSB), the most toxic DNA lesions, are either a consequence of cellular metabolism, programmed as in during V(D)J recombination, or induced by anti-tumoral therapies or accidental genotoxic exposure. One origin of DSB sources is replicative stress, a major source of genome instability, especially when the integrity of the replication forks is not properly guaranteed. To complete stalled replication, restarting the fork requires complex molecular mechanisms, such as protection, remodeling, and processing. Recently, a link has been made between DNA damage accumulation and inflammation. Indeed, defects in DNA repair or in replication can lead to the release of DNA fragments in the cytosol. The recognition of this self-DNA by DNA sensors leads to the production of inflammatory factors. This beneficial response activating an innate immune response and destruction of cells bearing DNA damage may be considered as a novel part of DNA damage response. However, upon accumulation of DNA damage, a chronic inflammatory cellular microenvironment may lead to inflammatory pathologies, aging, and progression of tumor cells. Progress in understanding the molecular mechanisms of DNA damage repair, replication stress, and cytosolic DNA production would allow to propose new therapeutical strategies against cancer or inflammatory diseases associated with aging. In this review, we describe the mechanisms involved in DSB repair, the replicative stress management, and its consequences. We also focus on new emerging links between key components of the nuclear envelope, the lamins, and DNA repair, management of replicative stress, and inflammation.

Keywords:  DNA replication stress; Hutchinson-Gilford progeria syndrome; aging; cGAS-STING pathway; cancer; double-strand break repair; genome instability; inflammation; lamins; senescence

DOI:  https://doi.org/10.3390/genes12040552
Cancers (Basel). 2021 Apr 19. pii: 1957. [Epub ahead of print]13(8):

The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition.

Luca Mattiello, Sara Soliman Abdel Rehim, Martina Musella, Antonella Sistigu, Andrea Guarracino, Sara Vitale, Francesca Corradi, Claudia Galassi, Francesca Sperati, Gwenola Manic, Ruggero De Maria, Ilio Vitale.

  Cancer stem cells (CSCs) drive not only tumor initiation and expansion, but also therapeutic resistance and tumor relapse. Therefore, CSC eradication is required for effective cancer therapy. In preclinical models, CSCs demonstrated high capability to tolerate even extensive genotoxic stress, including replication stress, because they are endowed with a very robust DNA damage response (DDR). This favors the survival of DNA-damaged CSCs instead of their inhibition via apoptosis or senescence. The DDR represents a unique CSC vulnerability, but the abrogation of the DDR through the inhibition of the ATR-CHK1 axis is effective only against some subtypes of CSCs, and resistance often emerges. Here, we analyzed the impact of druggable DDR players in the response of patient-derived colorectal CSCs (CRC-SCs) to CHK1/2 inhibitor prexasertib, identifying RAD51 and MRE11 as sensitizing targets enhancing prexasertib efficacy. We showed that combined inhibition of RAD51 and CHK1 (via B02+prexasertib) or MRE11 and CHK1 (via mirin+prexasertib) kills CSCs by affecting multiple genoprotective processes. In more detail, these two prexasertib-based regimens promote CSC eradication through a sequential mechanism involving the induction of elevated replication stress in a context in which cell cycle checkpoints usually activated during the replication stress response are abrogated. This leads to uncontrolled proliferation and premature entry into mitosis of replication-stressed cells, followed by the induction of mitotic catastrophe. CRC-SCs subjected to RAD51+CHK1 inhibitors or MRE11+CHK1 inhibitors are eventually eliminated, and CRC-SC tumorspheres inhibited or disaggregated, via a caspase-dependent apoptosis. These results support further clinical development of these prexasertib-based regimens in colorectal cancer patients.

Keywords:  DNA damage; chromosomal instability; colorectal cancer; targeted therapy; tumor-initiating cells

DOI:  https://doi.org/10.3390/cancers13081957
DNA Repair (Amst). 2021 Apr 20. pii: S1568-7864(21)00082-3. [Epub ahead of print]103 103126

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches.

Mingrui Duan, Rachel M Speer, Jenna Ulibarri, Ke Jian Liu, Peng Mao.

  Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.

Keywords:  CPD-seq; CSB; DNA damage; Mutagenesis; RNA Pol II

DOI:  https://doi.org/10.1016/j.dnarep.2021.103126
J Biol Chem. 2021 Apr 23. pii: S0021-9258(21)00496-8. [Epub ahead of print] 100707

CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model.

Jianping Ren, Yan Wu, Ya Wang, Yuqin Zhao, Youhang Li, Shuailin Hao, Lixiu Lin, Shuyuan Zhang, Xingzhi Xu, Hailong Wang.

  MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression. The post-transcriptional maturation of miRNAs is controlled by the Drosha-DGCR8 microprocessor. Dysregulation of miRNA biogenesis has been implicated in the pathogenesis of human diseases, including cancers. CtIP is a well-known DNA repair factor that promotes the processing of DNA double-strand break (DSB) to initiate homologous-recombination-mediated DSB repair. However, it was unclear whether CtIP has other unknown cellular functions. Here, we aimed to uncover the roles of CtIP in miRNA maturation and cancer cell metastasis. We found that CtIP is a potential regulatory factor that suppresses the processing of miRNA primary transcripts (pri-miRNA). CtIP directly bound to both DGCR8 and pri-miRNAs through a conserved Sae2-like domain, reduced the binding of Drosha to DGCR8 and pri-miRNA substrate and inhibited processing activity of Drosha complex. CtIP depletion significantly increased the expression levels of a subset of mature miRNAs, including miR-302 family members which are associated with tumor progression and metastasis in several cancer types. We also found that CtIP-inhibited miRNAs, such as miR-302 family members, are not crucial for DSB repair. However, increase of miR-302b levels or loss of CtIP function severely suppressed HCT116 tumor cell metastasis in a mouse xenograft model. These studies reveal a previously unrecognized mechanism of CtIP in miRNA processing and tumor metastasis that represents a new function of CtIP in cancer.

Keywords:  CtIP; DGCR8; Drosha; metastasis; microRNA maturation; microprocessor

DOI:  https://doi.org/10.1016/j.jbc.2021.100707
Front Genet. 2021 ;12 635808

Human ALKBH6 Is Required for Maintenance of Genomic Stability and Promoting Cell Survival During Exposure of Alkylating Agents in Pancreatic Cancer.

Shengyuan Zhao, Rodan Devega, Aaliyah Francois, Dawit Kidane.

  Alpha-ketoglutarate-dependent dioxygenase (ALKBH) is a DNA repair gene involved in the repair of alkylating DNA damage. There are nine types of ALKBH (ALKBH1-8 and FTO) identified in humans. In particular, certain types of ALKBH enzymes are dioxygenases that directly reverse DNA methylation damage via transfer of a methyl group from the DNA adduct onto α-ketoglutarate and release of metabolic products including succinate and formaldehyde. Here, we tested whether ALKBH6 plays a significant role in preventing alkylating DNA damage and decreasing genomic instability in pancreatic cancer cells. Using an E. coli strain deficient with ALKB, we found that ALKBH6 complements ALKB deficiency and increases resistance after alkylating agent treatment. In particular, the loss of ALKBH6 in human pancreatic cancer cells increases alkylating agent-induced DNA damage and significantly decreases cell survival. Furthermore, in silico analysis from The Cancer Genome Atlas (TCGA) database suggests that overexpression of ALKBH6 provides better survival outcomes in patients with pancreatic cancer. Overall, our data suggest that ALKBH6 is required to maintain the integrity of the genome and promote cell survival of pancreatic cancer cells.

Keywords:  Alkb E. coli; DNA repair; alkbh6; alkylating DNA damage; pancreatic cancer

DOI:  https://doi.org/10.3389/fgene.2021.635808
Semin Cancer Biol. 2021 Apr 24. pii: S1044-579X(21)00113-9. [Epub ahead of print]

Critical DNA damaging pathways in tumorigenesis.

Jake A Kloeber, Zhenkun Lou.

  The acquisition of DNA damage is an early driving event in tumorigenesis. Premalignant lesions show activated DNA damage responses and inactivation of DNA damage checkpoints promotes malignant transformation. However, DNA damage is also a targetable vulnerability in cancer cells. This requires a detailed understanding of the cellular and molecular mechanisms governing DNA integrity. Here, we review current work on DNA damage in tumorigenesis. We discuss DNA double strand break repair, how repair pathways contribute to tumorigenesis, and how double strand breaks are linked to the tumor microenvironment. Next, we discuss the role of oncogenes in promoting DNA damage through replication stress. Finally, we discuss our current understanding on DNA damage in micronuclei and discuss therapies targeting these DNA damage pathways.

Keywords:  DNA damage; Genome instability; Tumorigenesis

DOI:  https://doi.org/10.1016/j.semcancer.2021.04.012
Cancers (Basel). 2021 Apr 14. pii: 1866. [Epub ahead of print]13(8):

FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.

Katia A Mesquita, Reem Ali, Rachel Doherty, Michael S Toss, Islam Miligy, Adel Alblihy, Dorjbal Dorjsuren, Anton Simeonov, Ajit Jadhav, David M Wilson, Ian Hickson, Natalie J Tatum, Emad A Rakha, Srinivasan Madhusudan.

  FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin β. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLβ and XRCC1. FEN1i treatment was selectively toxic to POLβ deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

Keywords:  DNA repair inhibitor; FEN1; cisplatin sensitivity; ovarian cancer; synthetic lethality

DOI:  https://doi.org/10.3390/cancers13081866
DNA Repair (Amst). 2021 Apr 26. pii: S1568-7864(21)00071-9. [Epub ahead of print]103 103115

The molecular mechanism of nuclear signaling for degradation of cytoplasmic DNA: Importance in DNA damage response and cancer.

Erfan Mohammadi, Fatemeh Sadoughi, Simin Younesi, Ansar Karimian, Zatollah Asemi, Nader Farsad-Akhtar, Fahime Jahanbakhshi, Hamidreza Jamilian, Bahman Yousefi.

