bims-numges 2021-09-12 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒09‒12
thirty-two papers selected by
Sean Rudd
Karolinska Institutet

The RAD51 recombinase protects mitotic chromatin in human cells.
Role of 53BP1 in end protection and DNA synthesis at DNA breaks.
Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.
The novel nucleoside analogue ProTide NUC-7738 overcomes cancer resistance mechanisms in vitro and in a first-in-human Phase 1 clinical trial.
The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions.
Immune checkpoint inhibitor sensitivity of DNA repair deficient tumors.
Significance of base excision repair to human health.
The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining.
Genomic instability and metabolism in cancer.
Cancer testis antigens and genomic instability: More than immunology.
XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase.
Transcription-associated DNA breaks and cancer: A matter of DNA topology.
Niraparib Suppresses Cholangiocarcinoma Tumor Growth by Inducing Oxidative and Replication Stress.
Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.
MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA.
Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.
DNA double-strand break repair in cancer: A path to achieving precision medicine.
The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.
A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU.
Lymphoid-specific helicase in epigenetics, DNA repair and cancer.
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage.
Cyclin E expression is associated with high levels of replication stress in triple-negative breast cancer.
Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion.
TARG1 protects against toxic DNA ADP-ribosylation.
Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.
5-Aza-4'-thio-2'-deoxycytidine, a new orally bioavailable non-toxic "best-in-class" DNMT1 depleting agent in clinical development.
Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome.
Reactive oxygen species and DNA damage response in cancer.
Break-induced replication mechanisms in yeast and mammals.
A combination of PARP and CHK1 inhibitors efficiently antagonizes MYCN-driven tumors.
Inhibition of CK1ε potentiates the therapeutic efficacy of CDK4/6 inhibitor in breast cancer.
Integrated evaluation of telomerase activation and telomere maintenance across cancer cell lines.

Nat Commun. 2021 Sep 10. 12(1): 5380

The RAD51 recombinase protects mitotic chromatin in human cells.

Isabel E Wassing, Emily Graham, Xanita Saayman, Lucia Rampazzo, Christine Ralf, Andrew Bassett, Fumiko Esashi.

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

DOI: https://doi.org/10.1038/s41467-021-25643-y
Genes Dev. 2021 Sep 09.

Role of 53BP1 in end protection and DNA synthesis at DNA breaks.

Jacob Paiano, Nicholas Zolnerowich, Wei Wu, Raphael Pavani, Chen Wang, Hongzhi Li, Li Zheng, Binghui Shen, Barry P Sleckman, Bo-Ruei Chen, André Nussenzweig.

  Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

Keywords:  53BP1; end resection; homologous recombination

DOI:  https://doi.org/10.1101/gad.348667.121
J Biol Chem. 2021 Sep 04. pii: S0021-9258(21)00972-8. [Epub ahead of print] 101170

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Nicole E Bowen, Joshua Temple, Caitlin Shepard, Adrian Oo, Fidel Arizaga, Priya Kapoor-Vazirani, Mirjana Persaud, Corey H Yu, Dong-Hyun Kim, Raymond F Schinazi, Dmitri N Ivanov, Felipe Diaz-Griffero, David S Yu, Yong Xiong, Baek Kim.

  Elevated intracellular levels of deoxy-nucleotide triphosphates (dNTPs) have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multi-functional dNTPase, SAMHD1, have been reported in various cancers. Here we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely due to loss of interaction with γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating double-stranded DNA break repair, interacting with CtIP and Cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Keywords:  SAMHD1; cancer; dNTPs; enzymology; structure

DOI:  https://doi.org/10.1016/j.jbc.2021.101170
Clin Cancer Res. 2021 Sep 08. pii: clincanres.1652.2021. [Epub ahead of print]

The novel nucleoside analogue ProTide NUC-7738 overcomes cancer resistance mechanisms in vitro and in a first-in-human Phase 1 clinical trial.

Hagen Schwenzer, Erica De Zan, Mustafa Elshani, Ruud van Stiphout, Mary Kudsy, Josephine Morris, Valentina Ferrari, In Hwa Um, James Chettle, Farasat Kazmi, Leticia Campo, Alistair Easton, Sebastian Nijman, Michaela Serpi, Stefan Symeonides, Ruth Plummer, David J Harrison, Gareth Bond, Sarah P Blagden.