  This review summarizes and addresses non-coding RNAs (rRNA, tRNA, Vault and Y RNA, snRNA, and miRNA) cytoplasmic decay pathways, the molecules, enzymes, and modifications such as uridylation, which play vital roles in the degradation processes in various eukaryotic organisms. Plus, SIRT1's role in fundamental cellular processes, including autophagy, DNA repair, DNA damage response (DDR), and the molecular mechanisms, is explored. Further, the HuR (an RNA-binding protein) impact on the expression of genes following DNA damage, and the pathways that regulate HuR function, which is through phosphorylation by Chk1/Cdk1 and Chk2, are specified. Finally, the role of DIF1/ Rnr2-Rnr4 in DDR has been discussed.

Keywords:  DNA damage response; DNA repair; Enzymes; Molecular mechanisms

DOI:  https://doi.org/10.1016/j.dnarep.2021.103115
Nucleic Acids Res. 2021 Apr 27. pii: gkab271. [Epub ahead of print]

Inhibition of DNA replication initiation by silver nanoclusters.

Yu Tao, Tomas Aparicio, Mingqiang Li, Kam W Leong, Shan Zha, Jean Gautier.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

DOI: https://doi.org/10.1093/nar/gkab271
Cancers (Basel). 2021 Apr 25. pii: 2083. [Epub ahead of print]13(9):

Mouse Models for Deciphering the Impact of Homologous Recombination on Tumorigenesis.

Gabriel Matos-Rodrigues, Emmanuelle Martini, Bernard S Lopez.

  Homologous recombination (HR) is a fundamental evolutionarily conserved process that plays prime role(s) in genome stability maintenance through DNA repair and through the protection and resumption of arrested replication forks. Many HR genes are deregulated in cancer cells. Notably, the breast cancer genes BRCA1 and BRCA2, two important HR players, are the most frequently mutated genes in familial breast and ovarian cancer. Transgenic mice constitute powerful tools to unravel the intricate mechanisms controlling tumorigenesis in vivo. However, the genes central to HR are essential in mammals, and their knockout leads to early embryonic lethality in mice. Elaborated strategies have been developed to overcome this difficulty, enabling one to analyze the consequences of HR disruption in vivo. In this review, we first briefly present the molecular mechanisms of HR in mammalian cells to introduce each factor in the HR process. Then, we present the different mouse models of HR invalidation and the consequences of HR inactivation on tumorigenesis. Finally, we discuss the use of mouse models for the development of targeted cancer therapies as well as perspectives on the future potential for understanding the mechanisms of HR inactivation-driven tumorigenesis in vivo.

Keywords:  DNA repair; genomic instability; homologous recombination; mouse model; tumorigenesis

DOI:  https://doi.org/10.3390/cancers13092083
Cancers (Basel). 2021 Apr 24. pii: 2057. [Epub ahead of print]13(9):

The PARP Enzyme Family and the Hallmarks of Cancer Part 2: Hallmarks Related to Cancer Host Interactions.

Máté A Demény, László Virág.

  Poly (ADP-ribose) polymerases (PARPs) modify target proteins with a single ADP-ribose unit or with a poly (ADP-ribose) (PAR) polymer. PARP inhibitors (PARPis) recently became clinically available for the treatment of BRCA1/2 deficient tumors via the synthetic lethality paradigm. This personalized treatment primarily targets DNA damage-responsive PARPs (PARP1-3). However, the biological roles of PARP family member enzymes are broad; therefore, the effects of PARPis should be viewed in a much wider context, which includes complex effects on all known hallmarks of cancer. In the companion paper (part 1) to this review, we presented the fundamental roles of PARPs in intrinsic cancer cell hallmarks, such as uncontrolled proliferation, evasion of growth suppressors, cell death resistance, genome instability, replicative immortality, and reprogrammed metabolism. In the second part of this review, we present evidence linking PARPs to cancer-associated inflammation, anti-cancer immune response, invasion, and metastasis. A comprehensive overview of the roles of PARPs can facilitate the identification of novel cancer treatment opportunities and barriers limiting the efficacy of PARPi compounds.

Keywords:  angiogenesis; anticancer immunity; evasion of immune response; hallmarks of cancer; inflammation; invasion; metastasis; oncogenes; poly (ADP-ribose) polymerase

DOI:  https://doi.org/10.3390/cancers13092057
Biochemistry. 2021 Apr 30.

RNA Editing of the Human DNA Glycosylase NEIL1 Alters Its Removal of 5-Hydroxyuracil Lesions in DNA.

Jongchan Yeo, Elizabeth R Lotsof, Brittany M Anderson-Steele, Sheila S David.

Editing of the pre-mRNA of the DNA repair glycosylase NEIL1 results in substitution of a Lys with Arg in the lesion recognition loop of the enzyme. Unedited (UE, Lys242) NEIL1 removes thymine glycol lesions in DNA ∼30 times faster than edited (Ed, Arg242) NEIL1. Herein, we evaluated recognition and excision mediated by UE and Ed NEIL1 of 5-hydroxyuracil (5-OHU), a highly mutagenic lesion formed via oxidation of cytosine. Both NEIL1 isoforms catalyzed low levels of 5-OHU excision in single-stranded DNA, bubble and bulge DNA contexts and in duplex DNA base paired with A. Removal of 5-OHU in base pairs with G, T, and C was found to be faster and proceed to a higher overall extent with UE than with Ed NEIL1. In addition, the presence of mismatches adjacent to 5-OHU magnified the hampered activity of the Ed isoform. However, Ed NEIL1 was found to exhibit higher affinity for 5-OHU:G and 5-OHU:C duplexes than UE NEIL1. These results suggest that NEIL1 plays an important role in detecting and capturing 5-OHU lesions in inappropriate contexts, in a manner that does not lead to excision, to prevent mutations and strand breaks. Indeed, inefficient removal of 5-OHU by NEIL1 from 5-OHU:A base pairs formed during replication would thwart mutagenesis. Notably, nonproductive engagement of 5-OHU by Ed NEIL1 suggests the extent of 5-OHU repair will be reduced under cellular conditions, such as inflammation, that increase the extent of NEIL1 RNA editing. Tipping the balance between the two NEIL1 isoforms may be a significant factor leading to genome instability.

DOI: https://doi.org/10.1021/acs.biochem.1c00062
Int J Mol Sci. 2021 Apr 03. pii: 3740. [Epub ahead of print]22(7):

RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle.

Zuzana Nascakova, Barbora Boleslavska, Vaclav Urban, Anna Oravetzova, Edita Vlachova, Pavel Janscak, Jana Dobrovolna.

  R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

Keywords:  B02 inhibitor; G1 phase of the cell cycle; R-loop; RAD51; origin of replication; pre-replication complex

DOI:  https://doi.org/10.3390/ijms22073740
Cancers (Basel). 2021 Apr 23. pii: 2042. [Epub ahead of print]13(9):

The PARP Enzyme Family and the Hallmarks of Cancer Part 1. Cell Intrinsic Hallmarks.

Máté A Demény, László Virág.

  The 17-member poly (ADP-ribose) polymerase enzyme family, also known as the ADP-ribosyl transferase diphtheria toxin-like (ARTD) enzyme family, contains DNA damage-responsive and nonresponsive members. Only PARP1, 2, 5a, and 5b are capable of modifying their targets with poly ADP-ribose (PAR) polymers; the other PARP family members function as mono-ADP-ribosyl transferases. In the last decade, PARP1 has taken center stage in oncology treatments. New PARP inhibitors (PARPi) have been introduced for the targeted treatment of breast cancer 1 or 2 (BRCA1/2)-deficient ovarian and breast cancers, and this novel therapy represents the prototype of the synthetic lethality paradigm. Much less attention has been paid to other PARPs and their potential roles in cancer biology. In this review, we summarize the roles played by all PARP enzyme family members in six intrinsic hallmarks of cancer: uncontrolled proliferation, evasion of growth suppressors, cell death resistance, genome instability, reprogrammed energy metabolism, and escape from replicative senescence. In a companion paper, we will discuss the roles of PARP enzymes in cancer hallmarks related to cancer-host interactions, including angiogenesis, invasion and metastasis, evasion of the anticancer immune response, and tumor-promoting inflammation. While PARP1 is clearly involved in all ten cancer hallmarks, an increasing body of evidence supports the role of other PARPs in modifying these cancer hallmarks (e.g., PARP5a and 5b in replicative immortality and PARP2 in cancer metabolism). We also highlight controversies, open questions, and discuss prospects of recent developments related to the wide range of roles played by PARPs in cancer biology. Some of the summarized findings may explain resistance to PARPi therapy or highlight novel biological roles of PARPs that can be therapeutically exploited in novel anticancer treatment paradigms.

Keywords:  cell death; hallmarks of cancer; metabolic reprogramming; oncogenes; poly (ADP-ribose) polymerase; replicative immortality; tumor suppressors

DOI:  https://doi.org/10.3390/cancers13092042
Int J Mol Sci. 2021 Apr 28. pii: 4679. [Epub ahead of print]22(9):

Functional Roles of PARP2 in Assembling Protein-Protein Complexes Involved in Base Excision DNA Repair.

Inna Vasil'eva, Nina Moor, Rashid Anarbaev, Mikhail Kutuzov, Olga Lavrik.

  Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polβ, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD+ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.

Keywords:  PARP1; PARP2; base excision repair; dynamic light scattering; fluorescence techniques; poly(ADP-ribosyl)ation; protein–protein interaction

DOI:  https://doi.org/10.3390/ijms22094679
Trends Genet. 2021 Apr 22. pii: S0168-9525(21)00053-6. [Epub ahead of print]

DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.

Chaoyou Xue, Eric C Greene.

Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.

DOI: https://doi.org/10.1016/j.tig.2021.02.008
Int J Mol Sci. 2021 Apr 16. pii: 4134. [Epub ahead of print]22(8):

The Multifaceted Roles of Ku70/80.

Sayma Zahid, Murielle Seif El Dahan, Florence Iehl, Paloma Fernandez-Varela, Marie-Helene Le Du, Virginie Ropars, Jean Baptiste Charbonnier.

  DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.

Keywords:  DNA repair machinery; c-NHEJ; double-strand break; protein-DNA interactions; telomeres

DOI:  https://doi.org/10.3390/ijms22084134
Br J Pharmacol. 2021 Apr 26.

BBIT20 inhibits homologous DNA repair with disruption of the BRCA1-BARD1 interaction in breast and ovarian cancer.