PURPOSE: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a pre-activated nucleoside released. 3'-deoxyadenosine (3'-dA) is a naturally-occurring adenosine analogue with established anti-cancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738.EXPERIMENTAL DESIGN: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in cancer patients.
RESULTS: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide binding protein 1. PK and tumour samples obtained from the ongoing first-in-human Phase 1 clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective pro-apoptotic agent in cancer cells with effects on the NF-kappaB pathway.
CONCLUSIONS: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and support its further clinical evaluation as a novel cancer treatment within the growing pantheon of anti-cancer ProTides.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-1652
Elife. 2021 Sep 08. pii: e72286. [Epub ahead of print]10

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions.

Charanya Kumar, Sahil Batra, Jack D Griffith, Dirk Remus.

  R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Keywords:  S. cerevisiae; biochemistry; chemical biology; chromosomes; gene expression

DOI:  https://doi.org/10.7554/eLife.72286
Immunotherapy. 2021 Sep 08.

Immune checkpoint inhibitor sensitivity of DNA repair deficient tumors.

Pauline Rochefort, Françoise Desseigne, Valérie Bonadona, Sophie Dussart, Clélia Coutzac, Matthieu Sarabi, Christelle de la Fouchardiere.

  Faithful DNA replication is necessary to maintain genome stability and implicates a complex network with several pathways depending on DNA damage type: homologous repair, nonhomologous end joining, base-excision repair, nucleotide-excision repair and mismatch repair. Alteration in components of DNA repair machinery led to DNA damage accumulation and potentially carcinogenesis. Preclinical data suggest sensitivity to immune checkpoint inhibitors in tumors with DNA repair deficiency. Here, we review clinical studies that explored the use of immune checkpoint inhibitor in patient harboring tumor with DNA repair deficiency.

Keywords:  BRCA; DNA repair deficiency; biomarker; cancer; immune checkpoint inhibitors; immunotherapy; microsatellite instability

DOI:  https://doi.org/10.2217/imt-2021-0024
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00061-7. [Epub ahead of print]364 163-193

Significance of base excision repair to human health.

Shengyuan Zhao, Serkalem Tadesse, Dawit Kidane.

  Oxidative and alkylating DNA damage occurs under normal physiological conditions and exogenous exposure to DNA damaging agents. To counteract DNA base damage, cells have evolved several defense mechanisms that act at different levels to prevent or repair DNA base damage. Cells combat genomic lesions like these including base modifications, abasic sites, as well as single-strand breaks, via the base excision repair (BER) pathway. In general, the core BER process involves well-coordinated five-step reactions to correct DNA base damage. In this review, we will uncover the current understanding of BER mechanisms to maintain genomic stability and the biological consequences of its failure due to repair gene mutations. The malfunction of BER can often lead to BER intermediate accumulation, which is genotoxic and can lead to different types of human disease. Finally, we will address the use of BER intermediates for targeted cancer therapy.

Keywords:  Base excision repair; DNA damage; Genomic instability; Inflammation associated disease

DOI:  https://doi.org/10.1016/bs.ircmb.2021.05.002
DNA Repair (Amst). 2021 Sep 01. pii: S1568-7864(21)00173-7. [Epub ahead of print]108 103217

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining.

Bo-Ruei Chen, Yinan Wang, Zih-Jie Shen, Amelia Bennett, Issa Hindi, Jessica K Tyler, Barry P Sleckman.

  The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

Keywords:  DNA end resection; NHEJ; RNF168; RNF8; V(D)J recombination; XLF

DOI:  https://doi.org/10.1016/j.dnarep.2021.103217
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00063-0. [Epub ahead of print]364 241-265

Genomic instability and metabolism in cancer.

Haojian Li, Susan E Zimmerman, Urbain Weyemi.