Liliana Raimundo, Ângela Paterna, Juliana Calheiros, Joana Ribeiro, David Cardoso, Ilaria Piga, Susana Junqueira Neto, Denise Hegan, Peter M Glazer, Stefano Indraccolo, Silva Mulhovo, José Luís Costa, Maria-José U Ferreira, Lucília Saraiva.

   BACKGROUND AND PURPOSE: Advances in the treatment of triple-negative breast cancer (TNBC) and ovarian cancer (OC) remain challenging. In particular, resistance to the available therapy, by restoring or overexpressing the DNA repair machinery, has often been reported. New strategies to improve the therapeutic outcomes of these cancers are needed. Herein, we disclose the dregamine 5-bromo-pyridin-2-ylhydrazone (BBIT20), a natural monoterpene indole alkaloid derivative, as an inhibitor of homologous DNA repair (HDR).
EXPERIMENTAL APPROACH: To unveil BBIT20 antitumor activity and underlying molecular mechanism of action, two-dimensional (2D) and three-dimensional (3D) cell cultures, patient-derived cell lines and xenograft mouse models were used.
KEY RESULTS: BBIT20 disrupted the BRCA1-BARD1 interaction, triggering nuclear-to-cytoplasmic BRCA1 translocation, cell cycle arrest and downregulation of HDR-related genes and proteins, with subsequent enhancement of DNA damage, reactive oxygen species generation and apoptosis, in TNBC and OC cells. BBIT20 also displayed pronounced antitumor activity in patient-derived cells and xenograft mouse models of OC, with low toxicity in non-malignant cells and undetectable side effects in mice. Additionally, it did not induce resistance in TNBC and OC and displayed marked synergistic effects with cisplatin and olaparib (a poly (ADP-ribose) polymerase inhibitor), on 2D and 3D models of these cancer cells.
CONCLUSION AND IMPLICATIONS: These findings add an inhibitor of the BRCA1-BARD1 interaction to the list of DNA-damaging agents. Importantly, either as a single agent or in combination therapy, BBIT20 reveals great potential in the personalized treatment of aggressive and resistant cancers, particularly TNBC and advanced OC.

Keywords:  Anticancer targeted therapy; BRCA1; Homologous recombination; Indole alkaloids

DOI:  https://doi.org/10.1111/bph.15506
Cell Death Discov. 2021 Apr 30. 7(1): 87

Can small molecular inhibitors that stop de novo serine synthesis be used in cancer treatment?

Megan Jessica McNamee, David Michod, Maria Victoria Niklison-Chirou.

To sustain their malignancy, tumour cells acquire several metabolic adaptations such as increased oxygen, glucose, glutamine, and lipids uptake. Other metabolic processes are also enhanced as part of tumour metabolic reprogramming, for example, increased serine metabolism. Serine is a non-essential amino acid that supports several metabolic processes that are crucial for the growth and survival of proliferating cells, including protein, DNA, and glutathione synthesis. Indeed, increased activity of D-3-phosphoglycerate dehydrogenase (PHGDH), the enzyme rate-limiting de novo serine synthesis, has been extensively reported in several tumours. Therefore, selective inhibition of PHGDH may represent a new therapeutic strategy for over-expressing PHGDH tumours, owing to its downstream inhibition of essential biomass production such as one-carbon units and nucleotides. This perspective article will discuss the current status of research into small molecular inhibitors against PHGDH in colorectal cancer, breast cancer, and Ewing's sarcoma. We will summarise recent studies on the development of PHGDH-inhibitors, highlighting their clinical potential as new therapeutics. It also wants to shed a light on some of the key limitations of the use of PHGDH-inhibitors in cancer treatment which are worth taking into account.

DOI: https://doi.org/10.1038/s41420-021-00474-4
Cells. 2021 Apr 13. pii: 889. [Epub ahead of print]10(4):

FLYWCH1, a Multi-Functional Zinc Finger Protein Contributes to the DNA Repair Pathway.

Sheema Almozyan, James Coulton, Roya Babaei-Jadidi, Abdolrahman S Nateri.

  Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways.

Keywords:  ATM; CRC; Cys2-His2 (C2H2)-type zinc-finger; DNA-damage response; FLYWCH1; p53; γH2AX

DOI:  https://doi.org/10.3390/cells10040889
Oncol Lett. 2021 Jun;21(6): 451

Dual roles of SAMHD1 in tumor development and chemoresistance to anticancer drugs.

Zhangming Chen, Jie Hu, Songcheng Ying, Aman Xu.

  Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) has been identified as a GTP or dGTP-dependent deoxynucleotide triphosphohydrolase (dNTPase) and acts as an antiviral factor against certain retroviruses and DNA viruses. Genetic mutation in SAMHD1 causes the inflammatory Aicardi-Goutières Syndrome and abnormal intracellular deoxyribonucleoside triphosphates (dNTPs) pool. At present, the role of SAMHD1 in numerous types of cancer, such as chronic lymphocytic leukemia, lung cancer and colorectal cancer, is highly studied. Furthermore, it has been found that methylation, acetylation and phosphorylation are involved in the regulation of SAMHD1 expression, and that genetic mutations can cause changes in its activities, including dNTPase activity, long interspersed element type 1 (LINE-1) suppression and DNA damage repair, which could lead to uncontrolled cell cycle progression and cancer development. In addition, SAMHD1 has been reported to have a negative regulatory role in the chemosensitivity to anticancer drugs through its dNTPase activity. The present review aimed to summarize the regulation of SAMHD1 expression in cancer and its function in tumor growth and chemotherapy sensitivity, and discussed controversial points and future directions.