  Genomic instability and metabolic reprogramming are among the key hallmarks discriminating cancer cells from normal cells. The two phenomena contribute to the robust and evasive nature of cancer, particularly when cancer cells are exposed to chemotherapeutic agents. Genomic instability is defined as the increased frequency of mutations within the genome, while metabolic reprogramming is the alteration of metabolic pathways that cancer cells undergo to adapt to increased bioenergetic demand. An underlying source of these mutations is the aggregate product of damage to the DNA, and a defective repair pathway, both resulting in the expansion of genomic lesions prior to uncontrolled proliferation and survival of cancer cells. Exploitation of DNA damage and the subsequent DNA damage response (DDR) have aided in defining therapeutic approaches in cancer. Studies have demonstrated that targeting metabolic reprograming yields increased sensitivity to chemo- and radiotherapies. In the past decade, it has been shown that these two key features are interrelated. Metabolism impacts DNA damage and DDR via regulation of metabolite pools. Conversely, DDR affects the response of metabolic pathways to therapeutic agents. Because of the interplay between genomic instability and metabolic reprogramming, we have compiled findings which more selectively highlight the dialog between metabolism and DDR, with a particular focus on glucose metabolism and double-strand break (DSB) repair pathways. Decoding this dialog will provide significant clues for developing combination cancer therapies.

Keywords:  Cancer; DNA repair; Genomic instability; Glycolysis; Metabolism; Mitochondrial homeostasis

DOI:  https://doi.org/10.1016/bs.ircmb.2021.05.004
DNA Repair (Amst). 2021 Aug 17. pii: S1568-7864(21)00170-1. [Epub ahead of print]108 103214

Cancer testis antigens and genomic instability: More than immunology.

Ash Jay, Diedre Reitz, Satoshi H Namekawa, Wolf-Dietrich Heyer.

  Cancer testis antigens or genes (CTA, CTG) are predominantly expressed in adult testes while silenced in most or all somatic tissues with sporadic expression in many human cancers. Concerted misexpression of numerous CTA/CTGs is rarely observed. This finding argues against the germ cell theory of cancer. A surprising number of CTA/CTGs are involved in meiotic chromosome metabolism and specifically in meiotic recombination. Recent discoveries with a group of CTGs established that their misexpression in somatic cells results in genomic instability by interfering with homologous recombination (HR), a DNA repair pathway for complex DNA damage such as DNA double-stranded breaks, interstrand crosslinks, and single-stranded DNA gaps. HR-deficient tumors have specific vulnerabilities and show synthetic lethality with inhibition of polyADP-ribose polymerase, opening the possibility that expression of CTA/CTGs that result in an HR-defect could be used as an additional biomarker for HR status. Here, we review the repertoire of CTA/CTGs focusing on a cohort that functions in meiotic chromosome metabolism by interrogating relevant cancer databases and discussing recent discoveries.

Keywords:  BRCAness; Homologous recombination; Meiosis; Poly (ADP-ribose) polymerase inhibition; Replication fork protection

DOI:  https://doi.org/10.1016/j.dnarep.2021.103214
Nucleic Acids Res. 2021 Sep 09. pii: gkab785. [Epub ahead of print]

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase.

Abhishek Bharadwaj Sharma, Hélène Erasimus, Lia Pinto, Marie-Christine Caron, Diyavarshini Gopaul, Thibaut Peterlini, Katrin Neumann, Petr V Nazarov, Sabrina Fritah, Barbara Klink, Christel C Herold-Mende, Simone P Niclou, Philippe Pasero, Patrick Calsou, Jean-Yves Masson, Sébastien Britton, Eric Van Dyck.

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.

DOI: https://doi.org/10.1093/nar/gkab785
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00060-5. [Epub ahead of print]364 195-240

Transcription-associated DNA breaks and cancer: A matter of DNA topology.

Agnese Cristini, Mathéa Géraud, Olivier Sordet.

  Transcription is an essential cellular process but also a major threat to genome integrity. Transcription-associated DNA breaks are particularly detrimental as their defective repair can induce gene mutations and oncogenic chromosomal translocations, which are hallmarks of cancer. The past few years have revealed that transcriptional breaks mainly originate from DNA topological problems generated by the transcribing RNA polymerases. Defective removal of transcription-induced DNA torsional stress impacts on transcription itself and promotes secondary DNA structures, such as R-loops, which can induce DNA breaks and genome instability. Paradoxically, as they relax DNA during transcription, topoisomerase enzymes introduce DNA breaks that can also endanger genome integrity. Stabilization of topoisomerases on chromatin by various anticancer drugs or by DNA alterations, can interfere with transcription machinery and cause permanent DNA breaks and R-loops. Here, we review the role of transcription in mediating DNA breaks, and discuss how deregulation of topoisomerase activity can impact on transcription and DNA break formation, and its connection with cancer.