Keywords:  DNA damage repair; cancer; cell cycle; chemosensitivity; sterile α motif and HD-domain-containing protein 1

DOI:  https://doi.org/10.3892/ol.2021.12712
Int J Mol Sci. 2021 Apr 23. pii: 4422. [Epub ahead of print]22(9):

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

Wei-Che Tseng, Chi-Yuan Chen, Ching-Yuh Chern, Chu-An Wang, Wen-Chih Lee, Ying-Chih Chi, Shu-Fang Cheng, Yi-Tsen Kuo, Ya-Chen Chiu, Shih-Ting Tseng, Pei-Ya Lin, Shou-Jhen Liou, Yi-Chen Li, Chin-Chuan Chen.

  BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

Keywords:  BRCA-deficient; DNA repair; RAD52 inhibitor; curcumin; synthetic lethal

DOI:  https://doi.org/10.3390/ijms22094422
Nat Protoc. 2021 Apr 28.

Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells.

Zhiming Li, Xu Hua, Albert Serra-Cardona, Xiaowei Xu, Zhiguo Zhang.

Faithful duplication of both genetic and epigenetic information is essential for all eukaryotic cells. DNA replication initiates from replication origins and proceeds bidirectionally but asymmetrically, with the leading strand being synthesized continuously and the lagging strand discontinuously as Okazaki fragments by distinct DNA polymerases. Unraveling the underlying mechanisms of chromatin replication at both strands is crucial to better understand DNA replication and its coupled processes, including nucleosome assembly, sister chromatid cohesion and DNA mismatch repair. Here we describe the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method to analyze the enrichment of proteins of interest, including histones and their modifications at replicating chromatin in a strand-specific manner in mammalian cells. Briefly, cells are pulsed with the thymidine analog bromodeoxyuridine to label newly synthesized DNA. After cell permeabilization, the target proteins are sequentially bound by antibodies and protein A-fused transposase, which digests and tags genomic DNA of interest once activated by magnesium. The strand specificity is preserved through oligo-replacement. Finally, the resulting double-strand DNA is denatured and immunoprecipitated with antibodies against bromodeoxyuridine to enrich nascent DNA associated with proteins of interest. After PCR amplification and next-generation sequencing, the mapped reads are used to calculate the relative enrichment of the target proteins around replication origins. Compared with alternative methods, the eSPAN protocol is simple, cost-effective and sensitive, even in a relatively small number of mammalian cells. The whole procedures from cell collection to generation of sequencing-ready libraries can be completed in 2 days.

DOI: https://doi.org/10.1038/s41596-021-00520-6
Cancers (Basel). 2021 Apr 07. pii: 1769. [Epub ahead of print]13(8):

Treatment Strategies for ARID1A-Deficient Ovarian Clear Cell Carcinoma.

Kazuaki Takahashi, Masataka Takenaka, Aikou Okamoto, David D L Bowtell, Takashi Kohno.

  Ovarian clear cell carcinoma (OCCC) is a histological subtype of ovarian cancer that is more frequent in Asian countries (~25% of ovarian cancers) than in US/European countries (less than 10%). OCCC is refractory to conventional platinum-based chemotherapy, which is effective against high-grade serous carcinoma (HGSC), a major histological subtype of ovarian cancer. Notably, deleterious mutations in SWI/SNF chromatin remodeling genes, such as ARID1A, are common in OCCC but rare in HGSC. Because this complex regulates multiple cellular processes, including transcription and DNA repair, molecularly targeted therapies that exploit the consequences of SWI/SNF deficiency may have clinical efficacy against OCCC. Three such strategies have been proposed to date: prioritizing a gemcitabine-based chemotherapeutic regimen, synthetic lethal therapy targeting vulnerabilities conferred by SWI/SNF deficiency, and immune checkpoint blockade therapy that exploits the high mutational burden of ARID1A-deficient tumor. Thus, ARID1A deficiency has potential as a biomarker for precision medicine of ovarian cancer.

Keywords:  ARID1A; gemcitabine; molecular targeted therapy; ovarian clear cell carcinoma; precision medicine; synthetic lethality

DOI:  https://doi.org/10.3390/cancers13081769
Int J Mol Sci. 2021 Apr 15. pii: 4102. [Epub ahead of print]22(8):

Knockdown of RRM1 with Adenoviral shRNA Vectors to Inhibit Tumor Cell Viability and Increase Chemotherapeutic Sensitivity to Gemcitabine in Bladder Cancer Cells.

Xia Zhang, Rikiya Taoka, Dage Liu, Yuki Matsuoka, Yoichiro Tohi, Yoshiyuki Kakehi, Mikio Sugimoto.