Keywords:  Cancer; DNA break; DNA topology; Genome instability; Oncogenic translocation; R-loop; RNA polymerase; RNA/DNA hybrid; Topoisomerase; Transcription

DOI:  https://doi.org/10.1016/bs.ircmb.2021.05.001
Cancers (Basel). 2021 Aug 31. pii: 4405. [Epub ahead of print]13(17):

Niraparib Suppresses Cholangiocarcinoma Tumor Growth by Inducing Oxidative and Replication Stress.

Vladimir Bezrookove, John M Patino, Mehdi Nosrati, Pierre-Yves Desprez, Sean McAllister, Liliana Soroceanu, Ari Baron, Robert Osorio, Mohammed Kashani-Sabet, Altaf A Dar.

  Cholangiocarcinoma (CCA) is the second most common hepatobiliary cancer, an aggressive malignancy with limited therapeutic options. PARP (poly (ADP-ribose) polymerase) 1 and 2 are important for deoxyribonucleotide acid (DNA) repair and maintenance of genomic stability. PARP inhibitors (PARPi) such as niraparib have been approved for different malignancies with genomic alteration in germline BRCA and DNA damage response (DDR) pathway genes. Genomic alterations were analyzed in DDR genes in CCA samples employing The Cancer Genome Atlas (TCGA) database. Mutations were observed in various DDR genes, and 35.8% cases had alterations in at least one of three genes (ARID1A, BAP1 and ATM), suggesting their susceptibility to PARPi. Niraparib treatment suppressed cancer cell viability and survival, and also caused G2/M cell cycle arrest in patient-derived xenograft cells lines (PDXC) and established CCA cells harboring DDR gene mutations. PARPi treatment also induced apoptosis and caspase3/7 activity in PDXC and CCA cell lines, and substantially reduced expression of BCL2, BCL-XL and MCL1 proteins. Niraparib caused a significant increase in oxidative stress, and induced activation of DNA damage markers, phosphorylation of CHK2 and replication fork stalling. Importantly, niraparib, in combination with gemcitabine, produced sustained and robust inhibition of tumor growth in vivo in a patient-derived xenograft (PDX) model more effectively than either treatment alone. Furthermore, tissue samples from mice treated with niraparib and gemcitabine display significantly lower expression levels of pHH3 and Ki-67, which are a mitotic and proliferative marker, respectively. Taken together, our results indicate niraparib as a novel therapeutic agent alone or in combination with gemcitabine for CCA.

Keywords:  PAPR inhibitors; cholangiocarcinoma; oxidative stress; patient derived xenograft; replication fork stalling

DOI:  https://doi.org/10.3390/cancers13174405
Oncotarget. 2021 Aug 31. 12(18): 1763-1779

Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.

Mario Scarpa, Shivani Kapoor, Eric S Tvedte, Kshama A Doshi, Ying S Zou, Prerna Singh, Jonelle K Lee, Aditi Chatterjee, Moaath K Mustafa Ali, Robin E Bromley, Julie C Dunning Hotopp, Feyruz V Rassool, Maria R Baer.

  Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Keywords:  FLT3 internal tandem duplication; Pim kinase; alternative non-homologous end-joining DNA repair; genomic instability; topoisomerase 2 inhibitors

DOI:  https://doi.org/10.18632/oncotarget.28042
Int J Mol Sci. 2021 Aug 30. pii: 9392. [Epub ahead of print]22(17):

MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA.

Ming-Tsung Wu, Wei-Ting Ye, Yi-Cheng Wang, Po-Ming Chen, Jun-You Liu, Chien-Kuo Tai, Feng-Yao Tang, Jian-Rong Li, Chun-Chi Liu, En-Pei Isabel Chiang.

  Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = -0.81, p < 0.0001), MTHFR protein expression (r = -0.8; p = 0.01), and MTHFR enzyme activity (r = -0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion-induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion-induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight into the significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies.

Keywords:  MTHFR; folate depletion; hepatocellular carcinoma; micronuclei; nucleotide biosynthesis; uracil misincorporation

DOI:  https://doi.org/10.3390/ijms22179392
Mol Pharmacol. 2021 Sep 09. pii: MOLPHARM-AR-2021-000333. [Epub ahead of print]

Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.

Siennah R Miller, Meghan E McGrath, Kimberley M Zorn, Sean Ekins, Stephen H Wright, Nathan J Cherrington.

  Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs remdesivir, molnupiravir and its active metabolite, EIDD-1931 and four non-nucleoside molecules repurposed as antivirals for COVID-19. The study used 3D pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H] uridine uptake (ENT1 IC50: 38.65 mM; ENT2 IC50: 76.72 mM), followed by EIDD-1931 (ENT1 IC50: 258.9 mM; ENT2 IC50: 467.3 mM), while molnupiravir was a modest inhibitor (ENT1 IC50: 701.0 mM; ENT2 IC50: 851.4 mM). Other proposed antivirals failed to inhibit ENT-mediated [3H] uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of NBMPR by 30% in ENT1 cells (p = 0.0248) and 27% in ENT2 cells (p = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (p = 0.0463 ) and by 64% in ENT2 cells (p = 0.0132), supporting computational predictions that both are ENT substrates which may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. Significance Statement Significance statement: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.

Keywords:  Antiviral drugs; Transporter-mediated drug/metabolite disposition; Uptake transporters (OATP, OAT, OCT, PEPT, MCT, NTCP, ASBT, etc.); Uptake transporters (OATP, OAT, OCT, PEPT, MCT, NTCP, ASBT, etc.)s; antivirals; drug transport

DOI:  https://doi.org/10.1124/molpharm.121.000333
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00077-0. [Epub ahead of print]364 111-137

DNA double-strand break repair in cancer: A path to achieving precision medicine.

Taneisha Gillyard, Jamaine Davis.

  The assessment of DNA damage can be a significant diagnostic for precision medicine. DNA double strand break (DSBs) pathways in cancer are the primary targets in a majority of anticancer therapies, yet the molecular vulnerabilities that underlie each tumor can vary widely making the application of precision medicine challenging. Identifying and understanding these interindividual vulnerabilities enables the design of targeted DSB inhibitors along with evolving precision medicine approaches to selectively kill cancer cells with minimal side effects. A major challenge however, is defining exactly how to target unique differences in DSB repair pathway mechanisms. This review comprises a brief overview of the DSB repair mechanisms in cancer and includes results obtained with revolutionary advances such as CRISPR/Cas9 and machine learning/artificial intelligence, which are rapidly advancing not only our understanding of determinants of DSB repair choice, but also how it can be used to advance precision medicine. Scientific innovation in the methods used to diagnose and treat cancer is converging with advances in basic science and translational research. This revolution will continue to be a critical driver of precision medicine that will enable precise targeting of unique individual mechanisms. This review aims to lay the foundation for achieving this goal.

Keywords:  Artificial intelligence; DSB repair; Homologous recombination; Machine learning; Nonhomologous end joining; Precision medicine; Risk prediction

DOI:  https://doi.org/10.1016/bs.ircmb.2021.06.003
Cell Div. 2021 Sep 08. 16(1): 4

The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.

Muhseena N Katheeja, Shankar Prasad Das, Suparna Laha.

  BACKGROUND: The budding yeast protein Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This helicase is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage.RESULTS: G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with faster kinetics in chl1 mutants compared to wild-type cells. Also, more damage to DNA is observed in chl1 cells. The viability falls synergistically in rad24chl1 cells. The regulation of Chl1p on budding kinetics in G1 phase falls in line with Rad9p/Chk1p and shows a synergistic effect with Rad24p/Rad53p. rad9chl1 and chk1chl1 shows similar bud emergence as the single mutants chl1, rad9 and chk1. Whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24, rad53 and chl1. In presence of MMS induced damage, synergistic with Rad24p indicates Chl1p's role as a checkpoint at G1/S acting parallel to damage checkpoint pathway. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further, we have also confirmed that the checkpoint defect functions in parallel to the damage checkpoint pathway of Rad24p.
CONCLUSION: Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint activity in presence of damage. This confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1p thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p for Rad53p activation when damaging agents perturb the DNA. Apart from checkpoint activation, it also regulates the budding kinetics as a repair gene.

Keywords:  Bud-emergence; Checkpoint; Chl1p; DNA damage; DNA repair; G1/S phase; Yeast

DOI:  https://doi.org/10.1186/s13008-021-00072-x
Int J Mol Sci. 2021 Aug 30. pii: 9409. [Epub ahead of print]22(17):

A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU.

Thomas Eekhout, José Antonio Pedroza-Garcia, Pooneh Kalhorzadeh, Geert De Jaeger, Lieven De Veylder.