  RRM1-an important DNA replication/repair enzyme-is the primary molecular gemcitabine (GEM) target. High RRM1-expression associates with gemcitabine-resistance in various cancers and RRM1 inhibition may provide novel cancer treatment approaches. Our study elucidates how RRM1 inhibition affects cancer cell proliferation and influences gemcitabine-resistant bladder cancer cells. Of nine bladder cancer cell lines investigated, two RRM1 highly expressed cells, 253J and RT112, were selected for further experimentation. An RRM1-targeting shRNA was cloned into adenoviral vector, Ad-shRRM1. Gene and protein expression were investigated using real-time PCR and western blotting. Cell proliferation rate and chemotherapeutic sensitivity to GEM were assessed by MTT assay. A human tumor xenograft model was prepared by implanting RRM1 highly expressed tumors, derived from RT112 cells, in nude mice. Infection with Ad-shRRM1 effectively downregulated RRM1 expression, significantly inhibiting cell growth in both RRM1 highly expressed tumor cells. In vivo, Ad-shRRM1 treatment had pronounced antitumor effects against RRM1 highly expressed tumor xenografts (p < 0.05). Moreover, combination of Ad-shRRM1 and GEM inhibited cell proliferation in both cell lines significantly more than either treatment individually. Cancer gene therapy using anti-RRM1 shRNA has pronounced antitumor effects against RRM1 highly expressed tumors, and RRM1 inhibition specifically increases bladder cancer cell GEM-sensitivity. Ad-shRRM1/GEM combination therapy may offer new treatment options for patients with GEM-resistant bladder tumors.

Keywords:  RRM1; adenoviral vector; chemotherapeutic sensitivity; gemcitabine; gene therapy; shRNA

DOI:  https://doi.org/10.3390/ijms22084102
Genes (Basel). 2021 Apr 19. pii: 600. [Epub ahead of print]12(4):

Inosine in Biology and Disease.

Sundaramoorthy Srinivasan, Adrian Gabriel Torres, Lluís Ribas de Pouplana.

  The nucleoside inosine plays an important role in purine biosynthesis, gene translation, and modulation of the fate of RNAs. The editing of adenosine to inosine is a widespread post-transcriptional modification in transfer RNAs (tRNAs) and messenger RNAs (mRNAs). At the wobble position of tRNA anticodons, inosine profoundly modifies codon recognition, while in mRNA, inosines can modify the sequence of the translated polypeptide or modulate the stability, localization, and splicing of transcripts. Inosine is also found in non-coding and exogenous RNAs, where it plays key structural and functional roles. In addition, molecular inosine is an important secondary metabolite in purine metabolism that also acts as a molecular messenger in cell signaling pathways. Here, we review the functional roles of inosine in biology and their connections to human health.

Keywords:  RNA modification; adenosine deaminase acting on RNAs; deamination; inosine; translation

DOI:  https://doi.org/10.3390/genes12040600
Biochem Biophys Res Commun. 2021 Apr 25. pii: S0006-291X(21)00685-9. [Epub ahead of print]558 120-125

Characterization of deoxyribonucleoside transport mediated by concentrative nucleoside transporters.

Taiki Yamamura, Katsuya Narumi, Tsukika Ohata, Hiroshi Satoh, Takao Mori, Ayako Furugen, Masaki Kobayashi, Ken Iseki.

  Human concentrative nucleoside transporters (CNTs) are responsible for cellular uptake of ribonucleosides; however, although it is important to better characterize CNT-subtype specificity to understand the systemic disposition of deoxyribonucleosides (dNs) and their analogs, the involvement of CNTs in transporting dNs is not fully understood. In this study, using COS-7 cells that transiently expressed CNT1, CNT2, or CNT3, we investigated if CNTs could transport not only ribonucleosides but also dNs, i.e., 2'-deoxyadenosine (dAdo), 2'-deoxyguanosine (dGuo), and 2'-deoxycytidine (dCyd). The cellular uptake study demonstrated that dAdo and dGuo were taken up by CNT2 but not by CNT1. Although dCyd was taken up by CNT1, no significant uptake was detected in COS-7 cells expressing CNT2. Similarly, these dNs were transported by CNT3. The apparent Km values of their uptake were as follows: CNT1, Km ＝ 141 μM for dCyd; CNT2, Km ＝ 62.4 μM and 54.9 μM for dAdo and dGuo, respectively; CNT3, Km ＝ 14.7 μM and 34.4 μM for dGuo and dCyd, respectively. These results demonstrate that CNTs contribute not only to ribonucleoside transport but also to the transport of dNs. Moreover, our data indicated that CNT1 and CNT2 selectively transported pyrimidine and purine dNs, respectively, and CNT3 was shown to transport both pyrimidine and purine dNs.

Keywords:  2ʹ-deoxyadenosine; 2ʹ-deoxycytidine; 2ʹ-deoxyguanosine; Concentrative nucleoside transporter; Deoxyribonucleoside; Thymidine

DOI:  https://doi.org/10.1016/j.bbrc.2021.04.075
J Hematol Oncol. 2021 Apr 26. 14(1): 70

Targeting the metabolic vulnerability of acute myeloid leukemia blasts with a combination of venetoclax and 8-chloro-adenosine.

Ralf Buettner, Le Xuan Truong Nguyen, Corey Morales, Min-Hsuan Chen, Xiwei Wu, Lisa S Chen, Dinh Hoa Hoang, Servando Hernandez Vargas, Vinod Pullarkat, Varsha Gandhi, Guido Marcucci, Steven T Rosen.