  During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.

Keywords:  DNA damage; cell cycle checkpoint; replication stress

DOI:  https://doi.org/10.3390/ijms22179409
Br J Cancer. 2021 Sep 07.

Lymphoid-specific helicase in epigenetics, DNA repair and cancer.

Xiangyu Chen, Yamei Li, Karla Rubio, Bi Deng, Yuyi Li, Qinwei Tang, Chao Mao, Shuang Liu, Desheng Xiao, Guillermo Barreto, Yongguang Tao.

Lymphoid-specific helicase (LSH) is a member of the SNF2 helicase family of chromatin-remodelling proteins. Dysfunctions or mutations in LSH causes an autosomal recessive disease known as immunodeficiency-centromeric instability-facial anomaly (ICF) syndrome. Interestingly, LSH participates in various aspects of epigenetic regulation, including nucleosome remodelling, DNA methylation, histone modifications and heterochromatin formation. Further, LSH plays a crucial role during DNA-damage repair, specifically during double-strand break (DSB) repair, since murine LSH was shown to be essential for non-homologous end joining (NHEJ) and homologous recombination (HR). Accordingly, overexpression of LSH drives tumorigenesis and malignancy. On the other hand, LSH homologs stabilise the genome. Thus, LSH might be implemented as a biomarker for various cancer types and potential target molecule to develop therapeutic strategies against them. In this review, we focus on the role of LSH in orchestrating chromatin rearrangements, such as DNA methylation and histone modifications, as well as in DNA-damage repair. Changes in chromatin structure may facilitate gene expression signatures that cause malignant transformation. We summarise recent findings of LSH in cancers and raise critical open questions for further studies.

DOI: https://doi.org/10.1038/s41416-021-01543-2
J Vis Exp. 2021 Aug 21.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage.

Vasiliki Pantazi, Ivett Berzsenyi, Barbara N Borsos, Tibor Pankotai.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

DOI: https://doi.org/10.3791/62175
NPJ Breast Cancer. 2020 Sep 07. 6(1): 40

Cyclin E expression is associated with high levels of replication stress in triple-negative breast cancer.

Sergi Guerrero Llobet, Bert van der Vegt, Evelien Jongeneel, Rico D Bense, Mieke C Zwager, Carolien P Schröder, Marieke Everts, Rudolf S N Fehrmann, Geertruida H de Bock, Marcel A T M van Vugt.

Replication stress entails the improper progression of DNA replication. In cancer cells, including breast cancer cells, an important cause of replication stress is oncogene activation. Importantly, tumors with high levels of replication stress may have different clinical behavior, and high levels of replication stress appear to be a vulnerability of cancer cells, which may be therapeutically targeted by novel molecularly targeted agents. Unfortunately, data on replication stress is largely based on experimental models. Further investigation of replication stress in clinical samples is required to optimally implement novel therapeutics. To uncover the relation between oncogene expression, replication stress, and clinical features of breast cancer subgroups, we immunohistochemically analyzed the expression of a panel of oncogenes (Cyclin E, c-Myc, and Cdc25A,) and markers of replication stress (phospho-Ser33-RPA32 and γ-H2AX) in breast tumor tissues prior to treatment (n = 384). Triple-negative breast cancers (TNBCs) exhibited the highest levels of phospho-Ser33-RPA32 (P < 0.001 for all tests) and γ-H2AX (P < 0.05 for all tests). Moreover, expression levels of Cyclin E (P < 0.001 for all tests) and c-Myc (P < 0.001 for all tests) were highest in TNBCs. Expression of Cyclin E positively correlated with phospho-RPA32 (Spearman correlation r = 0.37, P < 0.001) and γ-H2AX (Spearman correlation r = 0.63, P < 0.001). Combined, these data indicate that, among breast cancers, replication stress is predominantly observed in TNBCs, and is associated with expression levels of Cyclin E. These results indicate that Cyclin E overexpression may be used as a biomarker for patient selection in the clinical evaluation of drugs that target the DNA replication stress response.

DOI: https://doi.org/10.1038/s41523-020-00181-w
PLoS Genet. 2021 Sep 09. 17(9): e1009763

Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion.

Pei-Shang Wu, Jan Grosser, Donald P Cameron, Laura Baranello, Lena Ström.

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

DOI: https://doi.org/10.1371/journal.pgen.1009763
Nucleic Acids Res. 2021 Sep 11. pii: gkab771. [Epub ahead of print]

TARG1 protects against toxic DNA ADP-ribosylation.

Callum Tromans-Coia, Andrea Sanchi, Giuliana K Moeller, Gyula Timinszky, Massimo Lopes, Ivan Ahel.

ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed 'DarT' from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation.

DOI: https://doi.org/10.1093/nar/gkab771
Cell Rep. 2021 Sep 07. pii: S2211-1247(21)01112-8. [Epub ahead of print]36(10): 109668

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

Tanima SenGupta, Konstantinos Palikaras, Ying Q Esbensen, Georgios Konstantinidis, Francisco Jose Naranjo Galindo, Kavya Achanta, Henok Kassahun, Ioanna Stavgiannoudaki, Vilhelm A Bohr, Mansour Akbari, Johannes Gaare, Charalampos Tzoulis, Nektarios Tavernarakis, Hilde Nilsen.

  Aging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Keywords:  C. elegans; DNA-glycosylase; NTH-1; Parkinson disease; aging; base excision repair; hydrogen peroxide; mitohormesis; neurodegeneration; oxidative DNA damage

DOI:  https://doi.org/10.1016/j.celrep.2021.109668
J Pharmacol Exp Ther. 2021 Sep 09. pii: JPET-MR-2021-000758. [Epub ahead of print]

5-Aza-4'-thio-2'-deoxycytidine, a new orally bioavailable non-toxic "best-in-class" DNMT1 depleting agent in clinical development.

William B Parker, Jaideep V Thottassery.

  DNA methyltransferase 1 (DNMT1) is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation dependent disorders. Two DNMT1 depleting agents aza-dCyd (5-aza-2'-deoxycytidine, decitabine) and aza-Cyd (5-aza-cytidine, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and have also been investigated for non-oncology indications such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment for long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models, but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1 depleting agent with respect to aza-dCyd, and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. Significance Statement Aza-T-dCyd is a potent DNMT1 depleting agent. Although similar in structure to decitabine (aza-dCyd) its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.

Keywords:  DNA methylation; Nucleoside/Nucleotide derivatives; nucleosides

DOI:  https://doi.org/10.1124/jpet.121.000758
Elife. 2021 Sep 06. pii: e71420. [Epub ahead of print]10

Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome.

Luka Bacic, Guillaume Gaullier, Anton Sabantsev, Laura C Lehmann, Klaus Brackmann, Despoina Dimakou, Mario Halic, Graeme Hewitt, Simon Boulton, Sebastian Deindl.

  The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.

Keywords:  chromosomes; gene expression; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.71420
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00049-6. [Epub ahead of print]364 139-161

Reactive oxygen species and DNA damage response in cancer.

Xavier Renaudin.

  Compared with normal cells, cancer cells often have an increase in reactive oxygen species (ROS) level. This high level of ROS allows the activation of different pathways essential for cellular transformation and tumorigenesis development. Increase of ROS can be due to increase of production or decrease of detoxification, both situations being well described in various cancers. Oxidative stress is involved at every step of cancer development from the initiation to the metastasis. How ROS arise is still a matter of debates and may vary with tissues, cell types or other conditions and may happen following a large diversity of mechanisms. Both oncogenic and tumor suppressor mutations can lead to an increase of ROS. In this chapter, I review how ROS are produced and detoxified and how ROS can damage DNA leading to the genomic instability featured in cancers.

Keywords:  Cancer; DNA damage; DNA repair; Genomic instability; Mutational signatures; Reactive oxygen species

DOI:  https://doi.org/10.1016/bs.ircmb.2021.04.001
Curr Opin Genet Dev. 2021 Sep 01. pii: S0959-437X(21)00102-7. [Epub ahead of print]71 163-170

Break-induced replication mechanisms in yeast and mammals.

Xiaohua Wu, Anna Malkova.