   BACKGROUND: BCL-2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60-70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML.
METHODS: Using AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan-Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML.
RESULTS: We here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone (p ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells.
CONCLUSION: Taken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.

Keywords:  Acute myeloid leukemia; Fatty acid oxidation; Metabolism; Nucleoside analog; Oxidative phosphorylation

DOI:  https://doi.org/10.1186/s13045-021-01076-4
Cancers (Basel). 2021 Apr 01. pii: 1656. [Epub ahead of print]13(7):

Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration.

Xian Chen, Dong Yang, Jason P W Carey, Cansu Karakas, Constance Albarracin, Aysegul A Sahin, Banu K Arun, Merih Guray Durak, Mi Li, Mehrnoosh Kohansal, Tuyen N Bui, Min-Jin Ha, Kelly K Hunt, Khandan Keyomarsi.

  The identification of biomarker-driven targeted therapies for patients with triple negative breast cancer (TNBC) remains a major clinical challenge, due to a lack of specific targets. Here, we show that cyclin E, a major regulator of G1 to S transition, is deregulated in TNBC and is associated with mutations in DNA repair genes (e.g., BRCA1/2). Breast cancers with high levels of cyclin E not only have a higher prevalence of BRCA1/2 mutations, but also are associated with the worst outcomes. Using several in vitro and in vivo model systems, we show that TNBCs that harbor either mutations in BRCA1/2 or overexpression of cyclin E are very sensitive to the growth inhibitory effects of AZD-1775 (Wee 1 kinase inhibitor) when used in combination with MK-4837 (PARP inhibitor). Combination treatment of TNBC cell lines with these two agents results in synergistic cell killing due to induction of replicative stress, downregulation of DNA repair and cytokinesis failure that results in increased apoptosis. These findings highlight the potential clinical application of using cyclin E and BRCA mutations as biomarkers to select only those patients with the highest replicative stress properties that may benefit from combination treatment with Wee 1 kinase and PARP inhibitors.

Keywords:  BRCA; DNA replication stress; Wee1 kinase; cyclin E; low molecular weight cyclin E (LMWE) PARP

DOI:  https://doi.org/10.3390/cancers13071656
ESMO Open. 2021 Apr 22. pii: S2059-7029(21)00084-3. [Epub ahead of print]6(3): 100125

Issues and limitations of available biomarkers for fluoropyrimidine-based chemotherapy toxicity, a narrative review of the literature.

K Hodroj, D Barthelemy, J-C Lega, G Grenet, M-C Gagnieu, T Walter, J Guitton, L Payen-Gay.

  Fluoropyrimidine-based chemotherapies are widely used to treat gastrointestinal tract, head and neck, and breast carcinomas. Severe toxicities mostly impact rapidly dividing cell lines and can occur due to the partial or complete deficiency in dihydropyrimidine dehydrogenase (DPD) catabolism. Since April 2020, the European Medicines Agency (EMA) recommends DPD testing before any fluoropyrimidine-based treatment. Currently, different assays are used to predict DPD deficiency; the two main approaches consist of either phenotyping the enzyme activity (directly or indirectly) or genotyping the four main deficiency-related polymorphisms associated with 5-fluorouracil (5-FU) toxicity. In this review, we focused on the advantages and limitations of these diagnostic methods: direct phenotyping by evaluation of peripheral mononuclear cell DPD activity (PBMC-DPD activity), indirect phenotyping assessed by uracil levels or UH2/U ratio, and genotyping DPD of four variants directly associated with 5-FU toxicity. The risk of 5-FU toxicity increases with uracil concentration. Having a pyrimidine-related structure, 5-FU is catabolised by the same physiological pathway. By assessing uracil concentration in plasma, indirect phenotyping of DPD is then measured. With this approach, in France, a decreased 5-FU dose is systematically recommended at a uracil concentration of 16 ng/ml, which may lead to chemotherapy under-exposure as uracil concentration is a continuous variable and the association between uracil levels and DPD activity is not clear. We aim herein to describe the different available strategies developed to improve fluoropyrimidine-based chemotherapy safety, how they are implemented in routine clinical practice, and the possible relationship with inefficacy mechanisms.

Keywords:  5-FU; dihydropyrimidine dehydrogenase; genotype; phenotype; toxicity; uracil

DOI:  https://doi.org/10.1016/j.esmoop.2021.100125
Redox Biol. 2021 Apr 15. pii: S2213-2317(21)00126-9. [Epub ahead of print] 101978

Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A.

Bess Yi Kun Yu, Maria-Armineh Tossounian, Stefan Denchev Hristov, Ryan Lawrence, Pallavi Arora, Yugo Tsuchiya, Sew Yeu Peak-Chew, Valeriy Filonenko, Sally Oxenford, Richard Angell, Jerome Gouge, Mark Skehel, Ivan Gout.

  The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3'-5' exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modifications, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A (CoA) binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1β cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions.

Keywords:  Coenzyme A; Metastasis suppressor; NDPK; Oxidative stress; Protein CoAlation; Redox regulation

DOI:  https://doi.org/10.1016/j.redox.2021.101978