Break-induced replication (BIR) is a pathway specialized in repair of double-strand DNA breaks with only one end capable of invading homologous template that can arise following replication collapse, telomere erosion or DNA cutting by site-specific endonucleases. For a long time, yeast remained the only model system to study BIR. Studies in yeast demonstrated that BIR represents an unusual mode of DNA synthesis that is driven by a migrating bubble and leads to conservative inheritance of newly synthesized DNA. This unusual type of DNA synthesis leads to high levels of mutations and chromosome rearrangements. Recently, multiple examples of BIR were uncovered in mammalian cells that allowed the comparison of BIR between organisms. It appeared initially that BIR in mammalian cells is predominantly independent of RAD51, and therefore different from BIR that is predominantly Rad51-dependent in yeast. However, a series of systematic studies utilizing site-specific DNA breaks for BIR initiation in mammalian reporters led to the discovery of highly efficient RAD51-dependent BIR, allowing side-by side comparison with BIR in yeast which is the focus of this review.

DOI: https://doi.org/10.1016/j.gde.2021.08.002
Oncogene. 2021 Sep 10.

A combination of PARP and CHK1 inhibitors efficiently antagonizes MYCN-driven tumors.

Stefano Di Giulio, Valeria Colicchia, Fabio Pastorino, Flaminia Pedretti, Francesca Fabretti, Vittoria Nicolis di Robilant, Valentina Ramponi, Giorgia Scafetta, Marta Moretti, Valerio Licursi, Francesca Belardinilli, Giovanna Peruzzi, Paola Infante, Bianca Maria Goffredo, Anna Coppa, Gianluca Canettieri, Armando Bartolazzi, Mirco Ponzoni, Giuseppe Giannini, Marialaura Petroni.

MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.

DOI: https://doi.org/10.1038/s41388-021-02003-0
Nat Commun. 2021 Sep 10. 12(1): 5386

Inhibition of CK1ε potentiates the therapeutic efficacy of CDK4/6 inhibitor in breast cancer.

Fabin Dang, Li Nie, Jin Zhou, Kouhei Shimizu, Chen Chu, Zhong Wu, Anne Fassl, Shizhong Ke, Yuangao Wang, Jinfang Zhang, Tao Zhang, Zhenbo Tu, Hiroyuki Inuzuka, Piotr Sicinski, Adam J Bass, Wenyi Wei.

Although inhibitors targeting CDK4/6 kinases (CDK4/6i) have shown promising clinical prospect in treating ER+/HER2- breast cancers, acquired drug resistance is frequently observed and mechanistic knowledge is needed to harness their full clinical potential. Here, we report that inhibition of CDK4/6 promotes βTrCP1-mediated ubiquitination and proteasomal degradation of RB1, and facilitates SP1-mediated CDK6 transcriptional activation. Intriguingly, suppression of CK1ε not only efficiently prevents RB1 from degradation, but also prevents CDK4/6i-induced CDK6 upregulation by modulating SP1 protein stability, thereby enhancing CDK4/6i efficacy and overcoming resistance to CDK4/6i in vitro. Using xenograft and PDX models, we further demonstrate that combined inhibition of CK1ε and CDK4/6 results in marked suppression of tumor growth in vivo. Altogether, these results uncover the molecular mechanisms by which CDK4/6i treatment alters RB1 and CDK6 protein abundance, thereby driving the acquisition of CDK4/6i resistance. Importantly, we identify CK1ε as an effective target for potentiating the therapeutic efficacy of CDK4/6 inhibitors.

DOI: https://doi.org/10.1038/s41467-021-25700-6
Elife. 2021 Sep 06. pii: e66198. [Epub ahead of print]10

Integrated evaluation of telomerase activation and telomere maintenance across cancer cell lines.

Kevin Hu, Mahmoud Ghandi, Franklin W Huang.

  In cancer, telomere maintenance is critical for the development of replicative immortality. Using genome sequences from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer Project, we calculated telomere content across 1,299 cancer cell lines. We find that telomerase reverse transcriptase (TERT) expression correlates with telomere content in lung, central nervous system, and leukemia cell lines. Using CRISPR/Cas9 screening data, we show that lower telomeric content is associated with dependency of CST telomere maintenance genes. Increased dependencies of shelterin members are associated with wild-type TP53 status. Investigating the epigenetic regulation of TERT, we find widespread allele-specific expression in promoter-wildtype contexts. TERT promoter-mutant cell lines exhibit hypomethylation at PRC2-repressed regions, suggesting a cooperative global epigenetic state in the reactivation of telomerase. By incorporating telomere content with genomic features across comprehensively characterized cell lines, we provide further insights into the role of telomere regulation in cancer immortality.

Keywords:  cancer biology; computational biology; human; systems biology

DOI:  https://doi.org/10.7554/eLife.66198